Neuromethods 191

Eirini Papagiakoumou Editor

All-Optical
Methods to
Study Neuronal
Function
NEUROMETHODS

Series Editor
Wolfgang Walz
University of Saskatchewan
Saskatoon, SK, Canada

For further volumes:

http://www.springer.com/series/7657
Neuromethods publishes cutting-edge methods and protocols in all areas of neuroscience as
well as translational neurological and mental research. Each volume in the series offers tested
laboratory protocols, step-by-step methods for reproducible lab experiments and addresses
methodological controversies and pitfalls in order to aid neuroscientists in experimentation.
Neuromethods focuses on traditional and emerging topics with wide-ranging implications to
brain function, such as electrophysiology, neuroimaging, behavioral analysis, genomics,
neurodegeneration, translational research and clinical trials. Neuromethods provides investi-
gators and trainees with highly useful compendiums of key strategies and approaches for
successful research in animal and human brain function including translational “bench to
bedside” approaches to mental and neurological diseases.
All-Optical Methods to Study
Neuronal Function

Edited by

Eirini Papagiakoumou
Institut de la Vision, Sorbonne Université, INSERM, CNRS, Paris, France
Editor
Eirini Papagiakoumou
Institut de la Vision
Sorbonne Université, INSERM, CNRS
Paris, France

ISSN 0893-2336 ISSN 1940-6045 (electronic)

Neuromethods
ISBN 978-1-0716-2763-1 ISBN 978-1-0716-2764-8 (eBook)
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Preface to the Series

Experimental life sciences have two basic foundations: concepts and tools. The Neuro-
methods series focuses on the tools and techniques unique to the investigation of the
nervous system and excitable cells. It will not, however, shortchange the concept side of
things as care has been taken to integrate these tools within the context of the concepts and
questions under investigation. In this way, the series is unique in that it not only collects
protocols but also includes theoretical background information and critiques which led to
the methods and their development. Thus, it gives the reader a better understanding of the
origin of the techniques and their potential future development. The Neuromethods
publishing program strikes a balance between recent and exciting developments like those
concerning new animal models of disease, imaging, in vivo methods, and more established
techniques, including, for example, immunocytochemistry and electrophysiological tech-
nologies. New trainees in neurosciences still need a sound footing in these older methods in
order to apply a critical approach to their results.
Under the guidance of its founders, Alan Boulton and Glen Baker, the Neuromethods
series has been a success since its first volume published through Humana Press in 1985. The
series continues to flourish through many changes over the years. It is now published under
the umbrella of Springer Protocols. While methods involving brain research have changed a
lot since the series started, the publishing environment and technology have changed even
more radically. Neuromethods has the distinct layout and style of the Springer Protocols
program, designed specifically for readability and ease of reference in a laboratory setting.
The careful application of methods is potentially the most important step in the process
of scientific inquiry. In the past, new methodologies led the way in developing new dis-
ciplines in the biological and medical sciences. For example, physiology emerged out of
anatomy in the nineteenth century by harnessing new methods based on the newly discov-
ered phenomenon of electricity. Nowadays, the relationships between disciplines and meth-
ods are more complex. Methods are now widely shared between disciplines and research
areas. New developments in electronic publishing make it possible for scientists that
encounter new methods to quickly find sources of information electronically. The design
of individual volumes and chapters in this series takes this new access technology into
account. Springer Protocols makes it possible to download single protocols separately. In
addition, Springer makes its print-on-demand technology available globally. A print copy
can therefore be acquired quickly and for a competitive price anywhere in the world.

Saskatoon, SK, Canada Wolfgang Walz

v
Preface

Control and monitoring of neuronal activity with light, what is often called all-optical
manipulation of neurons, is admittedly the most adequate method for addressing questions
regarding communication of neurons in a neural circuit or between different circuits. The
big and continuously expanding toolbox of molecular photosensitive probes that activate/
inhibit or image (through membrane voltage or calcium changes) neuronal activity, in
combination with the development of original light-microscopy methods for stimulating
these probes, has tremendously contributed to this direction and led to innovative experi-
mental concepts, where neurons can be manipulated either as entities or as ensembles.
Indeed, the use of light offers suitable spatiotemporal resolution to manipulate neurons at
single-cell specificity. At the same time, it gives access to a large population of cells simulta-
neously via scanless, parallel illumination methods.
Neural circuit studies are more conclusive for addressing biological questions when
performed in vivo. In this sense, they necessitate three-dimensional (3D) accessibility both
for activation and imaging, at physiological time scales (few-ms scale activation and imag-
ing). 3D imaging approaches enable today using complementary strategies to access
volumes extending up to hundreds of micrometers in the axial direction. On the contrary,
the development of 3D photoactivation methods is more recent. These systems use
computer-generated holography (CGH), a technique based on phase modulation of the
excitation beam’s wavefront, to create multiple excitation regions of interest. Thanks to
3D-CGH, used either solely (parallel methods) or in its diffraction-limit version in combi-
nation with scanning of the holographic beamlets, it is nowadays possible to simultaneously
activate multiple neurons providing both the adequate temporal resolution, as well as the
spatial resolution for near single-cell precision. High spatial resolution and selectivity is often
assured by implementing those methods with two-photon excitation.
Although the first experiments of all-optical manipulation of neuronal activity per-
formed activation of neurons via uncaging of caged glutamate, the term all-optical today
is mostly related to the combination of functional imaging and optogenetic activation. There
is a growing number of studies using optogenetics and calcium imaging to explore several
hypotheses in cellular and systems neuroscience, nevertheless a full optical neuronal control
remains a challenge in terms of achieving reliable delivery and expression of sensors and
actuators in the same neurons, eliminating the crosstalk between imaging and activation,
and recording and stimulating with single-neuron and single-action-potential precision.
In this volume, we opt to give an overview of the methods that have been used so far in
all-optical experiments, but also to present other promising approaches potentially useful in
this domain. The book is addressed to people experienced in different disciplines, such as
physicists, engineers, and neuroscientists; therefore, it starts by providing some basic but
fundamental background information in terms of both physiology and optics in the context
of all-optical two-photon neurophysiology experiments (Chap. 1), followed by some
prompts for the selection of appropriate actuators and sensors, and functional imaging
methodologies that drive the choice of both, together with the suitable laser sources for
two-photon excitation (Chaps. 1 and 2).

vii
viii Preface

We then present, in detail, optical methods that have been used for photoactivation and
imaging. The reader can find the design principles and, in some cases, hardware implemen-
tation for methods like generalized phase contrast (Chap. 1), computer-generated hologra-
phy and scanning approaches (Chaps. 3 and 4), temporal focusing (Chaps. 1 and 4), as well
as guidance to the entire workflow for an all-optical experiment in circuit neuroscience
(Chap. 5). In Chap. 6, possibilities and limitations of optogenetic actuators are discussed
within the context of an all-optical single-beam experiment by giving insights into the
photophysical properties of actuators. Detailed methods are provided in Chap. 7 on a
miniature head mounted two-photon fiber-coupled microscope for imaging neuronal activ-
ity in vivo in freely moving animals, while Chap. 8 discusses how 3D holographic optoge-
netics can be added to a home-built light sheet microscope.
This book also attributes a part on innovative imaging techniques that could be
implemented in the framework of an all-optical electrophysiology experiment. Chapter 9
pronounces the theories of temporal focusing in combination with single-pixel detection for
imaging of fast collective biological processes at depth, over a widefield and at high
spatiotemporal resolution. Chapter 10 presents alternative imaging methods at synaptic
resolution, such as two-photon fluorescence microscopy equipped with Bessel focus scan-
ning technology and widefield fluorescence microscopy with optical sectioning ability.
The implementation of simultaneous two-photon imaging and holographic optoge-
netics in conjunction with population analytical tools or with psychophysical measurements
of evoked synthetic percepts to confirm a precise relationship between optical manipulations
and behavior is presented in Chaps. 11 and 12.
Finally, in Chap. 13, an approach for label-free imaging is presented: stimulated Raman
scattering (SRS) microscopy, a non-linear imaging method for visualizing a molecule based
on its chemical properties, and the way to integrate it in a commercial multiphoton
microscope, eventually useful for label-free functional imaging.
The use of all-optical methods for studying the neural function is a multiparametric and
arduous project to setup. It entails a multidisciplinary know-how both for developing the
optical system and the adequate biological preparation, and sometimes expensive equip-
ment, especially when multiphoton excitation is considered. We hope that this book can
serve as a guide to facilitate the first requirement, establishing a useful reference for groups
starting their activity in this domain, and give insights on the optical systems, the choice of
actuators and sensors, but also stimulate ideas for ground-breaking configurations and
experiments.

Institut de la Vision Eirini Papagiakoumou

Sorbonne Université, INSERM, CNRS, Paris, France
Contents

Preface to the Series . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi

1 Optical Manipulation and Recording of Neural Activity with

Wavefront Engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Ruth R. Sims, Imane Bendifallah, Kris Blanchard, Dimitrii Tanese,
Valentina Emiliani, and Eirini Papagiakoumou
2 Balancing the Fluorescence Imaging Budget for All-Optical
Neurophysiology Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Peter Quicke, Carmel L. Howe, and Amanda J. Foust
3 Light-Based Neuronal Circuit Probing in Living Brains at High
Resolution: Constraints and Layouts for Integrating Neuronal Activity
Recording and Modulation in Three Dimensions . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
Matteo Bruzzone, Enrico Chiarello, Andrea Maset, Aram Megighian,
Claudia Lodovichi, and Marco dal Maschio
4 High-Speed All-Optical Neural Interfaces with 3D Temporally
Focused Holography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
Ian Anton Oldenburg, Hayley Anne Bounds, and Nicolas C. Pégard
5 An All-Optical Physiology Pipeline Toward Highly Specific and
Artifact-Free Circuit Mapping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
Hendrik Backhaus, Nicolas Ruffini, Anna Wierczeiko,
and Albrecht Stroh
6 Spatial and Temporal Considerations of Optogenetic Tools in
an All-Optical Single-Beam Experiment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
Damaris Holder and Matthias Prigge
7 Miniature Multiphoton Microscopes for Recording Neural Activity
in Freely Moving Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
Baris N. Ozbay, Gregory L. Futia, Ming Ma, Connor McCullough,
Michael D. Young, Diego Restrepo, and Emily A. Gibson
8 Optogenetics and Light-Sheet Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
Laura Maddalena, Paolo Pozzi, Nicolò G. Ceffa
Bas van der Hoeven, and Elizabeth C. Carroll
9 Widefield Multiphoton Imaging at Depth with Temporal Focusing . . . . . . . . . . . 263
Philip Wijesinghe and Kishan Dholakia
10 High-Speed Neural Imaging with Synaptic Resolution: Bessel
Focus Scanning Two-Photon Microscopy and Optical-Sectioning
Widefield Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293
Guanghan Meng, Qinrong Zhang, and Na Ji
11 Optical and Analytical Methods to Visualize and Manipulate
Cortical Ensembles and Behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331
Luis Carrillo-Reid, Weijian Yang, and Rafael Yuste
ix
x Contents

12 Illuminating Neural Computation Using Precision

Optogenetics-Controlled Synthetic Perception . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363
Jonathan V. Gill, Gilad M. Lerman, Edmund Chong,
Dmitry Rinberg, and Shy Shoham
13 Spectrally Focused Stimulated Raman Scattering (sf-SRS)
Microscopy for Label-Free Investigations of Molecular Mechanisms
in Living Organisms. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393
Tamás Váczi, Lászlo Himics, Matteo Bruzzone, Miklo s Veres,
and Marco dal Maschio

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 417
Contributors

HENDRIK BACKHAUS • Leibniz Institute for Resilience Research, Mainz, Germany

IMANE BENDIFALLAH • Wavefront-Engineering Microscopy Group, Photonics Department,
Institut de la Vision, Sorbonne Université, INSERM, CNRS, Paris, France
KRIS BLANCHARD • Wavefront-Engineering Microscopy Group, Photonics Department,
Institut de la Vision, Sorbonne Université, INSERM, CNRS, Paris, France; Luyef
Biotechnologies, Santiago, Chile
HAYLEY ANNE BOUNDS • Helen Wills Neuroscience Institute, University of California,
Berkeley, CA, USA
MATTEO BRUZZONE • Padua Neuroscience Center – PNC, University of Padua, Padova,
Italy; Padova Neuroscience Center, Universita’ degli Studi di Padova, Padova, Italy
LUIS CARRILLO-REID • Neurobiology Institute, National Autonomous University of Mexico,
Mexico City, Mexico
ELIZABETH C. CARROLL • Department of Imaging Physics, Delft University of Technology,
Delft, The Netherlands
NICOLÒ G. CEFFA • Department of Imaging Physics, Delft University of Technology, Delft,
The Netherlands
ENRICO CHIARELLO • Padua Neuroscience Center – PNC, University of Padua, Padova,
Italy
EDMUND CHONG • University College London, London, UK
MARCO DAL MASCHIO • Padua Neuroscience Center – PNC, University of Padua, Padova,
Italy; Department of Biomedical Sciences, University of Padua, Padova, Italy
KISHAN DHOLAKIA • SUPA, School of Physics and Astronomy, University of St Andrews,
Scotland, UK
VALENTINA EMILIANI • Wavefront-Engineering Microscopy Group, Photonics Department,
Institut de la Vision, Sorbonne Université, INSERM, CNRS, Paris, France
AMANDA J. FOUST • Department of Bioengineering, Imperial College London, London, UK;
Centre for Neurotechnology, Imperial College London, London, UK
GREGORY L. FUTIA • Department of Bioengineering, University of Colorado Denver, Aurora,
CO, USA
EMILY A. GIBSON • Department of Bioengineering, University of Colorado Denver, Aurora,
CO, USA
JONATHAN V. GILL • Neuroscience Institute, NYU Langone Health, New York, NY, USA
LÁSZLÓ HIMICS • Institute for Solid State Physics and Optics, Wigner Research Centre for
Physics, Budapest, Hungary
BAS VAN DER HOEVEN • Department of Imaging Physics, Delft University of Technology, Delft,
The Netherlands
DAMARIS HOLDER • Department Cellular Neuroscience, Leibniz Institute for Neurobiology,
Magdeburg, Germany
CARMEL L. HOWE • Department of Bioengineering, Imperial College London, London, UK;
Centre for Neurotechnology, Imperial College London, London, UK
NA JI • Department of Molecular and Cell Biology, University of California, Berkeley, CA,
USA; Department of Physics, University of California, Berkeley, CA, USA; Helen Wills
Neuroscience Institute, University of California, Berkeley, CA, USA; Molecular Biophysics

xi
xii Contributors

and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley,

CA, USA
GILAD M. LERMAN • Neuroscience Institute, NYU Langone Health, New York, NY, USA
CLAUDIA LODOVICHI • Padua Neuroscience Center – PNC, University of Padua, Padova,
Italy; Veneto Institute of Molecular Medicine, VIMM, Padova, Italy; Department of
Biomedical Sciences, University of Padua, Padova, Italy; Institute of Neuroscience,
CNR-IN, Padova, Italy
MING MA • Department of Cell and Developmental Biology, University of Colorado Denver,
Aurora, CO, USA
LAURA MADDALENA • Department of Imaging Physics, Delft University of Technology, Delft,
The Netherlands
ANDREA MASET • Padua Neuroscience Center – PNC, University of Padua, Padova, Italy;
Veneto Institute of Molecular Medicine, VIMM, Padova, Italy
CONNOR MCCULLOUGH • Department of Bioengineering, University of Colorado Denver,
Aurora, CO, USA
ARAM MEGIGHIAN • Padua Neuroscience Center – PNC, University of Padua, Padova, Italy;
Department of Biomedical Sciences, University of Padua, Padova, Italy
GUANGHAN MENG • Department of Molecular and Cell Biology, University of California,
Berkeley, CA, USA
IAN ANTÓN OLDENBURG • Department of Neuroscience and Cell Biology, Robert Wood
Johnson Medical School, and The Center for Advanced Biotechnology and Medicine,
Rutgers the State University of New Jersey, Piscataway, NJ, USA
BARIS N. OZBAY • Department of Bioengineering, University of Colorado Denver, Aurora,
CO, USA
EIRINI PAPAGIAKOUMOU • Wavefront-Engineering Microscopy Group, Photonics Department,
Institut de la Vision, Sorbonne Université, INSERM, CNRS, Paris, France
NICOLAS C. PÉGARD • Department of Applied Physical Sciences, University of North Carolina
at Chapel Hill, Chapel Hill, NC, USA; Department of Biomedical Engineering,
University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; UNC Neuroscience
Center, Carolina Stress Initiative, Chapel Hill, NC, USA
PAOLO POZZI • Biomedical, Metabolic and Neural Sciences, University of Modena and
Reggio Emilia, Modena, Italy
MATTHIAS PRIGGE • Research Group Neuromodulatory Networks, Center for Behavioral
Brain Sciences, Leibniz Institute for Neurobiology, Magdeburg, Germany
PETER QUICKE • Department of Bioengineering, Imperial College London, London, UK;
Centre for Neurotechnology, Imperial College London, London, UK
DIEGO RESTREPO • Department of Cell and Developmental Biology, University of Colorado
Denver, Aurora, CO, USA
DMITRY RINBERG • Neuroscience Institute, NYU Langone Health, New York, NY, USA;
Center for Neural Science, New York University, New York, NY, USA; Department of
Physics, New York University, New York, NY, USA
NICOLAS RUFFINI • Leibniz Institute for Resilience Research, Mainz, Germany
SHY SHOHAM • Neuroscience Institute, NYU Langone Health, New York, NY, USA;
Tech4Health Institute, NYU Langone Health, New York, NY, USA; Department of
Ophthalmology, NYU Langone Health, New York, NY, USA
RUTH R. SIMS • Wavefront-Engineering Microscopy Group, Photonics Department, Institut
de la Vision, Sorbonne Université, INSERM, CNRS, Paris, France
 Contributors xiii

ALBRECHT STROH • Leibniz Institute for Resilience Research, Mainz, Germany; Institute of
Pathophysiology, University Medical Center Mainz, Mainz, Germany
DIMITRII TANESE • Wavefront-Engineering Microscopy Group, Photonics Department,
Institut de la Vision, Sorbonne Université, INSERM, CNRS, Paris, France
TAMÁS VÁCZI • Institute for Solid State Physics and Optics, Wigner Research Centre for
Physics, Budapest, Hungary
MIKLÓS VERES • Institute for Solid State Physics and Optics, Wigner Research Centre for
Physics, Budapest, Hungary
ANNA WIERCZEIKO • Leibniz Institute for Resilience Research, Mainz, Germany
PHILIP WIJESINGHE • SUPA, School of Physics and Astronomy, University of St Andrews,
Scotland, UK
WEIJIAN YANG • University of California at Davis, Davis, CA, USA
MICHAEL D. YOUNG • Department of Bioengineering, University of Colorado Denver,
Aurora, CO, USA
RAFAEL YUSTE • NeuroTechnology Center, Columbia University, New York, NY, USA
QINRONG ZHANG • Department of Physics, University of California, Berkeley, CA, USA
 Chapter 1

Optical Manipulation and Recording of Neural Activity

with Wavefront Engineering
Ruth R. Sims, Imane Bendifallah, Kris Blanchard, Dimitrii Tanese,
Valentina Emiliani, and Eirini Papagiakoumou

Abstract
One of the central goals of neuroscience is to decipher the specific contributions of neural mechanisms to
different aspects of sensory perception. Since achieving this goal requires tools capable of precisely
perturbing and monitoring neural activity across a multitude of spatiotemporal scales, this aim has inspired
the innovation of many optical technologies capable of manipulating and recording neural activity in a
minimally invasive manner. The interdisciplinary nature of neurophotonics requires a broad knowledge base
in order to successfully develop and apply these technologies, and one of the principal aims of this chapter is
to provide some basic but fundamental background information in terms of both physiology and optics in
the context of all-optical two-photon neurophysiology experiments. Most of this information is expected to
be familiar to readers experienced in either domain, but is presented here with the aim of bridging the divide
between disciplines in order to enable physicists and engineers to develop useful optical technologies or for
neuroscientists to select appropriate tools and apply them to their maximum potential.
The first section of this chapter is dedicated to a brief overview of some basic principles of neural
physiology relevant for controlling and recording neuronal activity using light. Then, the selection of
appropriate actuators and sensors for manipulating and monitoring particular neural signals is discussed,
with particular attention paid to kinetics and sensitivity. Some considerations for minimizing crosstalk in
optical neurophysiology experiments are also introduced. Next, an overview of the state-of-the-art optical
technologies is provided, including a description of suitable laser sources for two-photon excitation
according to particular experimental requirements. Finally, some detailed, technical, information regarding
the specific wavefront engineering approaches known as Generalized Phase Contrast (GPC) and temporal
focusing is provided.

Key words All-optical neurophysiology, Light shaping, Temporal focusing, Generalized phase con-
trast, Computer-generated holography, Functional imaging, Optogenetics, Molecular tools, GECIs,
GEVIs

1 Introduction

Experiments in modern neuroscience require techniques capable of

monitoring (“reading”) and manipulating (“writing”) neural activ-
ity across a staggering range of spatiotemporal scales. For instance,

Eirini Papagiakoumou (ed.), All-Optical Methods to Study Neuronal Function, Neuromethods, vol. 191,
https://doi.org/10.1007/978-1-0716-2764-8_1, © The Author(s) 2023

1
2 Ruth R. Sims et al.

Fig. 1 Different spatiotemporal scales encountered in all-optical neurophysiology experiments. Relevant

spatial (a–d) and temporal (e–h) scales encountered in all-optical neurophysiology experiments. (a) Ion
channels and pumps, with nanometer dimensions, residing within the cell membrane are ultimately respon-
sible for the excitability of individual neurons. (b) All-optical neurophysiology experiments aiming for photo-
activation with single-cell resolution target the neuronal soma (~10 μm diameter). (c) Neurons distributed
within millimeter volumes that display coordinated activity are termed neural ensembles or engrams. The
primary goal of a growing number of all-optical neurophysiology experiments is to manipulate these
functionally defined circuits. (d) Neural activity governing a particular behavior is commonly distributed across
multiple, often non-contiguous, brain regions which can span mesoscale (mm–cm) distances. (e) Neurons are
depolarized by excitatory inputs (EPSPs) and hyperpolarized by inhibitory inputs (IPSPs) on timescales of tens
of milliseconds [1]. (f) Larger and longer changes in membrane potential are sometimes observed when
neurons receive multiple synaptic inputs. (g) Action potentials (APs) are fired when the somatic membrane
potential is depolarized beyond threshold (-55 mV). Action potentials invert the membrane potential on
millisecond timescales. (h) Individual neurons display characteristic patterns of AP firing. Many all-optical
neurophysiology experiments (i) simultaneously monitor the dynamic pattern of AP firing in different neurons
or (ii) record the firing response of particular neurons to external stimuli (Si) in trials before replaying and
manipulating these physiological activity patterns using photostimulation and inhibition

ion channels have nanometer dimensions (Fig. 1a) and undergo

conformational changes on micro- to millisecond timescales,
whereas neuronal circuits in human brains span decimeters
(Fig. 1d) and can be refined over the course of a lifetime. Due to
the minimally invasive nature of infrared photons in brain tissue, a
plethora of optical technologies, based on multiphoton excitation,
spanning these spatiotemporal scales have been developed and the
toolbox of optical actuators and indicators of neural activity has
continuously expanded and evolved. As a result of this rapid multi-
disciplinary progress, optical activation and inhibition of genetically
defined classes of neurons can now be achieved using a variety of
light-gated actuators (mainly channelrhodopsins) and neural activ-
ity can be detected using highly specific and sensitive fluorescent
probes; including calcium and voltage indicators. The field of
optogenetics in neuroscience has matured to such an extent that
 All-Optical Neuronal Manipulation with Wavefront Engineering 3

two-photon all-optical experiments can be performed in-vivo,

whereby signals from multiple neurons constituting neural circuits
distributed across different brain regions can be elicited and
recorded optically, with single-cell and sub-millisecond
precision [2].
However, since different opsins and reporters can exhibit vastly
different photophysical characteristics, it is necessary to optimize
all-optical neurophysiology experiments according to the specific
requirements of each biological question. All-optical experiments
are challenging, and their success relies on the careful selection and
co-expression of an appropriate actuator-sensor combination, a
suitable photoactivation approach for precise and efficient excita-
tion of the desired population of neurons, and a sufficiently sensi-
tive imaging method capable of recording neural activity without
spurious activation of the opsin expressing cells.
This chapter introduces and reviews some of the most impor-
tant molecular and optical technologies for manipulating and
recording neural activity and highlights critical parameters com-
mon to most all-optical neurophysiology experiments. Since many
of these technologies are described in greater detail in subsequent
chapters of this book, we refer the reader to these chapters and
instead provide specific technical details for implementing
generalized phase contrast (GPC) and temporal focusing. Finally,
a detailed protocol for the preparation of mice hippocampal orga-
notypic slices (a commonly used biological preparation) expressing
both optical actuators and indicators for all-optical interrogation of
neuronal circuits is included.

2 State-of-the-art Technologies for All-Optical Neurophysiology

All-optical neurophysiology experiments require appropriate

molecular tools such as light triggered actuators capable of
controlling ion fluxes through the cell’s membrane and thus the
electrical activity of neurons [3–8] and fluorescent probes which
provide optical readouts of neural activity [9–14]. A wide variety of
molecular tools, exhibiting different photophysical properties,
have been discovered and engineered to meet this requirement.
This chapter will focus on tools capable of optically manipulating
and recording of neuronal activity in scattering tissue, which
commonly rely on two-photon excitation (2PE) based on the
near-simultaneous absorption of two infrared (IR) photons
[15–17]. The necessity of exploiting non-linear optical phenomena
such as 2PE for performing spatially localized experiments in scat-
tering tissue is well documented [18]. 2PE laser scanning micros-
copy (2PE-LSM) by rapid displacement of a tightly focused, pulsed,
laser beam using galvanometric mirrors [19] is the gold-standard
technique for imaging in turbid biological tissue and has also been
4 Ruth R. Sims et al.

applied to manipulate neural activity [20]. However, since in some

cases this approach does not provide sufficient temporal resolution,
a large number of different methods have been developed for
optimized excitation of channelrhodopsins and indicators of neural
activity. This section will provide an overview of the photophysical
requirements of the molecular tools commonly used in all-optical
neurophysiology experiments, before reviewing some state-of-the-
art sequential and parallel 2PE approaches and evaluating their
suitability with respect to exciting and imaging these actuators
and indicators.

2.1 Photophysical Useful technologies for all-optical neurophysiology experiments

Properties of Common must be capable of eliciting, suppressing, and recording neural
Molecular Tools Used activity on physiologically relevant spatiotemporal scales, high-
for All-Optical lighted in Fig. 1a–h. In order to describe and relate the photophy-
Neurophysiology sical properties of molecular tools to specific physiological
benchmarks, some relevant properties of single neurons and neural
networks will first be reviewed.
Although the extracellular and cytoplasmic environment of any
neuron is electrically neutral, the immediate surrounding of plasma
membrane (an electrical isolator) has very thin clouds of negative
and positive ions that are differentially spread on its inner and outer
surfaces (Fig. 1a) [21]. At rest, the inner cytoplasmic surface has an
excess of negative charge with respect to the extracellular side. This
electrical gradient is actively generated and maintained by the
action of the sodium–potassium pump and the presence of passive
ion channels (Fig. 1a), which are ultimately responsible for cellular
excitability. The difference in charge distribution across the mem-
brane gives rise to a difference in electric potential, the membrane
potential (Vm), which for most neurons has a somatic value of
around -70 mV (Fig. 1b). During communication via synaptic
transmission between connected neurons (Fig. 1c, d), the Vm of a
particular neuron is altered by presynaptic excitatory (depolarizing)
and/or inhibitory (hyperpolarizing) inputs (Fig. 1e, f). These per-
turbations of the resting potential are the so-called post-synaptic
potentials (PSPs) and they are processed and integrated by the
soma of the cell. If the net sum of multiple input excitatory or
inhibitory PSPs, arriving within the membrane time constant,
exceeds a threshold value (~ -55 mV), an action potential (spike)
is triggered. Action potentials are highly stereotypical electrical
signals which re-orient the electric field across the neuronal mem-
brane on millisecond timescales (Fig. 1g). Action potentials typi-
cally lead to an elevation of the concentration of cytosolic calcium
through voltage-gated calcium channels, which can last an order of
magnitude longer than the action potentials themselves. Each spike
is communicated to post-synaptic neurons via local and long-range
synaptic connections: pre-synaptic neurons release neurotransmit-
ter onto postsynaptic targets, evoking unitary inhibitory or
 All-Optical Neuronal Manipulation with Wavefront Engineering 5

excitatory post-synaptic potentials (uPSPs). Typically, uPSPs are

small in amplitude and duration while PSPs resulting from the
integration of multiple synaptic inputs have been observed to give
rise to longer and larger variations of somatic membrane potential
(Fig. 1e, f) [22, 23], and ultimately may result in action potential
firing (Fig. 1g). A wide variety of precise patterns and frequencies of
action potential firing have been observed, both for individual
neurons responding to distinct stimuli and for different types of
neurons located in particular brain regions [24] (Fig. 1h). Further-
more, particular patterns of spike firing in many individual neurons
has been observed to be closely correlated with changes in the
external sensory world [25–27], and observations of highly coordi-
nated patterns of activity in ensembles of different neurons [28, 29]
have led to one of the central hypotheses of modern neuroscience:
that higher brain function arises from the interactions between
interconnected neurons [30] (Fig. 1c, d, h). Elucidating the causal
relationship between neural circuits and network function requires
methods capable of stimulating and silencing neurons to mimic
physiological patterns of network activity. This necessitates the
observation and subsequent manipulation of rate and spike timing
across an ensemble of neurons with sub-millisecond temporal
precision.
Following decades of heroic protein engineering efforts,
desired populations of neurons in virtually all genetically tractable
model organisms can now be engineered to express photosensitive
transmembrane proteins known as channelrhodopsins [31]. Chan-
nelrhodopsins are ion channels of microbial origin, which can be
excited into current-conducting states upon light absorption
[32, 33] (Fig. 2a). The first sets of experiments that demonstrated
optical control of neuronal activity using channelrhodopsin were
based on the heterologous expression of Channelrhodopsin-2 from
Chlamydomonas reinhardtii [33, 36, 37]. Since then, a dizzying
number of excitatory and inhibitory opsin variants with different
mechanistic and operational properties have been discovered and
engineered. While the optimal choice of opsin for a given experi-
ment depends on the specific preparation and biological question,
usually opsin variants exhibiting large photocurrents, selectivity for
relevant ions, high light sensitivity, appropriate channel kinetics,
and spectral compatibility are preferred.
Using light to modulate electrical activity in opsin-expressing
neurons generally requires generating photocurrents with sufficient
magnitudes, within the membrane time constant, to depolarize or
hyperpolarize the cells and evoke or inhibit action potentials,
although there are interesting and notable exceptions [38]. The
precise magnitude of photocurrent necessary to evoke or inhibit
spikes depends on the biophysical properties of the membrane such
as input resistance, capacitance, and action potential threshold,
which can vary significantly between neurons. Furthermore, the
6 Ruth R. Sims et al.

Fig. 2 Two-photon characterization of channelrhodopsins. (a) In the simplest conceptual model of the opsin
photocycle, ion channels reside in the closed state. Upon light absorption, the channels open, allowing the
exchange of ions between the cytosol and extracellular space. Depending on the ion selectivity of the channel
and the electrochemical gradient, this flow of ions will hyperpolarize or depolarize the cell membrane and
ultimately inhibit or excite the cell. (b) In reality, the opsin photocycle is more complex, but can reasonably be
approximated by the so-called four-state model. For more details refer to [34, 35]. (c) Opsins can be
characterized using whole-cell voltage patch clamp to measure the currents that flow across the cell
membrane under different conditions. Inset: visualization of a characteristic 12-μm diameter holographic
spot, typically used for parallel 2P- photoactivation. (d) Photocurrent traces recorded in whole cell voltage
patch clamp from CHO (Chinese Hamster Ovary) cells expressing ChRmine as a function of increasing 2P
excitation power (920 nm, 12-μm diameter excitation spot, 200 ms continuous illumination, incident powers
varied between 0 and 50 mW as indicated in the color bar). The characteristic features of the photocurrent
traces (kinetics and peak/stationary photocurrent) are labeled. The magnitude of the photocurrent increases
with power density to saturation. (e) 2P-LSM image of AAV9-CaMKIIa-somBiPOLES-mCerulean expressed in
hippocampal organotypic slice cultures by bulk infection (scale bar represents 50 μm). (f) Photostimulation
(upper, 1100-nm illumination, 0.44 mW/μm2, 5 ms continuous illumination (red bar)) and inhibition (lower,
920-nm illumination, 0.3 mW/μm2, 200 ms illumination during constant current injection (gray bar)) of a single
neuron expressing somBiPOLES with a 12-μm diameter holographic spot

absolute photocurrent magnitude that can be generated in a given

neuron itself depends on many factors – including the specific
properties of the opsin, the degree of expression, the efficiency of
membrane targeting, and the photostimulation modality (for
instance, single- or multi-photon excitation). Excitatory or inhibi-
tory effects can be elicited by expressing different sub-classes of
opsins with specific ion selectivity (for instance, sodium or protons
[39, 40] for excitation and chloride or potassium for inhibition)
[41, 42]. Since the single-channel conductance of most opsins
(~50 fS) is three to four orders of magnitude smaller than that of
ion channels endogenously expressed in neurons (~100 pS)
[32, 43, 44], optical control of neuronal activity relies on the
expression and subsequent excitation of sufficient opsin molecules
distributed over an extended region of the cell membrane. This
consideration is essential in the case of 2P excitation which is
 All-Optical Neuronal Manipulation with Wavefront Engineering 7

intrinsically spatially confined. Hence, one of the first challenges in

optogenetics experiments is achieving reliable, homogeneous, and
functional expression of the desired opsin in the membranes of
target neurons. As a result of intensive protein engineering efforts,
several successful strategies such as codon optimization [45, 46]
and membrane trafficking sequence optimization [47] have been
developed which enable sufficiently high functional expression
levels (~105 opsin molecules per neuron) without detectably per-
turbing membrane physiology [48]. In particular, soma-targeted
opsin variants which utilize the c-terminal targeting motif from the
soma localized potassium channel Kv2.1 have exhibited improved
membrane localization and enhanced photocurrents [49]. Addi-
tionally, variants of inhibitory opsins with similar soma targeting
sequences have been demonstrated to result in fewer antidromic
spikes [42]. The use of soma-targeted opsins has also been demon-
strated to significantly reduce off-target photoactivation [6, 7, 50],
which is a crucial consideration for certain applications.
At physiological membrane potentials, exposing channelrho-
dopsin expressing neurons to the light of an appropriate wave-
length causes the light-gated ion channels to open. This allows
the passage of specific ions across the cell membrane (according
to their electrochemical gradients) and generates photocurrents
(I) that can modulate neuronal activity (Fig. 2a, b). As highlighted
previously, enhancing or suppressing neural activity using optoge-
netics requires the excitation of a sufficient number of opsins within
the membrane time constant to induce adequate depolarization
(or hyperpolarization) of the soma to cause (or prevent) the open-
ing of voltage-gated ion channels. As a result, it is the macroscopic
photocurrent parameters that emerge due to the combined action
of functional, membrane-localized, channels that are relevant for
all-optical neurophysiology experiments and will be discussed
throughout this section.
It is possible to quantify and characterize these macroscopic
photocurrent parameters using electrophysiology, specifically, using
whole-cell voltage clamp (Fig. 2c). For example, in response to
continuous illumination, the photocurrent of a channelrhodopsin
expressing neuron exhibits a characteristic profile with three main
features: (i) an initial peak (Ip) which decays to reach, (ii) a steady
state, (stationary) plateau (Is), and finally (iii) a return to baseline in
the absence of light. Representative photocurrent traces are plotted
for increasing illumination power in Fig. 2d with these main fea-
tures highlighted. The transitions between these features of the
macroscopic photocurrent are commonly parametrized by time
constants τon, τin, and τoff for indicating respectively the time it
takes for the photocurrent to reach the peak when the channels
open, the time for inactivation, and the time to reach zero when the
channels close, which typically exhibit millisecond values [47] but
vary between channelrhodopsins and can also depend on the
8 Ruth R. Sims et al.

intrinsic membrane properties of the cell. The functional profile of

this macroscopic photocurrent has extremely important implica-
tions for 2P optogenetics experiments since it ultimately dictates
the optimum illumination strategy and imposes bounds on tempo-
ral resolution, temporal precision (jitter), and spiking rate [48]. Dif-
ferent characteristics of the macroscopic photocurrent are relevant
for different paradigms of optogenetic photostimulation. For
instance, opsins with fast “off” kinetics are critical for applications
which necessitate the induction of spike trains with high temporal
fidelity (e.g., sub-millisecond precision) and high firing frequencies
[49]. Inducing cell depolarization at faster rates than the kinetics
permit can cause prolonged depolarization to the so-called plateau
potential, induced by excessive cation influx, which can induce
non-uniformity in neuron responses to identical light pulses and,
in some cases, cessation of action potential firing [51]. However, it
is important to note that opsins with faster τoff kinetics generally
require higher light intensities to reach action potential threshold,
which might be an important consideration in experiments aiming
to simultaneously photostimulate large numbers of neurons [52]
where the power for photoactivation must be divided between
targets. On the other hand, to reliably inhibit action potential firing


during a prolonged interval, the macroscopic photocurrent must
exhibit a high steady-state to peak I s=I p ratio and high conductiv-
ity of anions throughout the entire photocycle. Influencing neural
activity over extremely long periods of time without causing photo-
damage, for instance to sensitize entire neuronal networks to native
activity patterns, benefits from the use of a class of opsins with
exceptionally slow kinetics known as step function opsins (SFOs).
SFOs can be photoactivated using a single, low intensity light pulse,
remain in the “open” state for extended timescales (minutes) and
can often be closed using a second pulse of light at a different
wavelength [53]. In conclusion, the photocycle kinetics, sensitivity,
selectivity, and photocurrent magnitude are commonly the primary
considerations when selecting an appropriate opsin for a particular
all-optical neurophysiology experiment. Having selected and suc-
cessfully expressed the channelrhodopsin, the intensity and dura-
tion of delivered light must be titrated until the desired neuronal
response is reliably elicited. To achieve inhibition and excitation of
the same neurons during the same experiment, with different exci-
tation wavelengths, bicistronic constructs such as BiPOLES [54]
can be used (Fig. 2e, f). Such constructs are constituted of excit-
atory and inhibitory channelrhodopsins expressed in tandem for
precise stoichiometry.
In all-optical neurophysiology experiments, photostimulation
is performed alongside functional imaging, both in order to iden-
tify the specific set of neurons to target according to their activity
patterns (in response to a particular stimulus) and also to observe
how the induced patterns of neural activity affect cellular or
 All-Optical Neuronal Manipulation with Wavefront Engineering 9

Fig. 3 Calcium and voltage indicators as reporters of neuronal activity. (a) Cytosolic calcium concentration
increases temporarily as a result of the change in membrane potential that occurs during an action potential.
The intensity-based fluorescent probe GCaMP binds to calcium. This alters the conformation of the circularly
permuted GFP chromophore and results in an increase in fluorescence intensity. (b) Left: 2P-LSM image of
AAV9-Syn-jGCaMP7s expressed in hippocampal organotypic slice cultures by bulk viral infection (scale bar
represents 25 μm). Right: fluorescent jGCaMP7s traces in response to trains of action potentials (5, 15, 40 Hz)
evoked by pulsed current injection into a patched neuron (indicated below). (c) In the case of voltage-sensing
domain (VSD)-based voltage indicators, a change in membrane potential causes a change in conformation of
the VSD, which is covalently linked to a circularly permuted fluorophore. The change in conformation of the
fluorophore typically results in a decrease in fluorescence intensity. (d) Left: 2P-LSM image of AAV8-hSyn-
ASAP3b expressed in hippocampal organotypic slice cultures by bulk viral infection (scale bar represents
25 μm). Right: simulated ASAP3b traces in response to trains of action potentials (5, 15, 40 Hz) as in (b)

network function. Fluorescent reporters sensitive to changes in

many different aspects of neuron physiology have been developed,
but those responsive to action potentials, such as calcium and
voltage indicators, are the most widely used.
Calcium imaging using fluorescent protein sensors has proved
particularly useful for all-optical neurophysiology since the activity
of large numbers of neurons can be recorded simultaneously [55–
60]; the same group of neurons can be imaged during extended
time periods and can also be compared across different recording
sessions. The allure of calcium imaging stems, in part, from the
photophysical properties of the optical signal. In mammalian neu-
rons, spiking activity results in a temporary increase of Ca2+ con-
centration throughout the soma via voltage-gated Ca2+ channels,
which open as a result of the change in membrane potential during
the action potential (Fig. 3a). This somatic Ca2+ influx may also be
amplified by calcium release from intracellular stores [61, 62]. As
such, a vast number of freely diffusing calcium indicators
distributed throughout the cytosolic volume can collectively report
on the occurrence of action potentials. Although action potentials
10 Ruth R. Sims et al.

only last a few milliseconds, the duration of the calcium elevation

lasts approximately 2 orders of magnitude longer, resulting in a
bright, slowly decaying fluorescent signal that can readily be
detected with high signal-to-noise ratio (SNR) as illustrated in
Fig. 3b. The GFP-based GCaMP family of genetically encoded
calcium indicators (GECIs) is used most commonly in all-optical
neurophysiology. Multiple rounds of mutagenesis have yielded the
latest suite of variants (jGCaMP8) which exhibit different proper-
ties optimized for particular applications [63]. While calcium imag-
ing is the most commonly used approach for imaging the activity of
large neural populations, the potential pitfalls associated with using
a second messenger that exhibits slow kinetics are also widely
acknowledged and must be considered [64, 65].
Voltage indicators generate optical signals with magnitudes
proportional to changes in membrane potential (Fig. 3c) and can
be used to provide a readout of precise action potential timing in
addition to sub-threshold depolarizations and hyperpolarizations.
At present, genetically encoded voltage indicators (GEVIs) may
broadly be divided into three categories: rhodopsin-based indica-
tors [66, 67], hybrid chemogenetic indicators [13], and sensors
based on the fusion of a fluorophore to a voltage-sensing domain
(VSD) [68, 69], though only the latter category of GEVIs have
been demonstrated to be compatible with 2P excitation [70]. Cal-
cium imaging is a much more prevalent technique than voltage
imaging, since optically monitoring changes in membrane potential
is fundamentally more challenging in terms of signal detection.
Firstly, only voltage-sensitive reporters located within a Debye
length can report on the membrane potential, and improperly
localized GEVIs reduce the sensitivity of optical measurements of
membrane potential by increasing background fluorescence. Simi-
larly, as for channelrhodopsins, it has been demonstrated that fus-
ing GEVIs with soma localization motifs improves membrane
trafficking and reduces off-target intracellular labeling. While a
typical neural soma constitutes around 60% of the entire cell vol-
ume, the somatic membrane only accounts for 2–7% of the total cell
surface area [71, 72]. As a result, the number of voltage indicators
that can report on the membrane potential is less than 0.1% of the
number of Ca2+ indicators in the cytosol [73, 74], which places an
upper bound on the signal-to-noise ratio of voltage imaging
(Fig. 3d) [75]. This is compounded by the fact that action poten-
tials occur on much shorter timescales than the consequent calcium
signal, and hence voltage imaging requires much faster sampling
rates (>500 Hz and in many cases > kHz, depending on the
specific application). Raster scanning is an inefficient approach for
detecting membrane-localized signals which account for a small
fraction of the field of view (FOV) – and the resulting frame rates
are insufficient for population-level voltage imaging. The unifying
feature of different approaches optimized for 2P voltage imaging is
 All-Optical Neuronal Manipulation with Wavefront Engineering 11

an increased illumination duty cycle of signal-generating pixels.

Such increases in temporal resolution are often achieved at the
cost of increased photobleaching, which is compounded by the
fact that voltage indicators are replenished slower than calcium
indicators because diffusion is much slower in the membrane lipid
bilayer than in the cytoplasm, necessitating the use of more robust
fluorophores [76]. Additionally, sample motion is more problem-
atic for voltage imaging. Though population voltage imaging is
technically more challenging than calcium imaging, it has the
potential to provide more physiologically relevant information
about the logic and syntax of the neural code and, indeed, is
necessary for a subset of all-optical neurophysiology experiments.

2.2 Combining Compatible actuators and indicators must be carefully selected in

Molecular Tools for order to simultaneously and independently monitor and control
All-Optical neural activity in a single preparation. Firstly, the fluorophore used
Neurophysiology to aide visualization of opsin-positive cells should generally be
Experiments spectrally separate from both the opsin and the activity reporter
and, should be chosen so as not to occupy precious spectral band-
widths. This is a particularly important consideration in the case of
voltage imaging, where any bleed-through, activity-independent,
fluorescence degrades precious signal-to-noise ratio and ultimately
reduces the detectability of neuronal signals. Most crucially,
all-optical experiments generally benefit from employing spectrally
orthogonal opsins and activity reporters. Spurious activation of
opsin-positive neurons while imaging neural activity can perturb
neural networks by altering excitability and inducing changes in
synaptic release and plasticity [77]. Imaging artifacts can also be
induced due to the excitation of activity reporters during opsin
photoactivation, though this is typically less severe since network
function is not affected and, ordinarily, these artifacts can be mini-
mized by precisely de-synchronizing photostimulation and imaging
(possible at low frame rates such as those used for calcium imaging)
or removed during subsequent analysis. Hence, the term “optical
crosstalk” is commonly used to describe artefactual photostimula-
tion, induced during imaging in all-optical neurophysiology experi-
ments (for a much more detailed discussion regarding crosstalk
during all-optical neurophysiology experiments refer to Chaps. 2,
4 and 5).
Although channelrhodopsin variants with peak single-photon
(1P) excitation wavelengths spanning the visible region of the
electromagnetic spectrum have been engineered [39, 78],
performing crosstalk-free, multi-color experiments is not trivial.
Evidently, variants of actuators and reporters from opposing ends
of the spectral palette should be chosen. Unfortunately, the action
spectra of channelrhodopsins commonly used for 2P optogenetics
are typically extremely broad [39]. Furthermore, so-called, red--
shifted opsins exhibit persistent “blue tails”, which coincide with
12 Ruth R. Sims et al.

wavelengths used for 2P imaging of activity reporters

(920–950 nm). A number of different approaches aiming to allevi-
ate this problem have been proposed (see also Chap. 2). Very
recently, implementation of spectrally independent excitation
beams enabled artifact-free all-optical experiments with GCaMP
and red-shifted channelrhodopsins (see also Chap. 4) [79]. Parallel
excitation methods have taken advantage of the different
sub-cellular distributions of GECIs and opsins [80], though of
course this is less applicable in the case of voltage imaging (where
both indicator and actuator are membrane localized), and further is
not intrinsically robust to sample motion which is problematic for
in-vivo applications. An alternative approach is to employ blue-
shifted opsins in combination with red-shifted reporters
[50, 81]. One benefit of this is that longer wavelength fluorescent
photons exhibit longer scattering lengths in biological tissue which
should facilitate deeper imaging. While this approach has found
success for 1P excitation [67], the two-photon counterpart of this
approach has thus far been limited. On the one hand, genetically
encoded, red-shifted activity indicators display lower 2P efficacies
than green ones and, furthermore, amplified lasers in the spectral
region adequate for photostimulating several cells expressing blue-
shifted opsins (920–950 nm) have only recently become available
[81]. Another approach to minimize crosstalk is to use opsins with
fast kinetics and optimize the raster-scanned trajectory used to
image GCaMP activity to minimize the accumulation of photocur-
rent during the membrane time constant. Although this method
does not eliminate sub-threshold network perturbation, the (rela-
tively) fast repolarization of neurons expressing opsins with short
τoff values means they are unlikely to fire due to depolarization
induced by the scanned imaging beam. Of course, successful
employment of this method requires careful titration of different
imaging conditions, including imaging power, frame rate, and field
of view as an interim approach until high efficacy blue-shifted
opsins, red-shifted activity indicators [82], and amplified lasers in
the appropriate spectral range are developed.
A final subtle point to note when combining actuators and
indicators in all-optical neurophysiology experiments is that sus-
tained opsin activation can alter the conditions of the intra- and
extracellular environment [83], which could impact the behavior of
the opsin, the excitability of the neuron, and also the fluorescent
yield of the activity reporter [84], while long-term effects such as
changing chloride concentration could influence the entire net-
work. Each of these factors should be considered when drawing
conclusions about neural activity based on fluctuations in the fluo-
rescent signal.
 All-Optical Neuronal Manipulation with Wavefront Engineering 13

2.2.1 Expressing To perform all-optical neurophysiology experiments, neurons must

Molecular Tools in Specific be genetically modified in order to induce the expression of actua-
Populations of Neurons for tors and indicators in specific populations of neurons, typically via
All-Optical promoter-operating expression specificity. Examples of ubiquitous
Neurophysiology promoters that can be used to drive expression of actuators and
Experiments indicators in a broad set of neurons and that are strongly and
persistently active in a wide range of cells are the hSyn (human
synapsin) promoter and the synthetic mammalian-specific pro-
moter CAG. A variety of approaches exist for gene delivery based
on the molecular signatures, projection patterns, anatomical orga-
nization, and functional activity of neurons [85]. Viral approaches,
electroporation, and constitutive expression in transgenic animals
have all been utilized. The most commonly used strategy to date is
viral transduction. Viral vectors can be delivered directly to specific
brain regions using stereotaxic, intracranial injections, yielding
long-term expression and high transgene levels which is especially
important in the case of promoters with low transcriptional activity
[86]. The degree of viral spread (and hence transgene expression)
from the injection site varies with both virus serotype and tissue
type [87]. In general, for rodent brains, opsin gene expression
reaches functional levels within 3 weeks after adeno-associated
virus (AAV) injection. Another approach, single-cell electropora-
tion, provides a much greater degree of control of protein expres-
sion patterns than viral transduction and can be used to deliver
longer segments of DNA. Using electroporation, an exact set of
neurons can be transfected with precise amounts of a single plasmid
or with mixtures of plasmids with well-defined ratios [88]. Alterna-
tively, specific cortical layers can be targeted with in utero electro-
poration [89]. Transgenic animals are also invaluable for all-optical
experiments but can be expensive and time-consuming to generate.
Before establishing transgenic lines, it is important to test, charac-
terize, and calibrate appropriate optogenetic actuators and repor-
ters. In vitro dissociated cell cultures represent an important tool
for characterizing actuators and indicators in single homogeneous
cell populations. However, because the brain’s architecture is lost in
the culture process, they are not suitable for studying brain function
[90]. Organotypic cultures are becoming a favored preparation for
testing new preparations for all-optical neurophysiology experi-
ments (such as new actuator/indicator combinations), since the
main network architecture is maintained (Sect. 3.4; Fig. 9d), and
it is possible to test many different conditions per animal (10–15 in
the case of hippocampal organotypic cultures). A protocol used to
produce hippocampal organotypic cultures and perform bulk viral
infection is presented in Sect. 3.4 of this chapter. In Fig. 4 we show
an all-optical experiment in mice hippocampal organotypic slices,
co-expressing the soma-targeted cation channelrhodopsin
ST-ChroME and the genetically encoded Ca2+ indicator GCaMP7s
(Fig. 4a). Neurons were photostimulated using two-photon
14 Ruth R. Sims et al.

Fig. 4 All-optical electrophysiology in mice hippocampal organotypic slices. (a) Two-photon fluorescence
image showing the co-expression of the high performance and soma-targeted cation channelrhodopsin
ST-ChroME, here tagged to the chromophore mRuby3 (red colour corresponds to the nuclear localization of
mRuby3 reporter) and the genetically encoded Ca2+ indicator GCaMP7s (green) in the CA3 region of a
hippocampal organotypic slice. White circles represent the two-photon temporally focused spots delivered
to excite 50 different neurons (12 μm spot diameter, 1040 nm wavelength, 0.26 mW/μm2 incident power).
Scale bar: 50 μm. (b) Two-photon imaging of GCaMP7s fluorescence signals evoked by the sequential
stimulation of the cells (interstimulus interval ~3 s). Gray bars represent the stimulation protocol which
consisted of a train of 5 pulses of 5-ms duration at 4 Hz. The identity of the cells during the sequential
stimulation is denoted by the blue numbers on top. In this experiment, 28 out of 50 cells yielded calcium
transients in response to stimulation (green horizontal arrowheads). During the acquisition time (~160 s) two
synchronous network-wide bursting events were observed (vertical arrowheads at the bottom), the first one
seemed to be triggered by the direct activation of a hub-like cell (cell number 15 in the sequence; see pink
inset), while the second network-wide event seemed to be triggered by the spontaneous activation of a
hub-like cell in the circuit. Pink and orange arrowheads denote the evoked or spontaneous nature of the
events, respectively. A single event (in only 1 neuron) with similar characteristics to the network-wide bursting
events in terms of amplitude and kinetics was observed near the end of the acquisition time (horizontal orange
arrowhead). The large amplitude of these events reflects the large number and/or frequency of action potential
firing in comparison to the fine-tuned control of firing activity evoked by single-cell resolution and
sub-millisecond precision patterned photostimulation as it is observed in the inset in (c)

excitation with temporally focused 12-μm diameter holographic

spots, and their responses were detected by imaging GCaMP
using 2P scanning imaging on a standard galvanometric-based
setup. 28 of 50 cells yielded calcium transients in response to
photostimulation (Fig. 4b, green horizontal arrowheads). During
the experiment (~160 s) two synchronous network-wide bursting
 All-Optical Neuronal Manipulation with Wavefront Engineering 15

events were observed (Fig. 4b, vertical arrowheads at the bottom),

the first triggered by the direct activation of a hub-like cell (Fig. 4b,
cell 15; pink arrowheads and inset) and the second a possible
spontaneous event (Fig. 4b, orange arrowhead at the bottom).
These events are typically seen in developing hippocampal networks
[91], and demonstrate that network function is maintained in
organotypic slices. Moreover, the large amplitude of these events
reflects the large number and/or frequency of action potential
firing in comparison to the fine-tuned control of action potentials
evoked by single-cell resolution and sub-millisecond precision of
patterned photostimulation, as evidenced by the inset shown in
Fig. 4c.

2.3 State-of-the-art An extraordinary number of different 2PE technologies have been

Two-Photon Excitation developed to precisely control neuronal activity using microbial
Approaches for All- channelrhodopsins and to provide high-fidelity readouts of activity
Optical with calcium and voltage indicators. In this chapter, these methods
Neurophysiology will be broadly categorized as either sequential or parallel methods.
While in sequential-2PE a tightly focused beam visits distinct voxels
consecutively, parallel-2PE encompasses all methods in which 2PE
occurs within a region larger than the diffraction-limited volume.
A wide variety of components capable of rapidly varying the
three-dimensional position of a tightly focused beam throughout a
volume of interest have been incorporated into 2PE-LSM instru-
ments to increase the temporal resolution of sequential, point-
scanned, 2PE. This includes devices such as resonant galvanometric
mirrors [92], rotating polygon mirrors [93, 94], acousto-optic
deflectors (AOD) [95–99], deformable mirrors [100], spatial
light modulators (SLMs) [101–104], piezoelectric scanners
[105], microelectromechanical systems (MEMS) scanners [106],
electrically-tunable lenses (ETL) [107], voice-coils [55, 108], and
tunable acoustic gradient (TAG) lenses [109]. Other interesting
approaches specifically designed to improve volumetric imaging
rates rely on the conversion of lateral beam deflections, typically
using galvanometric mirrors, into axial displacements at kilohertz
rates [110, 111]. Furthermore, in general, the temporal resolution
of sequentially scanned-2PE approaches can be improved by opti-
mizing the scan trajectory according to a pre-defined region of
interest (ROI) (e.g., Lissajous scanning [105]).
While successful single-cell optogenetic activation using
scanning-2PE based on galvanometric mirrors has been demon-
strated [3, 112, 113], photostimulation based on pure sequential
scanning is incompatible for use with channelrhodopsins with fast
kinetics since a large portion of the somal membrane of each
neuron must be scanned before the channels begin to close in
order to integrate sufficient photocurrent and successfully reach
the threshold for action potential firing. Purely sequential raster-
scanned-2PE approaches are not capable of high fidelity,
16 Ruth R. Sims et al.

co-incident excitation of multiple neurons [102, 114, 115]. Simi-

larly, sequentially scanned-2PE methods have only demonstrated
sufficient temporal resolution for voltage imaging by extreme
reductions of the field of view to a single line [116] or point [117].
The acquisition rates of scanning-2PE systems can be increased
by random-access approaches. These techniques use multiple
AODs to rapidly deflect a tightly focused beam to a set of
pre-defined three-dimensional locations [98, 118]. Random-access
scanning has been successfully applied to both calcium and voltage
imaging of up to 20 distinct three-dimensional positions at kilo-
hertz sampling rates [14, 119, 120]. In principle, it is possible to
achieve denser spatial sampling than has been demonstrated by
random-access scanning; the fundamental limit for unambiguous
signal assignment in fluorescence microscopy is the fluorescence
lifetime (~ns) [121]. Spatiotemporal multiplexing methods aiming
to approach this upper bound have been successfully applied to
ultrafast recording of neural activity with calcium and voltage indi-
cators [122, 123]. Furthermore, since the lifetime of common
fluorophores is shorter than the pulse separation of common
mode-locked lasers used for 2PE, single pulses can be divided
into multiple beamlets (diffraction-limited spots), each of which
can be laterally or axially displaced to illuminate distinct sample
regions at different (although, in some cases, almost simultaneous)
times. Fluorescence signals sequentially excited by different beam-
lets can be de-multiplexed by accurate synchronization of the beam
displacement approach with the detector using high-speed elec-
tronics [60, 108, 124]. Neglecting scattering, spatiotemporally
multiplexed fluorescence from different locations can be unambig-
uously assigned to its origin provided that the effective dwell-time
is longer than the excited state lifetime of the fluorophore.
An alternative approach to increase temporal resolution is to
modulate the electromagnetic field and increase the instantaneous
volume of excitation using so-called parallel methods. Since the
inception of laser scanning microscopy, efforts have been made to
increase the extent of the excitation beam and hence reduce the
dimensionality of the raster scan required to fully sample the region
of interest. For instance, voltage imaging at rates of 15 kHz has
been demonstrated by rapidly scanning holographically generated
foci using AODs to simultaneously excite large membrane areas
[14]. More common variants of this approach, such as line-
scanning, increase the excitation extent in a single direction and
capture two-dimensional images by scanning in the transverse
direction [125]. Widefield temporal focusing takes this concept to
its theoretical limit by performing line scanning at the speed of light
[126, 127]. Line-scanned tomography has also been used to
achieve millisecond-resolved recordings of voltage and calcium
indicators [128]. The dimensionality of the excitation beam has
also been increased axially to form Bessel and Airy beams [129–
132], for volumetric imaging based on lateral scanning (See also
 All-Optical Neuronal Manipulation with Wavefront Engineering 17

Chap. 10). In many cases, elongated foci result in the projection of

axial information onto a two-dimensional recording, which can
limit its applicability to sparsely labeled samples. This can be over-
come by spatial multiplexing to record stereoscopic
information [133].
Moving from one-dimension scanning approaches toward
scanless configurations, one class of parallel-2PE approaches use
phase modulation to spatially multiplex the excitation beam and
simultaneously project multiple foci in three-dimensions to spa-
tially separated sample regions. For instance, spatial light modula-
tors (SLMs) have been used to deflect beamlets to different three-
dimensional sample positions through Computer-Generated
Holography (CGH) [134, 135] and perform both photostimula-
tion and imaging [80, 104, 136, 137]. The number of beamlets
and their position can be dynamically updated up to the SLM
refresh rate (~420 Hz for the latest SLM models). Recent innova-
tions such as the combination of overdrive with phase reduction
[138], or the sequential illumination of two SLMs [8] have
achieved refresh rates in the kHz-range. SLM-based spatially multi-
plexed calcium imaging has been combined with both single pixel
[80] and camera detection [139]. Furthermore, calcium imaging at
1 kHz acquisition rates has been demonstrated by using a microlens
array rather than an SLM to generate a grid of beamlets [140]. A
common approach for 2P photostimulation combines SLM-based
multiplexing with a pair of galvanometric mirrors which laterally
sweep each focus in a spiral motion spanning the average soma
diameter [8, 102, 103, 115, 141–144]. This method can simulta-
neously excite large ensembles of neurons without compromising
temporal resolution with respect to the single-cell spiral scanning
case (see also Chap. 3). Similarly, as for purely sequential-2PE, the
temporal resolution of these hybrid parallel-sequential methods can
be improved by upgrading the component responsible for sequen-
tial scanning.
Another category of parallel-2PE approaches uses phase mod-
ulation to increase the lateral extent of the excitation beam and
perform scanless excitation [145]. For instance, CGH using SLMs
can be also used to sculpt light into arbitrary shapes. This is gener-
ally combined with temporal focusing [146, 147] to preserve axial
resolution, which scales linearly with lateral extent for holographic
beams and quadratically for loosely focused quasi-Gaussian beams
[148]. Techniques for distributing temporally focused light
throughout a three-dimensional volume have been developed
[114, 149, 150] and low-numerical aperture (NA) temporally
focused Gaussian beams [113, 151], CGH, and generalized phase
contrast (GPC) have all been applied to photostimulation and
imaging [152–158]. Since parallel (scanless) 2PE methods can
simultaneously excite opsins distributed throughout the soma
high photocurrents can be efficiently evoked independently of the
18 Ruth R. Sims et al.

off kinetics, which facilitates control of neuronal activity with

sub-millisecond jitter [158]. Moreover, in contrast to scanning
approaches, in parallel approaches, the temporal resolution of the
activation process is solely defined by the dwell time of the physio-
logical process, i.e., the necessary time for the beam to remain
on-site for evoking the desired physiological effect.
Having excited an indicator of neural activity using one of the
methods outlined above, the next challenge is to detect fluorescent
emission. Unfortunately, popular calcium and voltage reporters
fluoresce in the visible region of the electromagnetic spectrum,
although development of activity reporters fluorescent in the infra-
red (IR) is an active area of research [159–161]. Thus, visible
photons emitted from fluorophores located deep in scattering tis-
sue will typically experience multiple scattering events prior to
detection. This is least problematic for sequentially scanned-2PE
methods since all collected fluorescence can reasonably be assumed
to have been generated by ballistic photons at the focal region.
Hence any signal recorded at a given time can be correctly assigned
to the correct spatial location (again provided that the dwell time is
longer than the fluorescence lifetime). 2PE imaging methods
which record fluorescence from different voxels simultaneously
are typically less robust against scattering. Beyond a few scattering
lengths, the origin of fluorescent photons becomes ambiguous,
which limits the depth of spatially multiplexed methods. Crosstalk
can be reduced by increasing the spatial separation between excita-
tion foci, but this is achieved at the cost of maximum acquisition
rate for full-frame scanning [140]. Computational methods have
also been developed to overcome scattering-induced ambiguity by
exploiting priors such as high-resolution spatial maps [144, 162],
temporal signatures [163–165], or adaptive optics [166–
168]. Finally, to correctly identify signals from different neurons
excited using three-dimensional, spatially multiplexed methods, the
effective depth of field of the detection axis must be extended with
respect to the widefield case. Common extended DOF approaches
include multi-focal plane microscopy [169] and point spread func-
tion engineering [170], which encodes information about axial
position as lateral changes in intensity.
In spite of the number of technological developments outlined
in this section, many 2P all-optical optogenetic studies performed
to date have used parallel excitation via CGH (either extended
holographic spots or spiral scanning) for photoactivation and gal-
vanometric scanners (both resonant and not), occasionally com-
bined with an ETL for calcium imaging across multiple axial planes
[8, 103, 115, 141–143, 154, 156, 157, 171]. These studies have
already provided novel insights into the principles of neural coding,
and it is anticipated that the wider adoption of newer technologies
will enable further progress. To assist in this dissemination, the next
section will provide specific details about: laser sources required for
 All-Optical Neuronal Manipulation with Wavefront Engineering 19

all-optical neurophysiology experiments, the implementation of

Generalized Phase Contrast and Temporal Focusing, and a proto-
col for preparing hippocampal organotypic slices.

3 Implementation of Methods

3.1 Laser Sources The feasibility of two-photon all-optical neurophysiology projects

is largely contingent on the first element in the optical path, the
laser, which ultimately dictates experimental parameters such as the
number of neurons that can be probed simultaneously, the maximal
speed of interrogation, and which probes can be excited (according
to their action spectrum). This section will provide a general review
of the different laser characteristics that impact the efficiency of
two-photon excitation and describe how the choice of laser can
be optimized based on specific experimental parameters.
To review, two-photon excitation occurs when two photons,
with sufficient combined energy, are absorbed quasi-simultaneously
and a molecule is excited into a higher energy level [172]. The
number of photons absorbed per molecule, per unit time, via
two-photon absorption (N2P) is proportional to the two-photon-
cross section (σ 2P) and to the square of the instantaneous intensity
(N2P / < I(t)2>). The low values of typical 2PE cross-sections
necessitate the use of high time-averaged photon fluxes to excite
actuators and indicators at sufficient rates. This can be achieved
using mode-locked lasers which generate femtosecond (fs) pulses of
light. It is intuitive that, at a given average power, shorter pulses
and fewer pulses per unit time result in a greater concentration of
photons, which ultimately leads to a higher probability of quasi-
coincident two-photon absorption. More formally, the concentra-
tion of photons in time can be parametrized according to the laser
duty cycle which is defined as the product of repetition rate (frep)
and pulse duration (τpulse) and corresponds to the fraction of time
per unit interval during which there is irradiance. Prior to satura-
tion, and at a given average power, the rate of two-photon absorp-
tion is higher for pulsed lasers as compared with their continuous
wave (CW) counterparts by a factor proportional to the inverse
duty cycle:
g p <I ðtÞ> 2
<N 2P > / <I ðtÞ2 > /
f rep τpulse
where gp ~0.558–0.664 [173] is a unitless factor which accounts
for the fact that real pulses emitted from mode-locked lasers are not
rectangular.
In fact, the wide adoption of 2P-LSM was aided by the devel-
opment and commercialization of reliable, mode-locked lasers
which provided enough energy to achieve sufficient rates of
20 Ruth R. Sims et al.

two-photon excitation of common fluorophores [174–176]. Ti:

Sapphire oscillators exhibiting 100 fs pulse widths and 80 MHz
repetition rates (12.5 ns pulse separation) have become the work-
horses of sequential 2P-LSM since these lasers provide an
~100,000-fold increase in the rate of two-photon excitation as
compared with CW excitation at the same average power, allowing
2P-LSM imaging to be performed using much more palatable
average powers (milliwatts in comparison to kilowatts). However,
these lasers no longer represent the gold standard for all-optical
neurophysiology experiments, particularly those in which multiple
neurons are probed simultaneously. The larger instantaneous
extent of the excitation area in parallel methods, or the division of
the original laser beam into a certain number of beamlets for
parallel spiral scanning necessitates the use of much higher peak
pulse intensities. Two obvious strategies for increasing the pulse
energy while maintaining average power are decreasing the pulse
width or repetition rate. In practice, some reduction of the pulse
width below the standard 100 fs value is possible [177, 178],
provided that the spectral width remains narrower than the action
spectra of the actuators and indicators (to maintain excitation effi-
ciency). However, this approach requires careful dispersion man-
agement, particularly when elements such as SLMs and diffraction
gratings are employed in the optical path. Much larger gains can be
achieved using amplified lasers with low repetition rates.
Ytterbium-doped fiber lasers with central wavelengths in the region
of 1030–1040 nm [179] are now commonly used for in-vivo
imaging and photostimulation, offering instantaneous powers
that are orders of magnitude higher than conventional tunable
lasers. The use of Ytterbium-doped fiber amplifiers with microjoule
pulse energies is necessary in order to simultaneously photostimu-
late neural ensembles composed of tens of neurons [5, 6, 103,
143]. Nevertheless, since these systems emit light at fixed wave-
lengths, the choice of opsin is constrained and multiple lasers with
different wavelengths must be used to excite different sensors and
actuators. Solutions that offer greater flexibility in terms of wave-
length while delivering high energy (microjoule) pulses can be
found in systems using optical parametric amplification (OPA) for
the generation of the excitation beam [79, 81].
When probing biological preparations with such high irra-
diances (which can often exceed 1024 photons cm-2 s-1) it is of
course necessary to consider the possibility of physiological pertur-
bations. Photoperturbations based on linear absorption processes
(N1P / < I(t)>), such as heating (via single-photon absorption) or
optical trapping [180, 181], occur throughout the excitation beam
while higher-order processes (NnP / < I(t)n>), for instance,
photolysis, ablation, and optical breakdown [182–184], are con-
fined to the focal region. This is particularly important to consider
when choosing the appropriate excitation approach for
 All-Optical Neuronal Manipulation with Wavefront Engineering 21

photostimulation [185]: parallel methods generally use lower

power density than spiral scanning but higher average powers.
Since the optimum excitation parameters and signal to photoper-
turbation ratio are likely to be highly dependent on the specific
characteristics of the sample preparation, it is advisable to vary the
repetition rate, pulse width, and average power in each case if
possible [186]. The optimal excitation parameters are likely to be
different for different excitation modalities.

3.2 Beam Shaping As outlined in Sect. 2.3, many parallel two-photon excitation
with Generalized approaches rely on lateral beam sculpting. A correspondingly wide
Phase Contrast variety of methods based on amplitude or phase modulation have
been conceived of and demonstrated experimentally. Phase modu-
lation is generally preferable since it is more power efficient than
amplitude modulation. Computer-generated holography (CGH) is
currently the most common phase modulation method used for
photoactivation or imaging in all-optical neurophysiology experi-
ments. Since CGH is described in detail in other chapters of this
book (Chaps. 3, 4, and 11), this section will focus on the principles
and implementation of an alternative phase modulation approach:
generalized phase contrast (GPC) [187].
GPC is an efficient approach for transverse beam shaping and
has been applied to imaging [188, 189], photomanipulation [190–
192], and atom trapping [193]. GPC patterns have smooth,
speckle-free intensity profiles and can be combined with temporal
focusing for depth-resolved, robust excitation, deep in scattering
tissue [147, 194]. As demonstrated in Fig. 5a, in GPC, the phase
imprinted on a beam (using a phase mask or an SLM) is mapped to
intensity variations in a conjugate image plane by engineered con-
structive and destructive interference. The simplest implementa-
tions of GPC are based on 4f arrangements of lenses, constructed as
follows (Fig. 5a): the first phase modulating element (hereafter
SLM) is located a distance f1 prior to the first lens (L1), which has
focal length f1, and is referred to hereafter as the Fourier lens. The
necessary SLM phase (ϕxy(x,y)) depends on the spatial profile of the
desired pattern. For binary GPC, ϕxy = ϕ1 for SLM pixels inside the
pattern and ϕxy = ϕ2 for SLM pixels outside of the pattern, ϕ1 = π
and ϕ2 = 0 is a simple (and useful) choice. An element known as a
phase contrast filter (PCF) is located in the Fourier plane (FP) of
L1, and a distance f2 prior to the second lens (L2), which has focal
length f2. The PCF applies a selective phase shift to the field in the
Fourier plane. The phase shift imparted by the PCF depends on its
thickness (d) and refractive index of the substrate (n2): ϕPCF =
(2πd(n2-n1))/λ, where n1 is the refractive index of the medium
surrounding the PCF (usually n1 = 1 for air, and n2 = 1.45 for a
PCF fabricated with fused silica). For binary GPC, and ϕ1 = π, ϕ2 =
0, constructive interference in the output pattern occurs for ϕPCF =
π. The resulting interference pattern is formed in the image plane
(IP) of the second lens, a distance f2 from L2.
22 Ruth R. Sims et al.

Fig. 5 Wavefront engineering based on Generalized Phase Contrast. (a) (i) Schematic representation of a
common configuration for Generalized Phase Contrast. The beam is modulated using an SLM, which is used to
impart a phase shift to the portion of the beam corresponding to the desired pattern. The SLM phase should
match the desired pattern (up to a magnification factor according to the respective focal lengths of L1 and L2).
In the binary case, the SLM is usually used to impart a π phase shift to the pixels within the pattern and 0 to
those outside. The synthetic reference beam is the portion that is phase shifted by the phase contrast filter
(PCF), which typically imparts a π phase shift relative to the field that does not pass through the PCF, referred
to here as the modulated beam. The different portions of the beam are recombined by L2 in the Image Plane
(IP), where the modulated and synthetic reference fields interfere to form the desired pattern. (ii) Cartoon
representations of the ideal 2D amplitudes and phases of the electric fields in the input (SLM) plane and the
output (Image) plane. The phase profile of a typical PCF is shown centrally, with the filter diameter indicated by
dashed black lines. (iii) 1D cross sections of the amplitudes and phases of the electric fields in the case of
binary circle GPC. (b) 2-photon excited fluorescence from a thin rhodamine layer for two different patterns:
circle and ring GPC. Scale bars represent 10 μm

To some extent, the perceived complexity of GPC arises from

the number of different parameters that contribute to pattern
fidelity. To elucidate the effects of some of these parameters, their
impact on three important metrics of pattern quality relevant to
two-photon excitation: efficiency, uniformity, and contrast will be
discussed. In this context, efficiency is defined as the fraction of
total energy contained within the pattern, uniformity as the inverse
of the curvature of intensity within the pattern and contrast as
difference between the maximum and minimum intensity in the
pattern vicinity ((Imax + Imin)/(Imax-Imin). While it is generally
desirable that these metrics are maximized for two-photon excita-
tion based on sculpted light, this cannot be achieved using low NA
Gaussian beams, where uniformity throughout the region of inter-
est (typically the neuronal soma) necessitates use of a large beam
waist, resulting in low pattern efficiency. To explain how uniformity
and efficiency can be jointly maximized in GPC, we will consider a
simple example based on an input Gaussian beam and a simple
binary pattern commonly used for two-photon excitation: a circular
disk of uniform intensity (Fig. 5a, b).
 All-Optical Neuronal Manipulation with Wavefront Engineering 23

Consider the propagation of the field modulated by the SLM

through the system in the absence of the PCF (Fig. 6a, upper). In
the image plane, the modulated field is a magnified image (accord-
ing to the ratio of f2/f1), of the input field with the imprinted phase
profile ϕxy. Given the modulated field in the image plane, it is
possible to find the corresponding ideal “reference field”, which,
summed with the modulated field would generate the desired
pattern with maximal efficiency, uniformity, and contrast (Fig. 6a,
lower). This requires total constructive interference between the
reference and modulated fields at all positions in the image plane
within the pattern and total destructive interference at all positions
outside. Achieving this stringent condition requires that the modu-
lated and reference fields:
(a) Have identical amplitude outside of the pattern.
(b) Have complementary amplitude inside the pattern.
(c) Arrive exactly in phase (modulo 2π) within the pattern.
(d) Arrive exactly out of phase (modulo 2π) outside of the pattern.
In GPC, the reference field is derived from the input field itself:
the portion of the field that is phase shifted by the PCF can be
considered a so-called “synthetic reference field” (SRF). The prop-
agation of the SRF through the 4f system can be considered sepa-
rately from the rest of the field (hereafter referred to as the
modulated field), as demonstrated in Fig. 6b. The efficiency, uni-
formity, and contrast in the output pattern are maximized by
finding the properties of the PCF such that the SRF approaches
the ideal reference field while the modulated field is minimally
perturbed. The optimal characteristics of the PCF for satisfying
conditions (a)–(d) in the image plane can be deduced by comparing
the profiles of the ideal reference and modulated fields to the
synthetic reference field in the Fourier plane (Fig. 6b). For instance,
it is clear that for the particular binary example of a disk, the
synthetic reference field should be phase shifted by π in order to
resemble the ideal reference field (Fig. 6b). Secondly, the edges of
the PCF should coincide with the first zero-crossings of the modu-
lated field, and thirdly the form of the phase contrast filter should
reflect the symmetry of the desired intensity pattern (for instance,
the highest fidelity circular patterns are obtained using circular
filters, whereas elliptical patterns would benefit from correctly ori-
ented elliptical filters). More complex patterns would benefit from
more complex filter shapes, although high efficiencies (>60%) can
still be achieved by using more common circular or rectangular
filters. In the case of the circular disk pattern with an appropriately
sized PCF, the efficiency of the output pattern is theoretically
70–80% and the maximum intensity is 3× higher than the
de-magnified input Gaussian beam [195].
24 Ruth R. Sims et al.

Fig. 6 Intuitive optimization of phase contrast filter for GPC. (a) The ideal reference field would generate total
constructive interference at all positions in the image plane within the desired pattern and total destructive
interference at all positions outside. The ideal reference field can be calculated by subtracting the modulated
field (i.e., the magnified image of the field at the SLM plane) from the field corresponding to the desired
pattern. The colors of the field profiles represent their phase ϕ, (blue: ϕ = 0 and red: ϕ = π). (b) The Fourier
transform (denoted F ) of this ideal reference field gives its profile in the Fourier plane, where the PCF is
located. The profiles of the ideal reference field and modulated field in the Fourier plane are used to guide the
 All-Optical Neuronal Manipulation with Wavefront Engineering 25

The 30% loss in efficiency is mainly a result of the differences

between the synthetic and ideal reference fields. Firstly, the SRF has
a narrower diameter and shorter amplitude than the ideal reference
field in the Fourier Plane (Fig. 6b). Consequently, the SRF in the
image plane is broader and of lower amplitude than the modulated
field and condition (a) is not met, resulting in a dim halo of light
(Fig. 6c) surrounding the pattern due to partial destructive inter-
ference. Note that the extraneous light can be blocked using an iris
in a conjugate image plane if problematic. Secondly, since the SRF
generated using a PCF to transmit the central lobe is constituted of
low spatial frequency components, any sharp features in the syn-
thetic reference wave are precluded. This reduces the uniformity of
the pattern with respect to the ideal case – since the SRF retains the
Gaussian envelope of the input beam (unless a beam shaper is used
prior to the first SLM such that it is illuminated with a top-hat beam
[195]). For small filters, the SRF approaches the “DC component”
of the incident field – a Gaussian envelope for most experimental
configurations. As the filter size increases, the SRF more closely
resembles a magnified version of the input field, while the modu-
lated field only contains the high spatial frequency components of
the input pattern – for instance, the pattern edges and small fea-
tures. The best pattern (highest efficiency, uniformity, and contrast)
is achieved when the edges of the PCF coincide with the first zero
crossings of the modulated field.
The concept of minimizing the differences between the syn-
thetic and ideal reference fields to maximize efficiency, uniformity,
and contrast is more general than the simple case of a circular disk
example presented and has been verified for a variety of analytically
tractable patterns [195, 196]. Experimentally, the properties of the
synthetic reference and modulated fields depend on interdependent
system parameters such as the diameter and profile of the input
beam, the spatial profile of the phase imparted by the SLM (i.e., the
desired output pattern), and the focal length of L1. Since these
parameters are interrelated, it is useful to introduce a level of
abstraction and optimize the efficiency, uniformity, and contrast

Fig. 6 (continued) choice of an optimal PCF filter in GPC. The optimal PCF parameters are those for which the
synthetic reference field most closely matches the ideal reference field. It is clear that this occurs when the
PCF imparts a π phase shift and its edges coincide with the first zero crossings of the modulated field
(indicated by black dashed lines). (c) Since the synthetic reference field cannot completely match the ideal
reference field, there exist some differences between the ideal output field and that which is obtained. In most
cases, there is a mismatch between the beam waists of the synthetic reference and the modulated fields,
resulting in a “ring-of-light” surrounding the output pattern (highlighted by gray arrows). This is normally
blocked by an iris positioned in a conjugate image plane. Furthermore, since the synthetic reference field is
typically composed of the low spatial frequency components of the field, there are no small features and the
Gaussian profile of the input beam is not compensated for (highlighted by black arrows)
26 Ruth R. Sims et al.

as a function of ξ and η, where ξ is defined as the ratio of the pattern

radius at SLM to the waist of the input Gaussian beam and η is
defined as the ratio of the radius of the focused beam in the Fourier
plane to the radius of the PCF [196]. For certain patterns, the
optimal values of ξ and η can be found analytically, or numerically
via simulations for more complex patterns.
The best approach for achieving the theoretically optimal values
of ξ and η experimentally depends on the precise constraints of the
experimental setup:
(i) ξ can be tuned by varying either the waist of the input beam or,
by changing the diameter of the pattern on SLM1.
(ii) η can be changed by varying the waist of the input beam, the
focal length of L1 and the physical diameter of the PCF.
In optical systems necessitating volumetric two-photon excita-
tion, GPC is generally combined with CGH for flexible 3D pattern
projection [197] and temporal focusing to improve the axial reso-
lution [147, 150]. In such systems, the downstream parameters are
tightly constrained to achieve a field of excitation with a particular
extent, and to meet the conditions described in the following
section, necessary to achieve optimal temporal focusing. Hence,
the extent of the SLM phase pattern for GPC is typically set
according to the desired size of the pattern at the focal plane of
the microscope objective and L1 kept fixed, while the input beam
and PCF diameters are varied in order to optimize efficiency uni-
formity and contrast in the output pattern. The optimization pro-
cess for a given set of experiments is eased by making it possible to
tune the diameter of the incident beam without altering its diver-
gence (for instance by having a variety of suitable telescopes
mounted on switchable magnetic bases) and additionally by
imprinting a selection of suitable PCFs (with a range of diameters
and shapes) on a phase mask which is then mounted on a three-axis
micrometer stage in order to easily be able to transition between
PCFs. For a given experiment, and desired sculpted light pattern,
the initial choice of PCF diameter is generally guided by simula-
tions. In lieu of simulations, a sensible starting point is to choose a
PCF diameter matched to the beam waist of the unmodulated
Gaussian beam in the Fourier plane and then to test several PCFs
with similar diameters to maximize efficiency, uniformity, and con-
trast. Using this strategy, efficiencies greater than 70% can be
routinely obtained experimentally.

3.3 Implementing Due to their interferometric character, GPC patterns suffer from a
Temporal Focusing lack of axial confinement and optical sectioning [147] (this is in
notable contrast to patterns generated using CGH). Temporal
focusing has been used to restore axial resolution for GPC, and
other extended light patterns that have been used for 2P
 All-Optical Neuronal Manipulation with Wavefront Engineering 27

optogenetics [147, 153, 171, 194]. The implementation of tem-

poral focusing in combination with Gaussian beams has been exten-
sively described in the literature [127, 198–200] and more detailed
descriptions may be found in this book (Chaps. 4 and 9).
In summary, an optical element placed in a conjugate image
plane of the optical path is used to separate the spectral frequencies
(hereafter “colors” for simplicity) of the femtosecond laser pulses.
While both diffusers or scatterers and diffraction gratings are suit-
able optical elements, diffraction gratings offer a more efficient
directional separation of the different colos and can be used in
conjunction with lasers commonly used for multiphoton micros-
copy (which exhibit characteristic pulse durations of hundreds of
femtoseconds, corresponding to pulse bandwidths of tens of nan-
ometers). Beam expanders are commonly used prior to the scatterer
or diffraction grating to adjust the beam diameter in order to
achieve the desired Gaussian beam size at the sample. The orienta-
tion of the grating is usually chosen such that the first order is
diffracted perpendicular to the grating (by convention, this corre-
sponds to θdiff = 0° ) (Fig. 7) to avoid tilted illumination of the
image plane at the sample in the case of a large ROI illumination or
a large field of excitation. However, this is not generally the same
angle that would maximize the light throughput for a blazed
grating (the so-called Littrow configuration) (see Note 1).
The groove density of the diffraction grating, G, and the focal
length f of the lens used as the tube lens of the microscope (Fig. 7)
should be chosen according to the properties of the microscope
objective: achieving the tightest axial confinement requires meeting
two conditions. Firstly: the extent of the chirped beam L should fill
the diameter of the back focal plane (dbfp); the extent of the chirped
beam due to linear dispersion induced by the diffraction grating is:
dλ f
L= Δλ = Δλ, ð1Þ
dx dG
with dλ
dx the linear dispersion induced by a grating with groove
density d G = G1 (lines/mm), and Δλ the spectral bandwidth. The
second condition for maximal axial confinement is that the instan-
taneous illuminated area of the scatterer/diffraction grating should
be imaged to a diffraction-limited spot at the focal plane of the
objective, which occurs when:
cτ Mλ
≈ , ð2Þ
sin θ 2NA
where c is the speed of light in vacuum, τ is the laser pulse duration,
θ is the incident angle of the light beam on the grating, λ is the
wavelength, NA the numerical aperture of the objective, and M, the
effective magnification between the scatterer/diffraction grating
and the sample. When temporal focusing is combined with light
patterning (such as CGH, GPC, or other approaches for intensity
28 Ruth R. Sims et al.

Fig. 7 Implementation of temporal focusing with light-shaping methods. (a) Temporal focusing of a Gaussian
beam. A diffraction grating placed in a conjugate image plane of the optical path is used to separate the
spectral frequencies (“colors”) of the femtosecond laser pulses. The grating is illuminated with a parallel
Gaussian beam of the appropriate size adjusted through a beam expander, for giving the desired beam size at
the sample plane. The orientation of the grating is usually chosen such that the 1st order is diffracted
perpendicular to the grating (θdiff = 0° ) to avoid tilted illumination of the image plane. Conjugation of the
grating (image) plane to the sample image plane is realized by a telescope consisting of a lens and the
microscope objective. (b) In temporal focusing of CGH beams the grating is placed at the image plane of CGH,
illuminated with the holographic pattern generated by addressing the corresponding phase on the SLM (inset).
(c) Similarly as in CGH, in temporal focusing of GPC beams the grating is illuminated with the intensity pattern
generated at the output (image) plane of the GPC configuration, when addressing the SLM with the
appropriate, in the simplest case binary, pattern (inset). In all panels θ denotes the incident angle of the
light-shaped beam onto the grating. In (b) and (c) the beam expander prior to the SLM is omitted for simplicity.
(Adapted from Ref. [201])

modulation), the grating is generally positioned in the output plane

of the patterning method. In this case, the conditions outlined
above for optimum axial confinement remain valid. However, the
dimensions of the beam at the back aperture of the objective
depend on the patterning method and may vary with respect to
the case of a Gaussian beam. Patterned light generated using GPC
(or amplitude modulation) resembles Gaussian beams in the sense
that they exhibit smooth phase profiles and large depths of focus
(confocal parameters). Changing the pattern changes the illumina-
tion of the back aperture but does not improve the axial resolution
since the linear dispersion of the diffracted beam is unaffected.
The illumination of the back aperture is different in the case of
CGH. CGH setups are usually designed such as to illuminate the
entire back aperture of the objective – hence extended CGH spots
have intrinsically better axial confinement than Gaussian beams of
similar lateral extent. Addition of a diffraction grating at a conju-
gate image plane in the CGH setup for temporal focusing affects
the illumination of the objective back aperture in the dispersive
direction. Because in CGH configurations the scatterer/grating is
illuminated with a focusing beam, the linear dispersion of the field
at the back aperture is strongly dependent on the focal length of the
lens used prior to the grating/scatterer. A detailed analytical
description of the full-width-at-half-maximum (FWHM) of the
 All-Optical Neuronal Manipulation with Wavefront Engineering 29

illumination distribution along the x and y axis at the back aperture

(more precisely the back focal plane) of the objective in a CGH-TF
setup may be found in Ref. [149]:
sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
pffiffiffiffiffiffiffiffiffiffiffi 2σ 2 cos ðθÞ2 f 2 2f 2 Δλ2
FWHMxBFP = 2 2 ln 2 2
þ 2 2 ð3Þ
f 21 dG
pffiffiffiffiffiffiffiffiffiffiffi f
FWHMyBFP = 2 2 ln 2 2 σ ð4Þ
f1
where 2σ is the waist of the Gaussian beam illumination at the SLM,
and f1, f2 are the focal lengths of the lenses used to conjugate the
SLM at the back focal plane of the objective (Fig. 7b). To optimally
illuminate the back aperture and maximize axial confinement, focal
lengths f1, f2 ought to be chosen according to the constraints set by
equations (3), which replaces equation (1), and (4), while also
aiming to satisfy equation (2). Since these lenses also dictate the
extent of the field of excitation, it may be necessary to compromise
field of view for desired axial resolution (or vice versa). For more
details refer to Note 2.
Temporal focusing microscopy is inherently a two-dimensional
method, since two-photon excitation only occurs in the vicinity of
the focal plane of the objective, which, by design, is conjugate to
the grating plane. Even if CGH was used to project patterns onto
different axial planes, two-photon excited fluorescence would only
be excited by the patterns projected onto the grating, the rest being
suppressed by temporal focusing. Extending temporally focused
excitation to three-dimensional (3D) space requires spatial multi-
plexing using an SLM in a conjugate Fourier plane following the
grating to modulate the phase of each monochromatic beam. Con-
volution of the temporally focused pattern projected on the grating
with the 3D configuration of beamlets at the sample plane creates
multiple temporally focused patterns at the position of the beam-
lets, which are replicas of the original pattern on the grating. A
detailed description of the 3D holographic spatial multiplexing
implementation on temporally focused Gaussian beams can be
found in Chap. 4, describing the technique 3D-SHOT (3D-Scan-
less Holographic Optogenetics with Temporal focusing) [5, 114].
For greater flexibility in the choice of the excitation shape and
size, 3D holographic spatial multiplexing of temporally focused
CGH, GPC patterns, or patterns created with amplitude modula-
tion techniques can be used [150]. In those cases what changes in
the optical setup is the way the beam is modulated before the
grating (Fig. 8). The shape of the beam that is projected onto the
grating is the one that is next replicated by 3D point-cloud CGH.
For applications where projection of different shapes or spot sizes is
necessary, a further variant of the above approaches is to perform
light shaping in two dimensions (2D) with a first SLM that is
30 Ruth R. Sims et al.

Fig. 8 Multiplexed temporally focused CGH patterns. Projection of temporally focused patterns in multiple
planes consists in a 3-step-approach: 1. beam amplitude shaping, here by CGH, 2. performing temporal
focusing, and 3. spatial multiplexing by using a SLM (SLM2) and 3D point-cloud CGH, at a Fourier plane after
dispersion of the spectral frequencies on the grating. The pattern generated through phase modulation (inset
SLM1) is projected onto the grating (F(X,Y) inset) and replicated by 3D point-cloud CGH to different 3D
positions (G(X,Y,Z) inset SLM2). The way SLM2 is illuminated is also shown in the inset. The resulting pattern
at the sample is a convolution of patterns F and G. (Reproduced from Ref. [201])

vertically tiled in regions, each one encoding a different pattern,

and use the second SLM, also tiled in the same number of regions
addressed with different phase profiles that independently control
the position in which each pattern is going to be projected at the
sample. Such an example is described in reference [150] where the
regions of SLM2 are addressed with phase profiles that control only
the axial position of the different patterns. A similar approach for
projecting different shapes at different positions is also presented
in [150].
The choice of the different lenses on this kind of configuration
is constrained by the requirement of filling the back aperture of the
microscope objective used to project patterns into the sample. All
telescopes between the grating and the objective must be
accounted for. Thus, in the case of CGH (Fig. 9), for instance,
equations (3) and (4) are modified as follows:
sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
pffiffiffiffiffiffiffiffiffiffiffi f 2σ 2 cos ðθÞ2 f 22 2f 22 Δλ2
FWHMxBFP = 2 2 ln 2 4 þ ð5Þ
f3 f 21 dG 2
pffiffiffiffiffiffiffiffiffiffiffi f f
FWHMyBFP = 2 2 ln 2 2 4 σ ð6Þ
f1 f3

Implementation of multiplexed temporally focused light shap-

ing (MTF-LS) either with CGH or GPC, or any other kind of
amplitude modulation is in general more demanding in terms of
 All-Optical Neuronal Manipulation with Wavefront Engineering 31

Fig. 9 Organotypic slices. (a) Organization of the hood for the dissection of organotypic slices. (b) Dissected
organotypic slices on PTFE membranes placed on inserts in a 6-well plate. (c) Transmitted light image of a
patched cell in a hippocampal organotypic slice. (d) Expression of a nuclear targeted fluorescent protein
(mRuby) following bulk infection with an AAV. The architecture of the hippocampus is maintained

alignment and equipment than multiplexing temporally focused

Gaussian beams. Care should be taken to align the beam on the
two SLMs used, one for light shaping (SLM1; Fig. 8) and the other
for 3D point-cloud CGH (SLM2; Fig. 8).
In MTF-LS methods, including Gaussian beams (3D-SHOT),
the excitation field is defined by the properties of the SLM used for
3D point-cloud CGH (pixel size and number of pixels or the size of
the SLM) and the telescope used to magnify this to the back
aperture of the objective (ff 4 in our schematic). Calibration of the
3
spot position between the SLM and the camera is achieved in an
identical manner as for CGH (see Sect. 3.2) and, similarly, the spot
intensity over the entire excitation field must be homogenized by
calibrating the diffraction efficiency SLM2 both laterally in the
image plane (xy) and axially throughout the excitation volume (z)
(refer to Note 3 for further details). Depending on the light
shaping method used, different calibration procedures for compen-
sating diffraction efficiency in light intensity may be needed. For
instance, in methods using SLM1 for controlling the lateral posi-
tion of the spots on a plane, diffraction efficiency calibration of
SLM1 is also necessary, and when the SLMs are tiled in different
regions, since light diffracted from each of them is not illuminating
the round back aperture of the objective in the same way, a special
32 Ruth R. Sims et al.

calibration of the intensity is needed for each zone of illumination

of the objective back aperture [149]. Finally, for systems using
MTF-LS with two SLMs and a single pattern projected in the
center of the diffraction grating it might be useful to orient the
grating close to the Littrow configuration to maximize light
throughput.

3.4 Preparation of While the benefits of using organotypic slice cultures for prototyp-
Organotypic ing preparations for all-optical neurophysiology experiments were
Hippocampal Slice described in Sect. 2.2.1 of this chapter, it is important to highlight
Cultures that this preparation can also be used to address many fundamental
questions central to modern neuroscience.
All animal experiments must comply with national regulations.
Organotypic hippocampal slices are prepared from mice (here from
Janvier Labs, C57Bl6J WT) at post-natal day 8 (P8).

3.4.1 Solutions Table 1 presents the quantities of reagents required for the solu-
tions used during the preparation of organotypic hippocampal
slices (their product references are also listed).

3.4.2 Equipment The following equipment is necessary to prepare the organotypic

slices:
– Tissue culture hood
– Incubator (37 °C; 5% CO2)
– Dissection stereomicroscope
– Tissue Chopper (McIlwain tissue chopper, Model TC752)
– Culture plates (six-wells; Corning 3516 or Sarstedt 83.1839)
– Millicell Cell Culture Insert (30 mm, hydrophilic Polytetrafluor-
oethylene (PTFE), 0.4 μm; Sigma PICM03050)
– PTFE membranes (hydrophilic, 0.45 μm; Millipore
FHLC04700)
– Tissue culture dishes (35 mm, sterile; Corning 353001)
– Filter paper or Whatman paper (Fisher 11392935)
– Transfer pipettes, narrow and wide bore (plastic, disposable,
sterile, e.g., Sarstedt 86.1171.001, wide bore pipettes can be
prepared by cutting the tip)
– Razor blade (two-sided)
– Sterilized dissection tools
• Large scissors (Fine Science Tools 14110-17)
• Fine scissors (Fine Science Tools 15003-08)
• Double-Ended Micro Spatula × 2 (Fine Science Tools
10091-12)
• Curved forceps (Dumont #7, Fine Science Tools 11274-20)
 All-Optical Neuronal Manipulation with Wavefront Engineering 33

Table 1
Solutions for organotypic hippocampal slice cultures preparation

Reagent Quantity (mL) Product reference

Dissection medium
Gey’s Balanced Salt Solution 500 Sigma G9779
D-glucose solution 45% 5 Sigma A4403
Vitamin E (500 μL (≈550 mg) diluted in 4.5 mL of sterile water) 1 Sigma T3001
Na-Pyruvate 100 mM 5 Sigma S8636
Hepes 1 M 5 Sigma H3537
Ascorbic acid 0.1 mM 0.1 Sigma A4403
a
Penicillin/Streptomycin (5000 U/mL) 2 Fisher 11528876
Opti-MEM culture medium (used during the first few days of culture)
Opti-MEM 50 Fisher 15392402
HBSS 25 Fisher 15266355
D-glucose solution 45% 1 Sigma A4403
Na-Pyruvate 100 mM 1 Sigma S8636
Vitamin E (500 μL (≈550 mg) diluted in 4.5 mL of sterile water) 0.2 Sigma T3001
Heat-inactivated horse serum 25 Fisher 10368902
Ascorbic acid 0.1 mM 0.02 Sigma A4403
a
Penicillin/Streptomycin (5000 U/mL) 1 Fisher 11528876
Neurobasal-A culture medium
Neurobasal-A 150 Fisher 11570426
Glutamin 200 mM 0.75 Fisher 15430614
Na-Pyruvate 100 mM 1.5 Sigma S8636
Ascorbic acid 0.1 mM 0.03 Sigma A4403
Vitamin E (500 μL (≈550 mg) diluted in 4.5 mL of sterile water) 0.3 Sigma T3001
B27 supplement 3 Fisher 11530536
Heat-inactivated horse serum 25 Fisher 10368902
Penicillin/Streptomycin (5000 U/mL)a 1.5 Fisher 11528876
a
Be aware that antibiotics can affect some cellular properties [202]. It is important to establish that their use will not
perturb or introduce any bias into the system under investigation. Organotypic hippocampal slices can also be prepared
without antibiotics by maintaining a strict asepsis throughout the entire process
34 Ruth R. Sims et al.

• Straight forceps (Dumont #5, Fine Science Tools 11254-

20)
• Scalpel handle and blades (#23) (Fine Science Tools 10023-
00)
– 15 mL tubes x2 (sterile, Corning 352095)
– Micropipette 200 μl (Eppendorf, 3124000083)
– Tips (sterile, 200 μL, Sorenson Bioscience 14220)
Always use sterile gloves and change them between each
dissection.
1. Prepare the solutions in a sterile environment.
2. Put 1 mL of Opti-MEM culture medium in each well of the
6-well plates. Place a membrane insert in each well using sterile
forceps. Make sure there are no air bubbles beneath the inserts.
3. Cut the PTFE membrane into small squares (5 × 5 mm) using a
scalpel (#23) and place them in the inserts (a maximum of
5 membranes per insert is advised for ease of retrieval). Put
the plates in the incubator.
4. Prepare 4 culture dishes (35 mm) per pup. Place some filter
paper in one of them. Half fill each culture dish with the
dissecting medium. Place them at 4 °C.
5. Place the following items under the hood (Fig. 9a):
(a) Tissue chopper set to cut 300 μm slices.
(b) Dissection stereomicroscope.
(c) Sterile transfer pipettes held in 15 mL tubes.
(d) Previously sterilized tools in ethanol 70%.
(e) Sterile PBS.
6. Thoroughly wipe the microscope, tissue chopper, razor blade,
and stage with ethanol 70%.
Before starting the dissection for each pup, place 4 petri dishes
under the hood.
7. Anesthetize pups according to local regulations and decapitate
using the large scissors.
8. Flush the head with 70% ethanol and transfer it to the first petri
dish. Insert the curved forceps into the eye sockets and remove
the skin.
9. Insert the lower part of the fine scissors into the foramen
magnum and cut the skull along the midline to the front to
the midpoint between the eyes.
10. Cut bilaterally starting from the midline towards the sides.
 All-Optical Neuronal Manipulation with Wavefront Engineering 35

11. Gently take apart the skull and transfer the brain in the Petri
dish containing the filter paper using a short and rounded
spatula. Place the brain on the filter paper.
12. Insert the spatula between the two hemispheres and gently
separate them.
13. Separate the cortex with the underlying hippocampus from the
brainstem, midbrain, and striatum using the spatula without
touching the hippocampus.
14. Place the cortex so that the hippocampus is exposed. Flip the
hippocampus over and out using the spatula.
15. Repeat steps 13–14 for the hippocampus of the remaining
hemisphere.
16. Use the wide-bore pipette to transfer the hippocampi to the
stage of the tissue chopper and align them perpendicularly to
the blade.
17. Remove any excess dissection medium from the stage to mini-
mize any motion of the hippocampi during the slicing process.
18. Cut 300 μm thick slices.
19. Using the narrow-bore pipette, flush the slices with some
dissection medium and transfer them to the 3rd petri dish.
20. Gently separate the slices with the pipette.
21. Transfer the best slices to the 4th petri dish and incubate at 4 °
C (or on ice) for at least 30 min (refer to Fig. 3 from Gogolla
et al. 2006 [203] for criteria of selection). Another pup can be
dissected during this time.
22. Following a minimum of 30 min of incubation at 4 °C, retrieve
the 6-well plate from the incubator and place each selected slice
in the middle of each square membrane, using the narrow-bore
pipette (Fig. 9b).
23. Remove any excess dissection medium around the slice using a
200 μL micropipette and put the plate back in the incubator
immediately.
Excess dissection medium affects gas exchange and, conse-
quently, slice health.
24. After 3 days, remove all culture medium below the insert and
replace it with 1 mL of fresh, warm neurobasal-A culture
medium.
25. Replace the culture medium every 3–4 days.

3.4.3 Bulk Infection Before infecting, it is essential to let the slices recover and adhere to
the membrane for at least 3 days in the incubator following slicing.
If possible, opt for AAVs as they are non-pathogenic, non-cyto-
toxic, and do not integrate in the host genome. Alternatively, to
avoid the use of viruses, one can utilize electroporation of plasmids.
36 Ruth R. Sims et al.

When testing a virus for the first time on organotypic slices, it is

wise to test different dilutions of the virus, as different combina-
tions of serotypes and promoters can lead to different optimal
windows of expression.
1. Prepare the virus solutions (if necessary, dilute the virus in PBS
or NaCl 0.9%) and keep them on ice.
2. Retrieve the 6-well plate from the incubator and put it under
the hood.
3. Put 1 μL of virus solution on top of the slice. Make sure that the
solution covers the entire slice.
4. Repeat the previous step for each slice to be infected.
As the virus diffuses in the medium, be careful to infect all
the slices in the same well with the same virus preparation, to
avoid undesired expression.
5. Put the plate back into the incubator immediately and wait for a
few days for expression.

3.4.4 Troubleshooting – The slices should flatten and become transparent after a few days
in culture (Fig. 9c). Dead slices remain whitish and opaque.
– To know: WT slices exhibit autofluorescence.
– Depending on type of experiment to conduct, it is essential to be
aware that the use of antibiotics can affect the physiology of the
cells.

3.4.5 What Is Essential – Use an upright microscope because the membrane will make it
on the Day of Experiment? hard to see the cells.
– For optimal results, oxygenate the external solution.
– pH should be kept around 7.4 (with HEPES or bicarbonate &
bubbling).
– Take the slice out of the incubator only once everything on the
setup is ready.
– Under optimal conditions the slices may be re-used across mul-
tiple experimental sessions.

4 Notes

1. Tilted illumination of the grating in relatively large angles

compared to Littrow configuration is also used in order to
increase the difference in optical path between the different
colors diffracted [126]. In this way the temporal focusing effect
is enhanced because the depth of focus where all colors arrive in
 All-Optical Neuronal Manipulation with Wavefront Engineering 37

phase to recreate the ultrashort pulse gets smaller. This helps as

well in using temporal focusing with pulses of the range of 100s
of fs.
2. The field of view, or more correctly field of excitation (FOE), in
CGH is defined by the size of the SLM pixel magnified at the
λf
back focal plane of the objective (α) [137, 149]: FOExy = 2 αobj,
f2
where α = d f , with d the physical SLM pixel size. Thus, the
1
shortest is f2 or the longest is f1, the biggest the FOE at the
sample. However, for optimizing the depth of focus in tempo-
ral focusing we often need to use long f2 values for increasing
the linear dispersion at the back aperture of the objective. In
other words, FOE and axial resolution in CGH-TF systems do
not change in the same direction, and a compromise needs to
be done in some cases. A particular situation is when objectives
with very big apertures are used. While these objectives are
commonly used to increase the accessible field of view for
f
imaging, the magnification f 2 that we need to use for CGH to
1
fill the objective back aperture is ≤1 and so this does not help
neither for increasing the FOE for CGH, nor for achieving the
optimum axial resolution for temporal focusing. In that case
the physical SLM pixel size d is critical, and it has to be chosen
according to the application needs.
3. For a precise spot intensity calibration, diffraction efficiency in
the xy plane must be characterized for different z positions, for
instance, every 5–10 μm of axial displacement. However, this is
a very cumbersome procedure, unless it is automatized, and it is
often omitted. Nevertheless, it can be crucial and necessary for
applications using volumetric excitation.

5 Outlook

All-optical neurophysiology is evolving as a useful approach in

neuroscience to decode patterns of neuronal activity and under-
stand how these patterns contribute to neural disorders, to cogni-
tive tasks, or to specific behaviors. Important achievements in
molecular biology and development of advanced optical methods
are contributing to elucidating the neural code. A plethora of
optogenetic constructs with variable properties in terms of excita-
tion spectrum, kinetics, and sensitivity can be used in combination
with optical methods that provide high spatial specificity and tem-
poral precision. It is now possible to manipulate brain activity at
different spatiotemporal scales, throughout large excitation
volumes, and, also, to reach deep brain regions [204–206]. One
of the latest developments in the field is a bidirectional tool,
BiPOLES, based on two potent channelrhodopsins: the inhibitory
GtACR2 and excitatory Chrimson [207]. Bidirectional tools have
38 Ruth R. Sims et al.

been developed almost since the advent of optogenetics [45, 208,

209] and BiPOLES builds upon twenty years of developments in
opsin engineering and trafficking. As a result, both the excitatory
and inhibitory opsins are efficiently trafficked to the membrane,
with equal sub-cellular distributions and hence a tightly controlled
ratio between excitatory and inhibitory action at specific wave-
lengths and membrane potentials is achieved. This means that
neuronal activation and silencing can be controlled precisely and
predictably in all transduced cells within a particular population.
BiPOLES ought to facilitate a large number of loss- and gain-of
function experiments, which are necessary for proving the necessity
and sufficiency of a particular circuit for a specific disease, for
precisely controlling spike timing without changing firing rates,
and also the possibility of the long sought-after optical voltage
clamp [210].
In terms of the next steps for optical technology development,
all-optical experiments will continue to benefit from the use of
lower laser powers, increased acquisition or modulation speeds
(for faster acquisition rates/higher temporal precision or larger
fields of observation/manipulation), higher spatial resolution and
access to deeper brain regions. 2P excitation microscopy with scan-
ners or scanless parallel illumination through spatial light modula-
tors, though fibers or fiber bundles for endoscopic applications,
with point spread function engineering for achieving mm3 excita-
tion volumes, or particular configurations allowing mesoscopic
imaging, will continue to be developed for studying neural circuits.
3P microscopy has been already used for morphological and func-
tional imaging beyond a depth of 1 mm, but it has not yet been
explored for photoactivating neurons in deep brain regions. More-
over, although multiphoton excitation approaches have advanced
separately for imaging or photostimulation of neurons, their com-
bination for all-optical manipulation has evolved at a much slower
rate and is limited to a handful of laboratories. All-optical neuro-
physiology experiments presented so far, usually involve sophisti-
cated photostimulation approaches using wavefront-engineering
techniques, combined with standard galvanometric scanning
microscopes and electrically tunable lenses for recording responses
from neurons in different axial planes. Combining high-speed exci-
tation and recordings throughout large, continuous volumes
would have a great impact in the field.
The optical manipulation of neural circuits has the potential to
be an extremely potent approach for understanding brain function
but requires carefully chosen and calibrated tools and methodology
in order to be capable of addressing the specific question under
investigation. Now that all-optical manipulation of neurons has
become possible more than ever in an on-off basis of cell excitation,
finest control of the spatiotemporal characteristics of the excitation
patterns that leads to the detection of subtle characteristics in
 All-Optical Neuronal Manipulation with Wavefront Engineering 39

neuronal reaction, is needed. This can help in diversifying the role

of the circuit activity itself or in correlation with other circuits, and
to observe how this difference may alter network performance or
lead to a different behavior.

Acknowledgments

We thank Christianne Grimm for critical reading of the chapter. We

acknowledge financing from the ‘Agence Nationale de la
Recherche’ (ANR) project ANR-17-CE16-0021 SLALLOM and
ANR-19-CE16-0026 HOLOPTOGEN, the IHU FOReSIGHT
grant (Grant P-ALLOP3-IHU-000), the National Institutes of
Health (Grant NIH 1UF1NS107574 – 01), the ERC Advanced
Grant HOLOVIS (ERC-2019-AdG; Award no. 885090), and the
FPSU-Chaire AXA-C18/1276.

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 Chapter 2

Balancing the Fluorescence Imaging Budget for All-Optical

Neurophysiology Experiments
Peter Quicke, Carmel L. Howe, and Amanda J. Foust

Abstract
The goal of this chapter is to establish a framework to evaluate imaging methodologies for all-optical
neurophysiology experiments. This is not an exhaustive review of fluorescent indicators and imaging
modalities but rather aims to distill the functional imaging principles driving the choice of both. Scientific
priorities determine whether the imaging strategy is based on an “optimal fluorescent indicator” or
“optimal imaging modality.” The choice of the first constrains the choice of the second due to each’s
contributions to the fluorescence budget and signal-to-noise ratio of time-varying fluorescence changes.

Key words Fluorescence, Calcium imaging, Voltage imaging, Neurophysiology, One-photon,

Multiphoton

1 Introduction

Optical methods provide powerful means to investigate the struc-

ture and function of many neurons simultaneously. Importantly,
photons can be focused to, and imaged from, multiple neurons in
parallel to control and detect their activity. Several new imaging
strategies are developed every year, and neurophysiologists must
navigate growing stacks of methods papers, microscope, and laser
adverts to identify which modality can best achieve the scientific
goals of their experiments. There are a broad set of competing
requirements on the techniques used to image neuronal activity
including speed, depth, spectral separation, robustness to scatter-
ing, and motion artifacts. The goal of this chapter is to distill the
trade-offs driving choice of imaging strategy for all-optical
experiments.
We begin by detailing two key challenges to imaging neuronal
activity in intact brains. We then define signal-to-noise ratio and the
concept of a “fluorescence budget,” which ultimately determines
how small and fast of a transient signal can be resolved by a given

Eirini Papagiakoumou (ed.), All-Optical Methods to Study Neuronal Function, Neuromethods, vol. 191,
https://doi.org/10.1007/978-1-0716-2764-8_2, © The Author(s) 2023

49
50 Peter Quicke et al.

imaging configuration. We then detail how the choice of fluoro-

phore, in particular indicators of calcium and membrane potential,
and imaging modality relate to the fluorescence budget, and the
trade-offs inherent to the choice of each.

2 Key Challenges to Imaging Neuronal Activity

2.1 Challenge 1: Brains are three-dimensional (3D), while conventional microscopes

Brains Are Three- image one plane at a time. A widefield fluorescence microscope
Dimensional (Fig. 1, left) excites fluorescence throughout a 3D volume and
images fluorescence from a single plane. Unless the fluorophore
itself is restricted to one plane (e.g., culture cell monolayers),
fluorescence is excited outside of the plane of focus (Fig. 2, left).
At the camera chip, this out-of-focus light reduces the contrast of
the in-focus image. Worse, in the context of imaging calcium or
voltage signals, the labeled out-of-focus cells have individually
varying fluorescence time courses that contribute to the in-focus
time course. This makes standard widefield fluorescence imaging
with single-cell resolution often untenable in densely labeled 3D
samples. Many techniques have been developed to restrict fluores-
cence excitation (two/three-photon, light-sheet microscopy)
and/or collection (confocal variants) to a single plane of focus

Fig. 1 Widefield, one-photon imaging (left) and multiphoton scanning (right) imaging modalities; not to scale
 Balancing the Fluorescence Budget April 2020 51

Fig. 2 The challenges of 3D imaging in brain tissue. (a) Techniques that are not optically sectioning suffer from
blur caused by the collection of light from out-of-focus planes. (b) Light rays can be scattered before exiting
the tissue, changing their apparent origin. In aggregate, this leads to a blurring effect that drastically increases
with depth. Reproduced from [88], CC BY-SA 4.0

and/or remove out-of-focus fluorescence in post-processing by

combining multiple images with structured illumination [22, 63,
73, 76]. The ability to localize fluorescence to a plane orthogonal
to the optical axis is termed “optical sectioning.” This “optical
section” can scan orthogonal to the plane of focus to build up a
volumetric image plane by plane.

2.2 Challenge 2: While certain model organisms (e.g., larval zebrafish, C. elegans)
(Most) Brains Scatter are transparent, most brains, especially mammalian brains, scatter
Light light strongly. Fluorescence excitation efficiency decreases with
increasing imaging depth as excitation light is scattered and
absorbed, resulting in the degradation of the excitation point-
spread function (PSF). Moreover, light excited in one spatial loca-
tion can be scattered before collection and detected as though
arising from somewhere else (Fig. 2, right). This degrades image
contrast and confuses analysis of fluorescence time courses in adja-
cent areas.
Together, out-of-focus fluorescence and scattered fluorescence
produce blurring, which drastically increases with imaging depth
[46]. The need for optical sectioning and robustness to scattering
has driven development and application of two- [30] and three-
photon [45, 121] (or “multiphoton”) point scanning modalities,
which achieve both. Due to non-linear dependence on excitation
52 Peter Quicke et al.

intensity, two- and three-photon fluorescence excited by high

numerical aperture (NA) focusing generates femtoliter fluorescence
volumes. Non-descanned fluorescence generated at the focus, scat-
tered and non-scattered, is collected through the objective onto a
large area detector (Fig. 1, right), commonly a photomultiplier
tube. Two-dimensional images or 3D volumes are built up point
by point by scanning the focus. This strategy enables calcium-based
imaging of action potentials (APs) at depths up to 1 mm in mouse
brains [59]. These techniques optically section and are robust to
scattering; for structural imaging in live brains, this is as close as we
have to perfect. Why, therefore, consider a different strategy for
functional imaging? The next section introduces the key concept
and guiding principle to designing and selecting a modality to
image neuronal activity: the “fluorescence budget.”

3 Signal-to-Noise Ratio Is King: Fluorescence Budget

The principal consideration guiding optical neurophysiology sys-

tems is the signal-to-noise ratio (SNR) of time-varying fluorescence
changes. Here the term signal-to-noise ratio most commonly refers
to the ratio of the amplitude of a time-varying signal (S) transient to
the “baseline noise” (σ), typically the root mean square of the signal
that precedes or follows a transient peak (Fig.3). Note that this
definition differs from how signal-to-noise ratio is often defined in
scientific and engineering disciplines (see Note 1). Note also that in
this chapter we are dealing specifically with the SNR of temporal,
not spatial, fluorescence changes.
The temporal SNR quantifies the ease with which a fluores-
cence transient can be resolved from a noisy time-varying signal.
The SNR is equal to the product of the fractional change in
fluorescence with respect to baseline, or “indicator sensitivity,”

Fig. 3 Signal-to-noise ratio can be defined as the ratio of the amplitude of a

time-varying signal (S) transient to the “baseline noise” (σ)
 Balancing the Fluorescence Budget April 2020 53

multiplied by the total fluorescence collected per location (pixel or

voxel), per integration period, the “fluorescence budget” (see
Note 2):
ΔF pffiffiffiffiffiffi
SN R = F 0: ð1Þ
F0
ΔF/F0 reflects the sensitivity of the indicator’s fluorescence to the
physiological signal (e.g., membrane potential or calcium). The
fluorescence budget, F0, is given by
F 0 = ϕ F gen , ð2Þ
where ϕ (unitless) is the fluorescence collection efficiency and Fgen
(in photons) is the fluorescence generated per location (pixel or
voxel), per integration period. ϕ is determined by an assortment of
factors dependent on the sample, such as imaging depth,
wavelength-specific absorption, scattering length, and anisotropy,
and specific to the collection optics, such as acceptance angle
(numerical aperture, NA) and detector efficiency [129]. ϕ quanti-
fies what fraction of fluorescence is collected by the imaging system.
The fluorescence generated is
F gen = RF l C F l V F l Δt, ð3Þ
where RFl is the fluorescence rate (photons/s/molecule), CFl is the
fluorophore concentration (molecules/m3), VFl is the per location
fluorescence excitation volume (m3), and Δt is the integration
period (s). The fluorescence rate is given by
RF l = σ n < I n > ð4Þ
in photons/s where σ n is the n-photon brightness (1-, 2-, or 3-
photon), the wavelength-dependent product of cross-section and
quantum yield (m2n sn-1/photonsn-1), and < In > is the time
average of the fluorescence excitation intensity raised to the n-th
power (photonsn/m2n/sn).1 Ideally, < In > is maximized to
increase SNR up to the limit of photobleaching, heating, and in
the case of ultrafast pulsed excitation, non-linear damage thresh-
olds. Substituting gives
ΔF pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
SN R = ϕ σ n < I n > C F l V F l Δt : ð5Þ
F0
The two imaging modalities introduced in the previous section,
widefield and multiphoton scanning, are at opposite extremes in
terms of fluorescence budget. Widefield integrates photons from all
locations in a two-dimensional frame simultaneously throughout
the frame period, and hence:

1
Intensity is often normalized to photon energy in the multiphoton imaging literature [130], different from the
standard definition of W/m2.
54 Peter Quicke et al.

1
Δt = , ð6Þ
Racq N Z
where Racq is the frame acquisition rate and NZ is the number of
planes imaged per volume. In contrast, multiphoton point rastering
modalities integrate fluorescence from each location for only a small
fraction of the acquisition period
1
Δt = , ð7Þ
Racq N X N Y N Z
where NX and NY are the lateral frame dimensions.
Consider the case where NZ = 1 to image a single plane, such
that Racq is the frame rate, and compare the widefield to the
multiphoton raster scan case. For the widefield case, Δt = 1/Racq.
For the multiphoton scanning case, digitizing each frame for exam-
ple as NX = NY = 256 pixels, we see that Δt = 1/(2562Racq). If we
base our SNR comparison solely on differences
pffiffiffiffiffiffiffiffiffiffiffi in Δt, we see that
the SNR for the widefield case is 2562 = 256 times that of
multiphoton scanning! Note that this factor could be even bigger
if the scanning modality involves significant “dead time” (i.e.,
galvonometric scanner turn around). Cameras also require read-
out time, during which they are not integrating photons; however,
this is typically around 10 μs per line for a modern sCMOS camera,
a small fraction of the frame period, while scanning dead time can
be around 10–20% of each frame.
The optical sectioning and robustness to scattering achieved by
2P and 3P point scanning modalities over widefield comes at high
cost to the fluorescence budget, F0. Note that in the multiphoton
point scanning case, F0 is inversely proportional to the product of
the number of locations (pixels or voxels, NXNYNZ) monitored,
and the rate at which they are monitored, Racq. This implies that F0
can be maintained by performing high Racq acquisition of few
locations or low Racq acquisition of many locations. For example,
at the high rate extreme, 10 kilohertz acquisition of voltage tran-
sients from a single “voxel” has been achieved in two photon by
parking the beam over a dendritic spine [3]. At the other extreme,
two-photon “mesoscopes” image FOVs multiple millimeters wide
at low frame rates (0.1–10 Hz) [102, 106] using slow and highly
sensitive calcium indicators [25, 29].
From Eq. 5, we see that SNR can be increased by: 1. use of a
high sensitivity (large ΔF/F0) fluorescence reporter and/or
2. increasing F0. F0 can be increased by: maximizing ϕ with efficient
photo-sensors, high collection NA, etc.; selecting a bright fluoro-
phore and exciting it at wavelengths for which its brightness, σ n, is
highest; maximizing fluorophore concentration, CFl, and exciting
and collecting fluorescence over the largest possible voxel, VFl;
exciting the fluorophore with the highest feasible intensity,
 Balancing the Fluorescence Budget April 2020 55

Table 1
Summary of variables determining temporal SNR for functional fluorescence imaging

Symbol, Meaning Depends on

ΔF/F0, Indicator sensitivity Fluorescent indicator
F0, Fluorescence budget Fluorescent indicator and imaging modality
ϕ, Fluorescence collection efficiency Imaging modality and sample properties
CFl, Fluorophore concentration Indicator expression/loading
VFl, Fluorescence excitation volume Imaging modality
Δt, Fluorescence integration time Imaging modality, constrained by transient decay constant
τOff
σ n, n-photon brightness Fluorescent indicator
< In > , Excitation intensity Imaging modality, constrained by tolerance to heating/
damage
Racq, frame/volume acquisition rate Inversely proportional to Δt
τOff, Fluorescence transient decay Fluorescent indicator
constant

< In > ; and maximizing integration time, Δt. Table 1 summarizes

the parameters contributing to SNR of functional fluorescence
signals. The next sections discuss trade-offs driving choice of fluor-
ophore and imaging modality.

4 Choosing a Fluorescent Indicator

A fluorescent indicator is an organic molecule or protein functio-

nalized to transduce biophysical changes into changes in fluores-
cence. Most commonly used are indicators that respond to changes
in membrane potential [11, 13] and calcium [7, 112], but others
exist that respond to pH [93, 94], sodium [56, 61], and neuro-
transmitter concentration including glutamate [69, 70], acetylcho-
line [52], or GABA [71]. With regard to temporal SNR, the key
parameters of such a molecule include its sensitivity (ΔF/F0) and
brightness (σ n) as described above and its temporal profile.

4.1 Temporal Here, indicator “temporal profile” refers to the convolution of the
Considerations time course of a biophysical process (e.g., fast membrane potential
variations vs. slow changes in calcium or neurotransmitter concen-
tration) with the kinetics of the fluorescent indicator. Here we refer
to τOff as a general term for the rate of decay back to baseline of the
fluorescence transient induced by an action potential or other brief
event. We note, however, that many indicators’ kinetics are not
described by a simple mono-exponential decay. Importantly, τOff
56 Peter Quicke et al.

determines the minimum Racq (and hence maximum Δt) able to

resolve the time-varying signal without aliasing. For example, some
highly sensitive Förster resonance energy transfer (FRET)-based
voltage indicators temporally low pass filter fast events such as
action potentials due to slow translocation of mobile charges in
the plasma membrane [41]. While considered a disadvantage in
most scientific contexts, this low pass filtering is an advantage for
imaging, as slower transient changes in fluorescence can be imaged
at lower rates Racq without aliasing, thereby increasing Δt and F0.
The minimum Racq is defined by the functional fluorescence tem-
poral profile and also by the goals of the experiment. For instance,
Racq can be relatively low for mere transient detection but must be
much higher to characterize transient timing and kinetics [90]. The
implications of differing τOff for imaging strategy design can be
readily appreciated through comparison of calcium and membrane
potential indicators for neuronal AP detection.

4.2 Membrane The majority of all-optical neurophysiology experiments monitor

Potential vs. Calcium neuronal membrane potential, either directly with voltage-sensitive
fluorophores or indirectly through fluorophores sensitive to cal-
cium concentration. In most cases, calcium transients are moni-
tored to detect when a neuron fires an action potential; so why not
always choose voltage-sensitive fluorophores to image membrane
potential directly? The particular challenges of imaging membrane
potential compared to calcium are summarized in Fig. 4. First, the
physiological signal kinetics are fast, milliseconds in the case of
action potentials, necessitating high Racq. High sampling rates
limit the available photon integration time, Δt, demanding brighter
(high σ n) indicators for adequate SNR as discussed in Note 2.
While calcium indicators are distributed throughout the cyto-
solic volume, voltage indicators are confined to the membrane. The
100-mV membrane potential fluctuation during an action potential
leads to an electric field change of around 3 × 107 V/m across the
3-nm-thick plasma membrane. Despite this large field, the fluores-
cent indicator’s sensor molecules must orient across the neuron’s
external plasma membrane to sense any changes. This causes mul-
tiple challenges. First, the potential for physiological disruption by
membrane indicator expression limits labeling density (and hence
CFl). Membrane capacitance also increases with the number of
charged or polarizable molecules in the plasma membrane, and
over-labeling can even abolish action potentials completely
[18]. Second, highly lipophilic dyes or poorly targeted genetic
constructs can label membranes non-specifically, including internal
membranes not exposed to changing fields. This increases back-
ground relative to the voltage-dependent signal, effectively reduc-
ing ΔF/F0 and proportionally the SNR. Third, overlapping
membranes from adjacent cellular processes in densely labeled
samples are indistinguishable in most microscope images. This
 Balancing the Fluorescence Budget April 2020 57

Fig. 4 The challenges of voltage imaging. Three issues make voltage imaging
more challenging than calcium imaging. First, faster intrinsic kinetics limit the
photon integration period. Second, voltage indicators must lie in the membrane
or degrade the signal; this limits the volume of indicators that can be integrated
to measure the signal. Lastly, membranes where signal arises are tightly packed
in the brain; fluorescent signals from overlapping membranes wash out single-
cell signals. Adapted from original in [88], CC BY-SA 4.0

results in signal mixing, especially with widefield single-photon

illumination. Voltage transients from individual cells are then
“washed out” by the bright background from adjacent cells.
Calcium indicators are widely used in neuroscience to indirectly
detect APs due to their relative ease of use compared to voltage
indicators. These indicators change brightness when bound to Ca2+
ions. As AP firing in most neurons is accompanied by a rapid,
transient increase in intracellular Ca2+ concentration, calcium sen-
sors report a proxy for neuronal spiking. The increase in intracellu-
lar Ca2+ on AP firing due to the opening of voltage-gated channels
can be “amplified” by release from internal stores [14] and results
in up to an order of magnitude change with a slow decay over
hundreds of milliseconds [14, 60]. This low pass filters AP wave-
forms and enables longer integration times (Δt) without signal
aliasing, increasing photon counts. Many more calcium indicators
can be loaded into the cell compared to voltage indicators as
calcium concentration changes throughout the cytosolic volume,
increasing signal brightness. Combined, these advantages have
made calcium imaging a popular technique enabling high SNR
optical recording of AP activity.
Despite these advantages, there remain significant drawbacks to
calcium indicators that limit their applicability. Calcium transients
are not exclusively linked to action potential firing in all neurons
[42, 68, 74]. AP-evoked calcium dynamics are also slow compared
58 Peter Quicke et al.

to APs, and indicator kinetics often further reduce the fluorescence

response speed. Although beneficial for imaging, this limits the
accuracy with which the timing of the underlying electrophysiolog-
ical activity can be estimated, even when the link between APs and
calcium transients is clear. Further complications confusing the link
between indicator brightness and electrophysiology include indica-
tor saturation, intrinsic and indicator buffering, and diffusion bio-
physics [95]. Increased calcium buffering due to high indicator
concentrations has also resulted in pathology [72, 105]. Possibly
the most important disadvantage to calcium indicators is their
inability to report subthreshold membrane potential changes. Cal-
cium indicators can be used to image calcium influx related to
synaptic events [17]; however, at the soma, they report suprathres-
hold AP activity. These factors have motivated development of
better optical, chemical, and biological techniques for imaging
voltage in neurons. Both the relatively low ΔF/F0 of these indica-
tors and high required RFl, however, necessitate careful consider-
ation of the photon budget (F0) when developing imaging
strategies.
In summary, voltage indicators feature small ΔF/F0 and fast
kinetics (short τOff) that require imaging with high fluorescence
budget, especially long Δt, modalities (e.g., widefield, light field) to
maintain SNR. Calcium indicators feature comparatively high
ΔF/F0 and calcium transients decay slowly, enabling imaging at
low Racq. These features render calcium indicators eminently com-
patible with low fluorescence budget modalities such as multipho-
ton raster scanning.

4.3 Spectral Of particular concern for all-optical experiments is avoiding spectral

Considerations crosstalk between photostimulation and fluorescence excitation
light. Such crosstalk comes in two types: imaging and physiological.
Imaging crosstalk occurs when photons meant to stimulate neu-
rons spuriously excite the functional fluorescent indicator. Imaging
crosstalk can be avoided altogether if the photostimulation wave-
length does not efficiently excite the indicator (e.g. [40]). If not,
fortunately, transient artefacts due to imaging crosstalk can be
predicted or measured, and subtracted, even in real time in some
configurations [35], and generally do not affect the scientific integ-
rity of the data. Physiological crosstalk occurs when light intended
to excite the functional indicator fluorophore is spuriously
absorbed by the opsin, causing neuronal de- or hyperpolarization.
In contrast with optical crosstalk, physiological crosstalk, even sub-
threshold, undermines the scientific validity of the data by
compromising the membrane potential, and in some cases action
potential rate and timing, of the imaged neurons. Indeed the
photocurrents spuriously generated by the imaging light cannot
be subtracted; they must be prevented to realize the scientific
potential of all-optical neurophysiology.
 Balancing the Fluorescence Budget April 2020 59

There are two ways to prevent physiological crosstalk in

all-optical experiments: spectral separation and spatial separation.
Spectral separation refers to exciting the indicator fluorophore at
wavelengths at which the opsin actuator cross-section is so small
that no photocurrents can be measured by whole-cell patch clamp
in the opsin-transfected cells. Importantly, this control must be
measured at the intensities and durations needed for imaging the
fluorescent indicator. For one-photon excitation, this has been
achieved by pairing blue–green-absorbing opsins with red-shifted
calcium dyes [104], voltage dyes [64, 114, 119], GEVIs [1, 34,
47], and GECIs [7, 124]. Alternatively, red-shifted opsins, such as
C1V1 [125], ReaChR [65], or Chrimson [57], can be combined
with green-emitting calcium indicators (see also Chapters 3 and 4),
although these red-shifted opsins exhibit 20%–30% actuation effi-
ciency under blue-light excitation [116] and are thus more suscep-
tible to spectral crosstalk than pairings where the opsin is excited at
shorter wavelengths than the indicator. Spatial separation refers to
limiting [107] or eliminating the fluorescence excitation light inci-
dent on opsin-expressing cells and substructures. For instance in
cases where the opsin is targeted to the soma [10, 67, 99] or to a
specific neuronal subpopulation, the fluorescence excitation light
could be patterned exclusively over non-opsin-expressing struc-
tures and cells. Efforts to reduce crosstalk in one-photon excitation
schemes with large spectral overlap between opsin actuators and
indicators (e.g., the actuator channelrhodopsin-2 [ChR2] +
GCaMP calcium reporters) have minimized read-out light intensi-
ties (< In > ) [44, 107] to the detriment of SNR.
Multiphoton schemes for opsin actuation and imaging have yet
to demonstrate a configuration completely free of physiological
crosstalk. Finding a scheme in which the imaging laser does not
evoke spurious photocurrents, producing sub- and/or suprathres-
hold membrane potential changes, is especially difficult due to
broad opsin two-photon action spectra. Spurious suprathreshold
activation has been reduced or avoided by using opsins and indica-
tors with partially separated absorption spectra (e.g., C1V1 with
GCaMP6s [83]; ChR2, GtACR2, or stCoChR with jRCaMP1a
[36, 37]) and by limiting the imaging dwell time, although sub-
threshold actuation may still occur. Broad multiphoton spectra can
provide an advantage for imaging-only configurations (without an
opsin) by exciting multiple fluorophores simultaneously with a
single wavelength (e.g., green-emitting OGB-1 or GCaMP with
red-emitting SR101 [16, 75]). Excitation to a higher-energy elec-
tronic excited state has also enabled multi-fluorophore three-pho-
ton imaging with a single wavelength [48].

4.4 Fluorophore Of critical importance to SNR is the fluorescent indicator spatial

Spatial Distribution distribution, both within a cell and within a population of cells. The
fluorescent indicator properties determine the maximum ΔF/F0,
60 Peter Quicke et al.

but this is effectively reduced in proportion to the amount of

“useless” or “background” fluorescence in the cell and surrounding
space. Background fluorescence reduces the SNR as the non-signal-
containing photons are collected from the same ROI as signal-
containing photons, reducing the fractional change in fluorescence.
If the baseline fluorescence rate from signal-containing molecules is
given by F0, and the rate of background fluorescence is given by FB,
then the fractional change in fluorescence is reduced to
ΔF ΔF FB
= ð1 - f B Þ , where fB= , ð8Þ
F0 þ FB F0 F0 þ FB
the fraction of fluorescence contributed by the background. The
SNR is then given by
ΔF pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi pffiffiffiffiffiffiffiffiffiffiffiffiffiffi ΔF pffiffiffiffiffiffi pffiffiffiffiffiffiffiffiffiffiffiffiffiffi
SNR = ð1 - f B Þ F0 þ FB = 1 - f B F 0 = 1 - f B SNR 0 ,
F0 F0
ð9Þ
as F 0 þ F B = F0
1-f B [58].
Dense fluorophore labeling poses problems especially for volt-
age indicators due to their membrane localization. Voltage signals
in densely labeled samples cannot be resolved without indicator
somatic restriction to reduce fluorescence contributions from over-
lapping adjacent processes (e.g., [2, 4, 117]) or imaging at
sub-micron resolution, which has yet to be demonstrated. This
problem is mitigated in preparations where the labeled cells are
non-adjacent or “sparse.” For example, single neuron, single-trial
action potential GEVI imaging has been achieved by expressing the
GEVI strongly and sparsely in a subpopulation of cortical layer 2/3
excitatory neurons [90] due to the high effective ΔF/F0.
In summary, the sensitivity, brightness, kinetics, and spatial
distribution of a fluorescent indicator determine which imaging
modalities can resolve the functional fluorescence transients.
Bright, slow, and sensitive indicators can boost temporal SNR for
low fluorescence budget imaging modalities. For example, if inter-
ested in membrane potential, but wanting to track the activity of
many cells with scattering-robust two-photon imaging, one can
compensate two photon’s low fluorescence budget with a slow,
bright calcium indicator.

5 SNR and Imaging Modality

Fluorescence imaging systems are comprised of two subsystems:

(1) the fluorescence excitation subsystem and (2) the fluorescence
detection subsystem. Here we detail how the characteristics of each
subsystem contributes to temporal SNR.
 Balancing the Fluorescence Budget April 2020 61

5.1 Fluorescence Regarding SNR, the fluorescence excitation subsystem can be char-
Excitation acterized in terms of light intensity, < In > , and the per pixel, per
integration period excited volume, VFl. Together with the fluoro-
phore cross-section (σ n), concentration (CFl), integration period
(Δt), and spatial distribution, these parameters determine the max-
imum available fluorescence excitation budget (Fgen; Eqs. 3, 4). The
illumination wavelength and fluorophore cross-sections determine
whether the system favors one-photon, two-photon, or three-
photon fluorescence excitation. Two- and three-photon excitation
requires ultrafast pulsed lasers with megahertz2 repetition frequen-
cies to achieve RFl sufficient for imaging. One-photon fluorescence
excitation rates, RFl, vary widely depending on indicator brightness
(σ 1) and illumination intensity (< I > ) and generally exceed RFl for
two- and three-photon modalities, which typically excite < 0.1
photons per laser pulse [101].

5.1.1 Fluorescence The fluorescence excitation rate critically depends on the degree to
Excitation Volume, VFl which the fluorescence excitation is parallelized. Widefield excita-
tion, which excites fluorescence in all locations throughout a vol-
ume simultaneously, features the highest degree of parallelization,
thus maximizing the fluorescence excitation budget. Focusing a
laser beam to a diffraction-limited point and serially scanning that
point correspond to lowest parallelization and excitation budget. In
between widefield and scanned point excitation, the excitation light
can be sculpted into many forms, including a large point (scanned-
temporal focusing; S-TeFo, [87, 118]), multiple scanned (spinning
disk confocal [108, 126], multifocal 2P [15, 55, 78, 89, 98, 120,
127]) or static (computer-generated holography, [23, 32, 85,
122]) points, a line (TeFo line scanning [28], SLAP [53], vTWINS
[103], Bessel beams [19, 66]), whole planes [5, 20, 51, 54, 82, 96,
97, 128], and extended shapes patterned directly onto structures of
interest [21, 39, 79, 109, 110]. It is important to note that not all
fluorescence photons contribute useful signal. For example, a
higher proportion of photons excited through two-photon point
scanning contribute to image formation compared to widefield
imaging, where photons excited outside the plane of focus are not
imaged and can smear the temporal signals extracted from in-focus
ROIs. It is also important to note that VFl introduced earlier in this
chapter refers to the per location fluorescence volume, not the total
spot, line, or sheet volume, and therefore depends on the spatial
discretization performed by the collection subsystem.

2
Imaging a 128 × 128 pixel FOV at 10 Hz with one pulse per pixel, the lower limit, requires a
10 × 1282 = 0.16 MHz repetition rate.
62 Peter Quicke et al.

Table 2
Δt for the different excitation volume shapes in scanning configurations

1
Scan type Δt = R acq ×
Single spot, rastered 1
NXNY NZ

Single spot, random access 1

N locs

k spots, rastered k
NXNY NZ

Line, scanned 1
NY NZ

Sheet, scanned 1
NZ

5.1.2 Fluorescence The fluorescence excitation subsystem determines the relationship

Integration Time, Δt between Racq and Δt. In particular, for excitation that does not
move or change shape during the acquisition period, Δt = 1/Racq.
Scanning generally reduces Δt in proportion to the number of
locations scanned. Table 2 summarizes Δt for the pdifferent
ffiffiffiffiffiffi scanned
excitation shapes. Bearing in mind that SN R / Δt, we appreciate
the power of parallelization to boost the fluorescence budget (F0),
enabling imaging of smaller and faster signals, and/or over larger
fields of view. Fluorescence parallelization, however, reduces
robustness to scattering, as discussed next.

5.2 Fluorescence The fluorescence detection system determines how the fluores-
Detection cence excitation budget is exploited to form images. Sensors for
fluorescence detection fall into two categories: single and multi-
channel.

5.2.1 Single-Channel Single-channel detectors, including photodiodes and photomulti-

Detectors plier tubes (PMTs), read out fluorescence intensity (or photons) as
a function of time. Imaging is achieved by combining single-
channel detectors with scanned fluorescence excitation through
the process of “temporal multiplexing”: the localization of fluores-
cence based on when it was detected. Moreover, temporal multi-
plexing can be used to scan multiple areas or z-planes by alternating
the focus of time sequential laser pulses [12, 24, 26, 49, 106, 118]).
The degree of temporal multiplexing, along with the total rate of
fluorescence excited from the sample (RFl), is ultimately deter-
mined by the indicator’s fluorescence lifetime [26].
Single-channel detection of point-scanned two- and three-
photon fluorescence excitation features the highest achievable
robustness to scattering and finest optical sectioning, owing to
temporal multiplexing. These advantages, as previously discussed,
are achieved at the cost of fluorescence excitation bandwidth, even
when fluorescence rates (RFl) are maximized through pulse ener-
gies and repetition rates increased to the maximum allowable by
 Balancing the Fluorescence Budget April 2020 63

photo-damage thresholds and fluorophore lifetimes, due to Δt’s

dependence on the number of voxels (NX × NY × NZ rastered, or
Nlocs randomly accessed).
Intermediate techniques excite fluorescence from extended
regions while collecting fluorescence on a single-channel detector
and postprocess the signal to recover a 2- or 3-dimensional image.
Notable examples include “Scanned Line Angular Projection”
(SLAP) [53], Bessel beam scanning [66], “volumetric
two-photon imaging of neurons using stereoscopy” (vTwINS)
[103], and multiplane imaging [123]. These can considerably
increase F0 for the same Δt compared to traditional single-point
scanning while remaining robust to scattering. This comes with the
caveat that the often complex and computationally expensive
reconstruction techniques typically require the imaged sample or
activity to be sparse.
With single-channel detection, the fluorescence excitation vol-
ume, VFl, is equal to the total volume excited by the spot, line, or
pffiffiffiffiffiffiffiffi that RFl remains constant, SNR increases in pro-
sheet. Assuming
portion to V F l . Hence, scanning with a large spot [87] increases
fluorescence excitation budget at the cost of spatial resolution,
which is also determined by the fluorescence spatial profile or
“point-spread function” (PSF). The ability to attribute fluores-
cence to individual neurons depends on the PSF and the sparsity
of the fluorescent indicator labeling.

5.2.2 Multi-channel Multi-channel detectors for optical neurophysiology include one-

Detectors or two-dimensional arrays of PMTs or photo diodes, and cameras,
primarily charge-coupled device (CCD) and complementary metal
oxide semiconductor (CMOS). Multi-channel devices enable “spa-
tial multiplexing”: the localization of fluorescence based on where
it was detected on the array. With the notable exception of compu-
tational reconstructions based on structural image priors [53],
imaging parallelized fluorescence excitation (multiple points,
lines, sheets, widefield) requires spatially multiplexed detection, in
most cases imaging a two-dimensional plane onto an array detector
or camera. For volumetric imaging, the imaging plane can be
scanned with the fluorescence excitation by moving the objective
or sample, with electrically or acoustic gradient tunable lenses, or by
remote focusing [8, 19]. Alternatively, the imaging depth of field
can be extended to encompass the entire volume through, for
example, wavefront coding [81, 92] or intentional spherical aber-
ration [113]. Volumetric imaging can also be achieved with light
field microscopy, which uses a microlens array to encode positional
and angular information, enabling reconstruction of full volumes
from a single two-dimensional frame [77]. Light field microscopy’s
high fluorescence budget has recently been exploited to image both
neuronal calcium [43, 50, 80, 84, 86, 100] and membrane poten-
tial [6, 27, 91].
64 Peter Quicke et al.

For multi-channel detection, VFl is equal to the volume of

excited fluorescence “seen” by each pixel of the detector. There-
fore, imaging at the lowest feasible magnification and/or binning
the fluorescence detected by pixels into regions-of-interest post
hoc, both benefit fluorescence budget and SNR at the cost of
spatial resolution.
All strategies that combine fluorescence parallelization with
multi-channel detection feature contrast that decreases quickly
with depth in scattering brain, because with multiple detectors
scattered photons can no longer be localized with certainty to a
single location. Thus increasing the photon budget by parallelizing
collection reduces the depth in scattering tissue at which functional
fluorescence signals can be imaged.

6 Summary of Key Points

The choice of fluorescent indicator and imaging strategy determine

SNR through each’s contributions to the fluorescence budget,
summarized in Fig. 5. Scientific priorities ultimately drive whether
the experiment is designed around an “ideal indicator” or “ideal
imaging modality,” which then constrains the choice of the other to
achieve sufficient SNR at the minimum required acquisition rate,
Racq. Figure 5 describes three example modality/indicator combi-
nations and situates them with respect to relative contributions of
each to SNR. For example, two-photon mesoscopy serially scans
many locations and hence features low Δt, which is compensated
through GCaMP6s’s high sensitivity (ΔF/F0), brightness (σ 2), and
long τOff, which accommodates low Racq [102]. In contrast, wide-
field imaging of fast voltage indicators, such as Di-4-ANEPPS
analogs [9], relies on widefield’s large fluorescence budget to
resolve membrane potential changes at kilohertz frame rates.
While this chapter has focused on SNR for shot noise-limited
imaging strategies, it is important to bear in mind other noise
sources. Importantly, instrument noise can dominate in low light
or low ΔF/F0 regimes. In vivo, noise arising from the sample,
including respiratory, cardiac, and other motion, can dominate
[33]. However, physiological noise occurs in distinct frequency
bands that can often be compensated or subtracted [38].
The problem of physiological crosstalk, in which imaging light
spuriously actuates changes in membrane potential due to broad
opsin action spectra, has not been fully addressed for multiphoton
excitation. Physiological crosstalk could be completely avoided, in
principle, by restricting fluorescence to structures or cell popula-
tions that are not illuminated by the imaging laser.
A key take home is that indicators and imaging modalities
featuring the highest fluorescence budgets enable the highest
acquisition rates over the largest number of locations. This concept
 Balancing the Fluorescence Budget April 2020 65

Fig. 5 Balancing indicator and imaging strategy contributions to SNR. The imaging modality (upper triangle)
and fluorescent indicator (lower triangle) properties together determine fluorescence transient SNR (horizontal
axis; equation, top). Importantly, the SNR of a “low fluorescence budget” imaging modality can be compen-
sated by a high ΔF/F0 or “high fluorescence budget” indicator and vice versa. The vertical dashed lines situate
three example indicator/modality pairings with respect to each’s relative contribution to SNR

is reviewed in detail for multiphoton modalities by [62]. However,

high budget modalities also generally do the least to mitigate light
scattering effects, limiting the depth at which functional fluores-
cence transients can be resolved. When comparing candidate imag-
ing modalities for all-optical experiments, careful inspection, in
particular of VFl and Δt, can enable reasonable prediction of how
66 Peter Quicke et al.

SNR would compare to that of alternative strategies. SNR is the

most important figure of merit to consider when designing an
optical physiology imaging strategy, as it encompasses a variety of
variables and ultimately determines whether or not the experiment
will be able to detect the biophysical phenomenon of interest.

7 Notes

1. Defining SNR: Variation Across Disciplines

Although a ubiquitous concept in many scientific and engi-
neering fields, the exact definition of SNR varies between fields.
In functional neuroscientific imaging particularly, SNR is often
defined in a way at odds with what is common in the signal
processing world. SNR is commonly understood as the ratio of
the level of the signal of interest to the level of the noise in the
measurement. Precisely defining what we mean by level, how-
ever, and how to report the ratio, is where different fields and
different studies within fields start to vary.
The canonical signal processing definition of the SNR is
given by [115]
PS 1
XN
SNR SP = , where Px = jx i j2 , ð10Þ
PN N i=0

where PS is the signal power, PN is the noise power, and Px

defines the power of a discrete signal of length N, xi. Calculat-
ing this SNR for a functional imaging trace requires measuring
or estimating a noise-free signal and the signal-less noise. This,
however, can often be difficult or near impossible for many
common functional imaging paradigms when there is no simul-
taneous electrophysiology. The functional imaging community
therefore often reports a different SNR measure (sometimes
called peak SNR or PSNR, not to be confused with the PSNR
measure used in image processing [111]), defined as
S F - F0
SNR N = , where S= , and σ 2 = VarðF 0 Þ: ð11Þ
σ F0
S is the amplitude of the fluorescence change during the
signal of interest, such as an AP, and σ is the estimated RMS
noise from a section of ptheffiffiffiffiffiffiffitime course without any signal,
approximately equal to P N from Eq. 10. This approach is
straightforward for most neuroscience signals, as the activity is
often temporally sparse, facilitating selection of time course
sections with and without activity. This SNR is often commonly
reported as a simple ratio, whereas SNRSP is often reported
in dB.
 Balancing the Fluorescence Budget April 2020 67

Fig. 6 Variation in SNR due to calculation method

These differences can result in different values for SNR for

the same traces, as the figure above demonstrates. Increasing
indicator sensitivity will scale SNRSP quadratically, while SNRN
scales linearly due to using signal amplitude, not power. Sec-
ond, and more importantly, SNRSP captures information about
signal duration which SNRN ignores. For signals that are tem-
porally sparse, SNRSP can seem surprisingly low compared to
SNRN as the noise is spread throughout the whole trace, while
the signal is concentrated into short periods (Fig. 6).
2. The Fundamental Limit on SNR in Optical Imaging
The physical nature of photon detection limits the theoret-
ical maximum SNR of functional optical imaging. We measure
the fluorescence intensity, F, of an indicator to infer something
about the underlying physiological process it reports. Poisson
noise due to a collection of fluorescence photons dictates how
well we can do this for a given number of photons collected.
The Poisson distribution gives the probability of detecting k
photons in an interval when the mean rate is F0 as
e - F 0 F k0 ð12Þ
PðF = kjF 0 Þ = :
k!
Both the expected value and the variance of F are equal to
F0. Traces are commonly normalized to the mean intensity, F0,
enabling easier comparison of structures with a different label-
ing brightness, and the variance of the normalized variable,
F/F0, can be simply calculated due to the linearity of the
expectation value as σ 2 = 1/F0. We assume here that the
change in brightness is small compared to the baseline bright-
ness, such that the noise during and outside the signal period is
68 Peter Quicke et al.

Fig. 7 Demonstration of the effect of Poisson noise on SNR. Reproduced from [88], CC BY-SA 4.0. Inspired by
[31]

the same. Our normalized signal, S, is given by ΔF/F0, the

change in fluorescence brightness, and so our signal-to-noise
ratio is given by
S ΔF pffiffiffiffiffiffi
SN R = = F 0: ð13Þ
σ F0
The figure above illustrates this by drawing samples from a
Poisson distribution to simulate fluorescent signals. The rate is
increased by 10% in the central samples, and the baseline
brightness is 10000 counts/sample in the top trace, and only
1000 counts/sample in the bottom. pffiffiffiffiffiffi This leads to an SNR
clearly increased by a factor of ≈ 10 ≈ 3 from the bottom to
the top trace. On the right, a graph shows the theoretical
maximum achievable SNR for a given brightness, for different
relative changes in fluorescence from the signal of interest,
ΔF/F0. This demonstration assumes our imaging system is
Poisson noise-limited, which is typically true for bright fluores-
cent samples (Fig. 7).

Acknowledgements

This work was supported by the Biotechnology and Biological

Sciences Research Council (BB/R009007/1), the Royal Academy
of Engineering under the RAEng Research Fellowships scheme
(RF1415/14/26), and the Engineering and Physical Sciences
Research Council (EP/L016737/1).
 Balancing the Fluorescence Budget April 2020 69

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 Chapter 3

Light-Based Neuronal Circuit Probing in Living Brains

at High Resolution: Constraints and Layouts for Integrating
Neuronal Activity Recording and Modulation in Three
Dimensions
Matteo Bruzzone, Enrico Chiarello, Andrea Maset, Aram Megighian,
Claudia Lodovichi, and Marco dal Maschio

Abstract
Understanding how the brain orchestrates neuronal activity to finely produce and regulate behavior is an
intriguing yet challenging task. In the last years, the progressive refinement of optical techniques and light-
based molecular tools allowed to start addressing open questions in cellular and systems neuroscience with
unprecedented resolution and specificity. Currently, all-optical experimental protocols for simultaneous
recording of the activity of large cell populations with the concurrent modulation of the firing rate at cellular
resolution represent an invaluable tool. In this scenario, it is becoming everyday more evident the
importance of sampling and probing the circuit mechanisms not just in a single plane, but extending the
exploration to the entire volume containing the involved circuit components. Here, we focus on the design
principles and the hardware architectures of all-optical approaches allowing for studying the neuronal
dynamics at cellular resolution across a volume of the brain.

Key words Optogenetics, Computer-Generated Holography, Volumetric Neuronal Imaging, 3d

photostimulation

1 Introduction

Light-based approaches have emerged as a powerful tool to inves-

tigate the circuit organization and the functional mechanisms
underlying the information processing in an intact brain [1]. This
stems from the fact that using light, with respect to other investi-
gation methods, allows recording the physiological variations, e.g.,
neuronal firing, membrane potential, and neurotransmitter release,
with enhanced cellular specificity and high spatial resolution, rang-
ing from extended circuits down to the subcellular compartments.
These optical techniques rely on engineered fluorescent reporters
like GECIs (Genetically Encoded Calcium Indicators) [2] or GEVI

Eirini Papagiakoumou (ed.), All-Optical Methods to Study Neuronal Function, Neuromethods, vol. 191,
https://doi.org/10.1007/978-1-0716-2764-8_3, © The Author(s) 2023

75
76 Matteo Bruzzone et al.

(Genetically Encoded Voltage Indicators) [3], whose absorption

efficiency or fluorescence emission yield depends on calcium ion
concentration or cell membrane potential. GCaMP is currently, by
far, the most commonly used GECI in neuroscience. The molecular
structure comprises a green fluorescent protein (EGFP) bound,
through a M13 fragment of a Myosin Light Chain, to Calmodulin
(CaM) [4]. CaM is a calcium-binding protein that, with the
increase of intracellular calcium concentration following the firing
of an action potential, induces a conformational change to the
overall configuration resulting in an increase in the fluorescence
intensity detected. In 2013, a family of ultrasensitive calcium repor-
ters, GCaMP6, was introduced [5] and later improved with the
family of GCamp7 [6]. These molecules are characterized by
changes in fluorescence that can reach up to 1100% and decay
times ranging from 950 to 250 milliseconds. In appropriate signal-
to-noise ratio conditions (SNR), these molecules enable the detec-
tion of a single AP with scores as high as 94%. Along with EGFP-
derived sensors, molecular variants with emission spectrum shifted
toward the red region have been engineered [7, 8]. RCaMPs and
R-GECOs (Red Genetically Encoded Calcium indicators for Opti-
cal imaging) are the main families of red reporters. They are
obtained by subtituting the fluorescence reporter with mRuby
and mApple, respectively. Compared to green indicators, typically
excited at 920–930 nm and emitting in the 490–560 nm interval,
RCaMPs are optimally excited around 1100 nm and emit around
590 nm. These reporters, in combination with optical approaches,
enable the recording of the neuronal activity in the brain circuit of
a living organism with cellular or even sub-cellular resolution.
From the circuit and systems neuroscience perspective, recon-
structing cellular activity offers a powerful tool to look into brain
mechanisms. However, the data obtained provide mostly correla-
tive information, i.e., the activation of a certain neuronal
sub-population occurring with the modification of a sensory or
behavioral parameter [9]. Indeed, in this case, the possibility of
validating the putative circuit mechanism and confirming its pre-
dictions on the expected dynamics of the system is still lacking.
Ideally, for this purpose, one would like to record the change in the
system activity when boundary conditions or network properties
are artificially controlled.
The recent development and the constant improvement of
light-activated modulators of the membrane potential offer, in
this sense, an additional tool to intervene non-invasively and
non-destructively into the circuits and probe the circuit mechan-
isms, i.e., to measure the circuit output under controlled condi-
tions, at cellular resolution [10, 11]. Such tools can be genetically
encoded to target designed neuronal populations with exogenous
light-sensitive ion channels or pumps (termed “actuators” or
“opsins”) [12]. These all rely on a retinal, a polyene chromophore
 Optical Approaches to Probe Neuronal Circuits in 3D 77

typically found in animal mechanisms of light conversion. When the

retinal absorbs a photon, it isomerizes and originates a series of
conformational changes that end up in the peculiar activity of these
proteins, e.g., ion diffusion or transport across the cellular mem-
brane. The physiological effect originating from light stimulation is
a change in the concentration of different ions, ultimately resulting
in the modulation of the polarization level of the cell membrane
potential. Several opsins are used in neuroscience, and are grouped
into two main classes, depending on the promotion or suppression
of the firing of action potential induced by light. On one side, one
can find actuators with depolarizing effects, like ChR2, C1V1,
ChrimsonR, and Chronos, typically used to kick the membrane
potential of excitable cells above the threshold for action potential
firing. In certain cases, the temporal kinetics of the elicited photo-
current is sufficiently fast to allow control of the neuronal firing
with submillisecond precision and repeated firing up to 100 Hz.
On the other side, there are light-gated molecules with hyperpolar-
izing effects, like eNpHR and GtACRs, whose light-based activa-
tion moves the membrane potential in a range preventing action
potential firing or strongly reducing its probability [13]. Depending
on their structure and properties associated with the photocycle,
these molecules present different photocurrent magnitudes, ion
selectivity, photocurrent kinetics, and spectral sensitivity. All these
parameters strongly impact either the kind of neuronal modulation
achievable or the optimal method to obtain it in combination with
concurrent activity recordings.
In this chapter, we describe the hardware components to probe
3D brain circuits at cellular resolution. We present currently
reported light-based architectures for 3D imaging and 3D photo-
stimulation based on multiphoton absorption. We highlight the
most relevant aspects associated with the integration of these two
components in the same experimental paradigm.

2 Methods

2.1 Molecular and In general, all-optical probing of brain circuits relies on the possi-
Technical Constraints bility of concurrently recording and modulating neuronal activity
with high resolution [14]. This assumes the compresence of light-
gated actuators and light-based activity reporters within the same
preparation and, in some cases, within the same excitation volume.
Then, combining these two families of tools together in the same
experimental scenario requires evaluating the biochemical and bio-
physical properties of these molecules along with technical aspects
and working constraints associated with their use.
78 Matteo Bruzzone et al.

2.2 Absorption In the design of all-optical experimental paradigms, one aims at

Characteristics and Its rendering the imaging and the photostimulation processes as much
Impact independent one from the other as possible: avoiding the imaging
process to drive the activation of the opsin, hence altering the state
of the expressing cells, and taking care that opsin photostimulation
does not compromise the expected functionality of the activity
reporter or the integrity of the signal extracted (see also Chap. 2).
While it is possible to control many of the experimental parameters
to keep such ideal working conditions, less straightforward is to
engineer the biophysical and chemical properties of the molecules
in use, that ultimately represent the first working constraint. Even
considering molecule pairs maximizing orthogonality, the overlap
between the absorption spectra is a condition frequently present,
and it can result in important effects of “crosstalk” [15] (Fig. 1).
Thus, the combination of an actuator with a reporter becomes

Fig. 1 Spectral properties of the most common actuator-reporter pairs, used for all-optical approaches. In the
upper part are reported the light-driven actuators. The range with a 2P action spectrum greater than 60% is
shown with a darkened mark indicating the wavelengths corresponding to the activation peak. On the right
side, the corresponding τOFF is reported. This indicates the photocurrent decay time at the offset of the
illumination. In the lower part, excitation and emission spectra are presented for the most common activity
reporters. Black lines highlight the actuator-reporter pairs reported [16–19]
 Optical Approaches to Probe Neuronal Circuits in 3D 79

possible at the condition of identifying suitable imaging or photo-

stimulation parameters, e.g., wavelength, pixel dwell time, power
density, and field of view, that minimize spurious activation of the
actuator or reporter signal contamination [20–25]. The identifica-
tion of a suitable molecular pair comes with the optimization of the
optical parameter based on light sources available on the market. In
general, the space of the optical parameters one can tune is suffi-
ciently large, allowing the design of experimental protocols also
with low orthogonality, where the extent of spectral overlap
becomes considerable.

2.3 Photocurrent In the design of an all-optical investigation protocol, one important

Integration and aspect to consider is the mechanism of photocurrent integration at
Spurious Opsin the cell membrane level and the impact the imaging process per se
Activation could have on the alteration of the network state [26]. This origi-
nates from the photocurrent generated from the simultaneous or
quasi-simultaneous activation of multiple molecules that spatio-
temporally add together their contributions. While photocurrent
spatio-temporal integration is the key element for an effective
photostimulation, this represents a constraint for imaging pur-
poses, with opsins absorbing significantly at wavelengths used for
imaging. Indeed, upon saturating excitation, net charge transfer
across the membrane is proportional to the channel conductance
and the opsin de-activation time constant, usually called τOFF, i.e.,
the time required after the light offset for the evoked current to
return back to zero (Fig. 1). The value of τOFF can almost cover two
orders of magnitude (from 1 to 2 ms for the fastest opsin, Chronos,
to several tens of milliseconds in the case of C1V1 and ReachR) and
can impact in the extent of opsin activation due to the imaging
process [27]. It is known that, keeping all the other parameters con-
stant, opsins with longer τOFF support more effectively a current
integration process in the temporal domain, resulting in greater net
charge transfer, potentially enhancing the effect of spurious opsin
activation associated with the imaging process. Along with the
imaging light power density at the focal position, it is then critical
to tune other imaging acquisition parameters to render the total
light dose sustainable and negligible the impact of the imaging
process on the network state. Currently, this is achieved by sparsen-
ing the pixilation matrix of the images or equivalently extending the
field of view and reducing the pixel dwell time or the line scan time.
On the other side, this solution for limiting the spurious activation
of the opsin impacts on SNR of the signal reporting the neuronal
activity. It becomes, then, a matter of properly balanced expression
levels of the molecules and experimental parameters to work on
optimal conditions.
80 Matteo Bruzzone et al.

2.4 Off-Target The possibility of using all-optical approaches to probe brain cir-
Activation and Somatic cuits critically depends, along with recording the neuronal activity
Opsin Targeting without perturbing the system, on the capability to target the
neuronal modulation with high spatial precision and selectivity.
This assumes that the photostimulation impacts exclusively or
mostly on the identified targets, i.e., sets of neuronal somata. This
is a goal not straightforward to achieve as dendrites and axons from
many different cells surround the targeted cell body and constitute
a dense mesh of neuronal processes, frequently expressing them-
selves the opsin. This represents a possible issue of off-target acti-
vation, i.e., indirect photostimulation of neuronal components
other than those targeted. This is beyond the limit of the hardware
design, independently from the specific optical implementation to
drive the light-based actuator. To overcome this limitation, in the
last years, many labs developed light-driven actuators specifically
targeted to the cell soma, importantly reducing the expression
along the axons and the other processes [28–30]. This frequently
results in more efficient stimulation (in terms of required power
density), and the strong reduction of indirect effects mediated by
passing-by neuronal processes.

3 Hardware Implementations for 3D Recordings of Neuronal Activity

There are many hardware layouts reported for recording neuronal

activity at different depths within the sample, either with sequential
scanning of diffraction-limited spots in raster or random schemes
either with longitudinally or laterally extended excitation profiles
[1, 31–33] (see also Chap. 10). Ideal methods may depend on
different factors, like the light scattering, the labeling sparseness
of the sample, and ultimately on the question addressed. Here we
focus on the general architecture implementing 3D raster-scanning
of diffraction-limited multiphoton excitation, as this is currently
the method offering the highest flexibility [34]. Three main com-
ponents generally characterize this scheme, in some cases partially
overlapping (Fig. 2): a first one, starting with the source and
including all the optical elements required for intensity modulation
and for the spatial conditioning of the beam; a second one devoted
to longitudinal scanning of the excitation spot along the light
propagation direction (Z); and a third one, for the deflection of
the excitation beam in the lateral direction (XY).
Ti:Sapphire-based sources are typically adopted for optical
recordings of neuronal activity. Fluorescence emission from the
activity reporter is excited deep in the brain tissue via multiphoton
absorption of 180–250 fs light pulses in the IR range of the spec-
trum and delivered at the sample at a repetition rate of
40–100 MHz. The fluorescence emitted is detected with photo-
multiplier tubes and solid-state detectors [35], resulting in
 Optical Approaches to Probe Neuronal Circuits in 3D 81

Fig. 2 General hardware layout for 3D imaging. This typically includes three elements: the first one with the
source and the intensity modulation unit, a component of the optical path designed for scanning the beam
along the longitudinal direction (in blue), and a module for scanning the excitation beam along the lateral
dimension (in red). The different elements are conjugated by means of relay optics

functional signals with a high SNR, as far as a sufficient photon

density of the ballistic excitation component is preserved, and the
scattered component does not contaminate the image background
level. A Pockels cell is usually used as an intensity modulation unit
to finely and rapidly control the imaging beam power at the sample.
As the beam diameter is typically below 2 mm at this stage, optical
elements for expanding the beam size are inserted, considering the
target size at the level of the objective pupil and the magnification
realized by the combination of the scan lens and the tube lens.
The more common design for functional recordings across a
3D brain volume is based on optical modules or elements enabling
the imaging beam to scan the sample along the light propagation
direction (longitudinal, Z). This has been traditionally implemen-
ted with the mechanical movement of the imaging objective by
means of a piezoelectric actuator [36]. More recently a series of
approaches, prevalently based on the control of the curvature of the
light wavefront at the Back Focal Plane (BFP) of the objective, have
been refined to remotely control the effective focal position
while keeping the objective still [31]. To reconstruct the activity,
82 Matteo Bruzzone et al.

the beam is deflected sequentially to different positions of the field

of view. To achieve the lateral scanning within the region of interest,
imaging systems adopt a pair of galvanometric mirrors or acousto-
optic deflectors.

3.1 Remote In general, scanning a diffraction-limited beam in a volume of the

Configurations for 3D sample relies on the possibility to modulate the light wavefront at
Beam Scanning the BFP of a lens or an objective. Indeed, superimposing a spatial
gradient or a curvature into the phase of the electric field of a planar
light wavefront leads upon propagation, respectively, to a lateral or
a longitudinal offset of the beam focus at the Frontal Focal Plane,
FFP (Fig. 5). These two planes are said to be Fourier conjugated,
and the light distribution at the FFP represents the results of the
diffraction of the wavefront modulated at the BFP. To easily impose
such modulation of the light wavefront, the BFP of the objective is
optically conjugated using relay optics to a remote plane, where the
element for introducing the wavefront modulation can be more
conveniently placed. This is, for instance, the design realized by the
tube lens-scan lens pair and the galvanometric scanner. If more than
one modulator has to be employed, e.g., to impose both a vertical
and horizontal tilt and a curvature, then either all the modulators
are placed very close together near the focal plane of the scan lens,
or they are distributed in multiple places and conjugated by addi-
tional relay optics to the BFP of the objective.

3.2 Methods for The most common way for scanning the sample along the lateral
Scanning the Sample dimension relies on a pair of galvanometric mirrors, conjugated to
Along the Lateral the BFP of the objective. These are 3–6 mm wide mirrors mounted
Dimension with an orthogonal optical axis whose orientations can be tuned by
proper control signals. A change in the orientation with respect to
the propagation of the incoming beam introduces a linear phase
gradient to the wavefront, resulting in the lateral offset of the beam
with respect to the center of the field of view (FOV). In a raster
scanning scheme, one mirror sweeps over a line, and the other
jumps from one line to the following one. With a typical pixel
dwell time of about 4 μs, this design results in a frame acquisition
time lower than a second for a mesh of 512  512 pixels. An
8–12 kHz resonant galvanometric mirror can replace the line scan-
ning galvo, reducing the line acquisition time to 62–41 μs, respec-
tively, and increasing the acquisition rates up to 30–45 frames per
second for a pixel matrix of 512  512 elements. In this second
scenario, there is no direct possibility to adjust the pixel dwell time
and its extremely short value, typically 120–80 ns, heavily impacts
the number of collected photons per pixel and, ultimately, the
image SNR. In parallel to the solutions described above, to steer
the light beams, it is possible to modulate the light wavefront in an
inertia-free approach using acousto-optic deflectors (AODs)
[37, 38]. These are active optical elements with a crystal window
 Optical Approaches to Probe Neuronal Circuits in 3D 83

bonded to a piezoelectric transducer. When this is driven by an

electrical signal at high frequency, it induces an acoustic wave
traveling along the crystal in a particular direction. Based on
the photoelastic effect, this generates a diffraction grating so that
an optical beam propagating through the crystal in the direction
orthogonal to the acoustic wave experiences an angular deviation
that is proportional to the driving frequency. A pair of consecutive
and orthogonal AODs can be used as a scanner to tilt the light
wavefront in both the lateral axes. AOD-based scanners are char-
acterized by bandwidths between 30 and 40 kHz, corresponding to
a typical beam resetting time between 15 and 25 μs that accounts
for the time needed for the acoustic wave to cover the distance
corresponding to the effective optical window. While in a random
access scheme, these numbers allow for extremely high and unpar-
alleled temporal resolution in the recordings, the acquisition rates
in full-frame raster scanning mode can be comparable with those
achieved with a resonant scanner, i.e., 30 Hz. Diffraction efficiency
and group delay dispersion are two other important figures that a
design with an AOD scanner should deal with to obtain good
imaging performances [39].

3.3 Methods for Adjusting remotely the longitudinal position of an excitation spot
Scanning the Sample generally relies on the possibility to impose a curvature on the
Along the Longitudinal wavefront profile in a plane optically conjugated to the BFP of the
Dimension objective. Considering the sole imaging purposes, possibly the
simplest implementation is to place a lens with a controllable focal
length, either at the level of the BFP downstream of the XY scanner
and the scan lens-tube lens pair or in a plane optically conjugated to
the BFP, upstream the scanner. An electrically tunable lens (ETL) is
an example of such a device, which is composed of a liquid volume
enclosed between a glass and an elastic polymer membrane, i.e.,
effectively a plano-convex liquid lens [40]. An electromagnetic coil
driven by an electric current exerts pressure on the liquid, increas-
ing the membrane curvature and thus the lens focal power. ETLs
are capable of relatively fast settling times, typically between 5 and
15 ms, in response to a step-like control signal and can easily reach,
depending on the objective properties, a few hundreds of microns
of focal range with limited distortion of the point spread function
(PSF) (Fig. 3). ETL driving, either with staircase-like or sawtooth
control signals, in coordination with the frame acquisition allows
for the plane-after-plane acquisition of a volume. According to a
similar scheme, a tunable acoustic gradient index of refraction lens
(or TAG lens) can be used to quickly scan the sample longitudinal
dimension [41, 42]. In this case, high-frequency driving of a piezo-
electric actuator generates in a viscous medium a cylindrical acous-
tic wave whose constructive interference produces periodic changes
in medium density and, consequently, in the lens optical power.
The resonance frequency of these devices, typically a few hundreds
84 Matteo Bruzzone et al.

Fig. 3 Multiphoton imaging across multiple planes in the olfactory bulb of a mouse expressing GCaMP6f. A
basic configuration for multiphoton imaging is complemented with an electrically tunable lens to sample
quasi-simultaneously different planes of the brain volume. Four different planes are shown with activity
profiles extracted from the corresponding cells

of kHz, results in axial scanning times in the order of a few micro-

seconds. In this case, volume information is acquired not plane-by-
plane moving along the longitudinal axis but section-by-section
aligned to it. A critical parameter to evaluate for the positioning
of an ETL or a TAG lens is the effective aperture of the optical
window with respect to beam diameter at the designed position
along the optical train.
Spatial light modulators (SLMs) based on a matrix of nematic
liquid crystal represent a valid alternative for imposing a curvature
profile [43–45]. These optical elements use the birefringent prop-
erties of the liquid crystal molecules to tune with a control voltage
the effective refractive index of the individual cells within the matrix
and control the phase delay of the different spatial components of a
propagating wavefront [46–50]. These devices can encode rela-
tively large wavefront curvature leveraging the large number of
pixels and the possibility of phase folding, resembling the scheme
of a Fresnel lens. For these systems, liquid crystal relaxation times
pose the major restriction to the refresh rate of the diffractive
optical element encoding the phase correction. Still, it can reach
300–400 Hz, allowing resettling times close to 2.5–5 ms. Faster
switching times can be obtained by moving to deformable or
tunable mirrors (DM) [51]. Indeed, these later devices can achieve
currently important maximal phase strokes with sub-millisecond
resettling times. As SLMs and DMs are more conveniently
operating in reflection mode, the preferred position of these remote
re-focusing devices is upstream of the XY scanner, where also more
space is available for beam conditioning according to the optical
window and the working mode of these devices. With respect to
 Optical Approaches to Probe Neuronal Circuits in 3D 85

ETL or TAG lens, an advantage of SLMs and DMs is that, along

with the wavefront correction required for the defocus, it becomes
possible to superimpose additional phase corrections to the wave-
front to compensate for aberrations induced by other optical ele-
ments, including the objective and the sample [44]. If a faster
longitudinal scanning speed is required, a variable curvature to
the wavefront can be added with an acousto-optic lens, which is
implemented with a combination of the same acousto-optic deflec-
tors (AOD) used for lateral scanning, as described previously
[39, 52]. When the acousto-optic deflector is driven with a fre-
quency ramp, the acoustic wave renders a gradient of local spatial
frequency which locally deviates the wave front by different
amounts, resulting in a focusing effect, with a focal length being
proportional to the rate at which the frequency changes. Since the
frequency changes over time, the position of the focus is not steady
but it drifts laterally with a constant speed. Employing a pair of
consecutive AODs, driven with the same frequency ramp but with
acoustic waves traveling in opposite directions, results in two drifts
that cancel each other out. Another way of longitudinally shifting
the excitation spot is by conjugating the focal plane of the imaging
objective with the focal plane of another “remote” objective, where
a fast translating mirror is placed in the FFP [53–55]. The beam
injected in this z-focusing arm enters the remote objective and gets
focused in a spot at its (fixed) focal plane. When it is then reflected
back by the mirror, it behaves as a point source which then gets
focused by the other objective at the sample, where an image of it is
formed. When the BFP of the remote objective is conjugated to the
XY scanner and to the BFP of the main objective, longitudinally
shifting the remote mirror shifts the actual position of the point
source and consequently shifts the position of its image at the
sample plane. This configuration leveraging the lightweight mirror
and the availability of accurate piezo actuators can achieve settling
time in the order of 1 ms and travel ranges of several hundreds of
micrometers. To further extend the bandwidth limits of the scan-
ning process, solutions based on temporal multiplexing have been
proposed to sample non-simultaneously points at different z-planes
without actually changing the setting of any optical components
[56]. This approach relies on the fact that the fluorescence decay
time of the reporter, typically 2–4 ns, is shorter than the interpulse
interval of the excitation sources, usually 10–12 ns. In these con-
ditions, an optical module can be designed to split the original
beam into a n-number of components which are temporally delayed
one from the other with an amount of time (0, τfl, 2 ∙ τfl, . . , n ∙ τfl,
with. . , n ∙ τfl  interpulse interval) covering the fluorescence decay
and are corrected with different degrees of wavefront divergence.
Upon recombination of the components, this results in a com-
pound beam encoding a set of n different curvatures of the beam
wavefront, each addressing a different z-planes in a different time
86 Matteo Bruzzone et al.

window corresponding to the delay introduced. Temporal demul-

tiplexing of the fluorescence signal with fast acquisition electronics
according to the same interleaving scheme allows to reconstruct the
neuronal activity from different planes with minimal interplane
interference.

4 Hardware Implementations for 3D Modulation of Neuronal Activity

The general architecture of an optical train for high-resolution 3D

photostimulation is designed after the principle of obtaining the
sufficient integration of the light-induced charge transfer across the
membrane. This results in layouts formed by two components: a
first one, starting with the source and including all the optical
elements required for beam intensity modulation and for the spatial
conditioning of the beam; a second one, downstream to the first
one, integrating optical components and systems for the generation
of the arbitrary distributions of the electric field at the sample
(Fig. 4). The scheme for the first part envisages the use of pulsed
and high peak energy laser sources to activate light-gated actuators
via a multiphoton absorption process. This is instrumental to access
components of the neuronal circuit laying in the deep layers of a
scattering preparation and to contain the spatial profile of excitation
along the light propagation direction, taking advantage of the
non-linear dependence of the multiphoton absorption on the exci-
tation power. Along with traditional Ti:Sapphire, more recently for
photostimulation are preferred laser sources based on oscillators
and amplifiers with high pulse energy, up to 100 μJ range, and low
repetition rate, 500 kHz – 5 MHz [26, 57, 58]. Indeed, for the
same average power at the sample, the probability of multiphoton
absorption scales with the inverse of the laser emission duty cycle,
allowing, with an increase in the available pulse photon density, a
more efficient activation of the molecules at saturation and target-
ing larger populations of cells. The basic optical train for the first
part includes, along with a light shuttering module, a stage for the
modulation of the source intensity. This is typically based on passive
optical components, like the combination of the half-wave plate
with a Polarizing Beam Splitter (PBS), or active elements based on
electro-optic or opto-acoustic mechanisms, such as Pockels cell or
Acousto-optic Modulators (AOM), respectively. The final stage of
this section includes the optical components for the conditioning of
the laser beam to match the constraints of the optical elements for
the generation of the intensity distribution in the following section.
It usually includes a set of lenses in a Galilean telescope configura-
tion to set the beam diameter and the beam divergence level and
waveplate for the rotation of the direction of the polarization of the
light according to the requirements of the downstream optical
elements.
 Optical Approaches to Probe Neuronal Circuits in 3D 87

Fig. 4 Scheme representing some of the hardware configurations for photostimulation in two- or three-
dimensions. Starting from the left side, we report the solution for sequential photostimulation with diffraction-
limited spot with spatially extended photostimulation patches (in gray). Then, the parallel configurations are
indicated, relaying on scanning of multiplexed beamlets (yellow), scanless simultaneous excitation of multiple
patches without (green), and with temporal focusing (red)

4.1 The General The core components for 3D light-assisted modulation of neuronal
Features of a circuits are optical configurations engineered to generate arbitrary
Photostimulation Train light distributions in the sample space and are generally grouped
into two main classes [26]: sequential and parallel excitation
approaches (Fig. 4). In the first design, a single beam, either
diffraction-limited or with an engineered PSF, quickly travels across
88 Matteo Bruzzone et al.

the investigation volume to excite sequentially multiple regions of

interest, corresponding for instance to a designated subset of neu-
rons or portions of the corresponding regions. In the second
scenario, the wavefront of the original beam is engineered to result
in the sample in arbitrary distributions of multiple beamlets, each of
these exciting at the same time a different cell within the targeted
subset. In the next sessions, we describe currently reported layouts
for both the photostimulation approaches and the critical para-
meters corresponding.

4.2 Sequential 3D In sequential or single-spot cell-resolution photostimulation, exci-

Photostimulation tation light is focused on the sample in a single spot at a time. This
spot can range from a diffraction-limited excitation volume scanned
sequentially over portions of the cell membrane or a single,
extended illumination profile tailored around the typical size of
the cell soma. Steering the beam along the lateral direction and
re-directing the excitation spot is then instrumental either to reach
sufficient photocurrent integration or to target multiple cells. This
design can be implemented with a pair of orthogonal galvanometric
mirrors or acousto-optic deflectors conjugated to the objective Back
Focal Plane (BFP) by a scan lens plus tube lens pair [59–61]. Both
these devices offer sufficient bandwidths to shift in a few micro-
seconds the excitation spot within the same cell body along a spiral
or raster trajectory and/or to jump from one cell to the other of the
identified subset. In principle, with the coordinated control of the
light intensity, the power could be distributed in an arbitrary spatial
pattern, and a group of cells could be stimulated quasi-
simultaneously with cell-matched excitation power density. As the
size of the excitation spot dictates the number of light-gated mole-
cules recruited, the same parameter ultimately impacts the integra-
tion time Ti, i.e., the total amount of time that the excitation spot is
addressed to a certain cell. Ti typically ranges within 1–15 milli-
seconds, and its optimization – depending on the cell membrane
time constant, the light power density, and the density of light-
gated molecules – is critical to elicit the expected physiological
effect. Extension of the sequential paradigms in three dimensions
has only been partially reported at the moment. Optically conjugat-
ing an upstream electrically tunable lens (ETL) to a galvanometric
scanner, makes it possible to realize a 3D point scanning manipula-
tion arm with 6–15 milliseconds of typical commutation time to
shift the beam from one plane to the other [62]. This figure could
be substantially improved to a few tens of microseconds consider-
ing a design relying on acousto-optic deflectors to achieve a shift of
the beam along the longitudinal direction, either as a standalone
z-module or integrated into a 3D acousto-optic lens. 3D-2P-AOD
systems with random access technology designed for functional
imaging are currently becoming commercially available. These
should be, in principle, capable of also supporting a sequential 3D
photostimulation paradigm, but so far, there is no related report.
 Optical Approaches to Probe Neuronal Circuits in 3D 89

Moving from scanning a diffraction-limited excitation volume

to scanning spatially extended excitation is an effective strategy to
enhance the photocurrent integration in the spatial domain and
ultimately improve the photostimulation bandwidth. Extending
the excitation volume is typically achieved using Gaussian beams
with reduced or low Numerical Aperture (NA) [61]. This solution
comes with the elongation of the excitation profile along the light
propagation direction that scales with the effective lateral size of the
excitation spot and that can quickly exceed substantially the typical
cell diameter. A way to limit the extension of the excitation spot
along the light propagation direction independently from the shape
lateral size is to integrate into the optical path an arm for Temporal
Focusing upstream of the beam XY scanner [25, 63]. This tech-
nique relies on a diffractive element, typically a grating, to intro-
duce a position-dependent delay into the diffracted spectral
components of the incoming light pulse. This leads to the temporal
stretching of the pulse envelope everywhere along the optical path
but the focal position, where the delayed components reconstitute
the original intensity distribution as a superposition of different
beamlets (see also Chaps. 1 and 9 of this volume). SLMs and
ETL, optically conjugated upstream of a galvanometric scanner,
can be used to control the position of photostimulation in 3D, with
typical repositioning times ranging from 3 to 15 milliseconds. In
this case, the resulting modulation of the wavefront at the BFP of
the objective would account for a first component associated with a
lateral offset introduced at the level of the galvanometric mirrors
and a second component for the axial offset introduced by the
SLM/ETL.

4.3 Parallel 3D Parallel photostimulation approaches are based on the possibility to

Photostimulation generate simultaneously a set of beamlets, each targeting a different
position in the sample volume. Most of the current strategies for
3D light shaping rely on Computer Generated Holography (CGH,
Fig. 5) [46–48, 64–66], a powerful technique to achieve patterned
illumination at the sample plane through phase modulation of the
laser beam in a plane conjugated to the BFP of the objective.
As described above, in the case of controlling the focal position
using SLMs, in CGH it is possible to impose spatial maps of phase
corrections on the light wavefront to render the desired distribu-
tion of the excitation. Basic phase modulation patterns imposed at
the objective BFP, like linear phase gradients (x, y) and parabolic
phase profiles (z), resemble the effects of a combination of prisms
and lenses and allow to split the original beam at different x, y, z
coordinates. Super-imposing these individual correction maps in
one single phase map, also called Diffractive Optical Element
(DOE), operates like an optical multiplexer that can encode alone
for hundreds of points in a designed 3D geometry at the sample
volume. DOEs can be superimposed to a propagating beam either
90 Matteo Bruzzone et al.

Fig. 5 Computer-Generated Holography. Engineering the light wavefront at the BFP allows for rendering
arbitrary light intensity distributions at the sample. Introducing a phase correction resembling a Fresnel lens
results in a change in the convergence properties of a propagating beam, moving the position of the focus
longitudinally. Similarly, with a linear gradient of phase delay applied at the BFP, the position of the focus is
moved laterally. Multiplexing different diffractive optical elements (DOEs) allows for rendering arbitrary light
distribution at the sample

with static phase masks engraved in glass or quartz material or using

SLMs to dynamically update the light distribution. One important
aspect of CGH is its versatility. An optical path based on this
approach can be used as a stand-alone photostimulation module
(3D-CGH) [20, 67] or, in combination with other components, to
integrate the multiplexing capabilities as a stage in more extended
photostimulation optical trains (MTF-CGH [68], 3D-SHOT [63]
and 3D-CGH spiral [69]). In the first scenario (3D-CGH), the
diffractive optical elements encode along with the positions of the
foci in the sample volume also for their actual lateral shape. Indeed,
iterative algorithms based on Fourier transforms can compute
phase maps to render a 3D distribution of bidimensional
 Optical Approaches to Probe Neuronal Circuits in 3D 91

illumination profiles tailored independently around the specific

structures of interest in the sample. These excitation foci can
range from ensembles of dots, patches of stimulation of any arbi-
trary geometries or their combinations. One aspect associated with
3D CGH is that, while the lateral (XY) dimension of the excitation
spots, supporting the process of photocurrent integration, can be
imposed simply by specifying the desired intensity mask, the longi-
tudinal (Z) extension of the illumination profile, being dictated by
the laws of diffraction, scales linearly with the lateral size of the
rendered shape. Depending on the experimental conditions, in
particular the characteristic cell size, sparseness of the light-gated
actuator expression, or its cellular localization, this feature can
impact the effective resolution achievable in targeting neuromodu-
lation. For neuronal modulation with 3D-CGH, it is then impor-
tant to identify a trade-off between the acceptable spatial resolution
and the photostimulation patch size to maximize the photocurrent
integration area. In order to relax these constraints and to extend
the performances and efficiency of the 2P-based photostimulation,
a few approaches have been developed, combining the power of
the 3D-CGH approach with other optical components. In one of
the first approaches reported, the beam multiplexing capability of
CGH is combined with the spiral scanning based on downstream
a galvanometric scanner [24]. In this implementation, similar to the
sequential approach described above, diffraction-limited spots are
quickly scanned with the same coordinated trajectory over different
cells. The objective wavefront at the BFP in this scheme accounts
for a fixed component, a DOE imposed with the SLM and encod-
ing a distribution of foci centered on the cell bodies, plus a time-
varying component encoded by the galvo pair to scan the spiral
over the designated cells. As far as the power density is kept under
control, this approach can offer the best longitudinal confinement
of the photostimulation pattern, corresponding to the PSF exten-
sion of a diffraction-limited spot. From the implementation with
2D parallel excitation, this approach can be extended in 3D, taking
advantage of control of the third dimension allowed by CGH
[59]. In alternative to the 3D-CGH spiral scanning, beam multi-
plexing supported by phase modulation has been combined with
temporal focusing. Independently from the particular type of
implementation, the general scheme consists of a step for shaping
the beam amplitude, followed by a path with a dispersive element
for Temporal Focusing (TF) [63], and finally completed with a
stage for the spatial multiplexing with multipoint CGH based on
a SLM. Shaping of the beam amplitude can be obtained either
directly with a low-NA Gaussian beam to get circular profiles [70]
or using CGH, Generalized Phase Contrast (GPC), and other
amplitude modulation methods for tailoring the illumination pat-
terns with top hat profiles [68] (see also Chap. 1). Most frequently
the beam shaping is used to generate a single intensity profile at the
92 Matteo Bruzzone et al.

level of the dispersive element for TF, resulting upon CGH-based

multiplexing in the rendering of a set of exact replicas of the original
shape. However, it is possible, by properly tiling and aligning the
optical window of the beam shaper and of the multiplexer, to
generate groups of replicas of different shapes [67, 68].

5 Setting Up an All-Optical 3D Investigation System

Combining light-based recording and modulation of neuronal

activity at high resolution is, in general, a delicate task. One should
identify and validate the experimental approach depending on the
tool’s molecular properties and available techniques’ capabilities.
Even if this is rarely the case, the optimal hardware configuration
definition should ideally come toward the end of a more extended
development pipeline. Identifying suitable light-based molecules is
the first phase in this kind of pipeline. Indeed, one should first
characterize the effective functionality and operativity ranges of
the light-based molecules, e.g., the molecules’ action spectrum,
the SNR ratio of the signal, or the change in membrane potential
induced with the excitation power. Even though most of these
characteristics could be found reported in the literature, these
features should be verified with the actual working conditions/
preparations of the planned experiments. After the initial phase
for the characterization of the intrinsic properties of the approach
(e.g., working parameters for photostimulation), in the following
phase, one should ideally evaluate its compatibility with the typical
working conditions of the second concomitant approach, e.g.,
GCaMP6s imaging. In particular, it should be assessed whether
the first approach perturbates the state and/or the functionality of
the molecules and the optimal working conditions required for the
second one. Here is typically where the impact of the molecule
crosstalk can be characterized and the effective capability of the
techniques estimated. Unsurprisingly, one will have to deal with a
set of experimental tradeoffs, gauging, for instance, between the
SNR of the recordings and the level of the spurious activation or
between the light power density of the photostimulation and the
acceptable signal contamination level due to the photostimulation
picked-up in the activity recordings. In this challenging task, even if
not strictly related to the hardware configurations or the molecular
properties, the optimization of the data acquisition chains and the
design of algorithms to filter and clean the recorded signals are
tools available to potentially relax the working conditions, at least
partially.

5.1 The Hardware Different can be the optical configurations to support an all-optical
Integration circuit probing framework. It is clear that integrating and coordi-
nating two components requires the identifying the appropriate
 Optical Approaches to Probe Neuronal Circuits in 3D 93

hardware solutions and the proper software capabilities. From the

point of view of the hardware integration, the photostimulation
train and the imaging train integrating the 3D scanning, once a
valid reporter-actuator pair is identified, can be considered as two
components, mostly independent: different sources, beam size dia-
meters, intensity modulation units, and working wavelengths. The
two components, on the other side, become dependent one from
the other when reaching the point of combining the two excitation
beams along a common segment of the optical path to finally reach
the objective back aperture. How and where multiplexing or com-
bining the two optical trains are two important aspects to consider.
Typically, the solutions adopted take advantage of either the polar-
ization state of the two beams or their spectral separation. In the
first scenario, the seeding beams, typically leaving the source with
horizontal polarization, are constrained and routed in such a way to
arrive at the point of combination with two linear and orthogonal
polarization states, one horizontally and one vertically aligned to
the respective propagation directions. At that point, a polarizing
beam splitter, properly oriented to reflect one of the beams and
transmit the other, acts as a beam combiner to launch them along
the same path (see Note 1). Alternatively, when flexibility in the
excitation wavelength is not required, a proper dichroic mirror,
either long-pass or short-pass, can serve as a beam combining
element. Working with a defined wavelength for the imaging
beam and the photostimulation beam allows for reducing the opti-
cal components required for conditioning the beams and, so, mini-
mizing undesired reflections. In general, defining the beam
combining architecture is not just a matter of the optical compo-
nent to use but also of identifying a convenient position along the
optical train where joining the two components. This should be
evaluated in terms of the available space, the minimization of the
introduced distortion in the light wavefront, and the constraints
dictated by the focal distances of the corresponding optical paths. It
is convenient to illustrate possible integration layouts, to consider
the most general scenario of an imaging system, presenting a XY
scanning assembly based on a pair of galvanometric mirrors, fol-
lowed by the scan lens-tube lens pair (Fig. 6). The most frequent
design envisages that the imaging and the photostimulation paths
run independently and merge downstream the XY scanner. This,
combined with a remote system for z-scanning the imaging spot,
allows the uncoupling of the two paths, where the imaging
z-scanning assembly is positioned upstream of the XY scanner and
the photostimulation fed into the train downstream to it. An ele-
ment for combining imaging and photostimulation beams can be
placed in three different positions: upstream (1), in between (2),
and downstream the scan lens–tube lens pair (3) (Fig. 6). While it
could be hard to precisely predict the impact of such element on the
propagation characteristic of the imaging beam, it is important to
94 Matteo Bruzzone et al.

Fig. 6 Layout of the integration scheme of the imaging and photostimulation arms. In cerulean is indicated the
path of the imaging beam with the assembly to control the z-position upstream of the XY scanner. In red are
shown the possible insertion points of the photostimulation path with respect to the optical components
required for imaging

consider the degree of flexibility associated with these three solu-

tions. Indeed, moving from solution (1) to (3), one gains progres-
sively more freedom to conjugate the photostimulation
modulation plane to the BFP. While with design (1) the photo-
stimulation beam is ultimately conjugated to the BFP using as relay
optics the scan lens–tube lens pair, in (3) one has all the flexibility to
design the relay component to the BFP of the objective indepen-
dently from the imaging path and potentially to accomodate the
requirements of the photostimulation train, independently by its
complexity.

5.2 Beam Co- The first aspect to consider is evaluating how the three light beams
registration used for the two approaches (excitation of the light-gated actuator,
Procedures excitation of the fluorescent reporter, and fluorescence emission by
the reporter) are reaching or leaving the sample, whether these
beams travel through the same objective, and whether the objective
is kept still during the acquisition. This is usually the most common
case [20, 23, 54, 69] but are also possible architectures relying on
independent arms, one for photostimulation and fluorescence col-
lection with a moving objective, the other for reporter excitation
[62]. Having one objective still assumes the use of remote optical
 Optical Approaches to Probe Neuronal Circuits in 3D 95

components for 3D control of both the excitation beams. Remote

control is instrumental in compensating for subtle differences in the
beam divergence characteristics of the different paths or chromatic
aberrations, and it facilitates the co-registration of the excitation
beams in the sample volume and the synchronization of the control
signals. For co-registration of the excitation beams, it is generally
considered the essential procedure allowing the calculation of the
affine transformations mapping the reference system for the photo-
stimulation into the reference system for the imaging. The goal is to
obtain a precise transformation that converts the position
information X, Y, and Z in the sample space into the corresponding
commands for targeting the photostimulation beam and the imag-
ing beam to that precise point. Typically, this relies on the use of a
detection arm equipped with a CMOS/CCD camera and is
achieved with the sequential illumination of a series of points in a
3D lattice, first with the imaging beam and then with the photo-
stimulation beam, while moving the objective to bring the current
excitation point in focus at the camera plane. Alternatively, without
using a camera but relying on the PMTs, it is possible to use
photobleaching of a fluorescent sample to measure the positions
of the points within the lattice (see Note 2).

5.3 Spatial In many optical systems, it is frequent to experience a progressive

Uniformity and degradation of the optical performances depending on the spatial
Addressable Field of distance from the center of the field of view. This appears, for
View instance, as an increase in the dimensions of the PSF for the
imaging path or a decrease in the effective light power density for
the photostimulation approach. This usually originates from a
non-uniform diffraction efficiency of the optical elements. On the
other side, mapping such non-uniformities is required to identify of
the effective volume addressable with the imaging and the photo-
stimulation beams under the constraints of the resolution, SNR
ratio requirements, and working conditions identified in the previ-
ous phases. This typically requires a procedure similar to the one
used for beam co-registration but refined in order to extract, along
with the XYZ positions of the points in the lattice space, the change
in the intensity, the eventual deformation of the excitation profiles,
and the presence of possible aberrations. It is important to note that
in a certain measure, it is possible to develop corrective strategies
and partially compensate for the optical components’ limitations.
This is normal for the diffraction efficiency of a SLM when included
in the optical arm for photostimulation based. Because of the
low-pass filtering effects originating from the SLM working princi-
ple [46], the efficiency curve measured at the sample shows a rapid
decrease with the increasing distance from the center of the optical
system (see Note 3).
96 Matteo Bruzzone et al.

6 Notes

1. The PBS-based solution offers the maximal flexibility for tun-

ing the excitation wavelengths but requires a careful evaluation
of the impact of a relatively bulky optical element like a PBS
introduced in a segment of the optical train where the beams
are usually not collimated. Indeed, it is expected that the
presence of a PBS could modify the characteristic of the origi-
nal light wavefront in that position or introduce, to a certain
extent, additional pulse dispersion. This could potentially result
in deterioration of the PSF, longitudinal shifts in the focal
position, and loss of multiphoton absorption efficiency.
2. The co-registration procedure also provides the information
about the accessible field of view implicitly for the two excita-
tion beams and the degree of spatial variation of the imaging/
photostimulation efficiency within the addressable volume. It is
then important to consider a solution where this procedure can
be easily and quickly performed, ideally in an automatic
approach.
3. Compensating for the non-uniformity: acting at the level of the
computational engine that is used to calculate the diffraction
optical elements loaded on the SLM, one, based on the
mapping obtained, can balance the weights of the
corresponding features, to control precisely the intensity and
counterbalance eventual losses due to the diffraction efficiency
curve [67].

7 Conclusions

In this chapter, we described the current state of the art of the

hardware configurations allowing all-optical investigation of the
neuronal circuits in vivo in three dimensions. This is a field where
the development of molecular tools and the technological advance-
ment continuously provide novel possibilities for designing and
refining experimental approaches that can extend the perspective
of investigating the neuronal dynamics in living organisms. Despite
the availability of technical solutions, the community has only
partially capitalized on these tools to explore brain mechanisms.
This is indeed not just a matter of the hardware required, but poses
a series of questions, and challenges, also from the point of view of
the design of the experimental protocol, the analysis of the data,
and the interpretation of the outcome.
 Optical Approaches to Probe Neuronal Circuits in 3D 97

Acknowledgments

The authors would like to acknowledge the support of the Depart-

ment of Biomedical Sciences (SID_DalMaschio2018) and the
Padua Neuroscience Center (ReTurnPD) at the University of
Padua, the support of EC Research Programs (VISGEN and
FLAMMES). The authors would like to thank the colleagues
providing comments and suggestions to the draft versions.

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 Chapter 4

High-Speed All-Optical Neural Interfaces with 3D

Temporally Focused Holography
Ian Antón Oldenburg, Hayley Anne Bounds, and Nicolas C. Pégard

Abstract
Understanding brain function requires technologies that can monitor and manipulate neural activity with
cellular resolution and millisecond precision in three dimensions across large volumes. These technologies
are best designed using interdisciplinary approaches combining optical techniques with reporters and
modulators of neural activity. While advances can be made by separately improving optical resolution or
opsin effectiveness, optimizing both systems together matches the strengths and constraints of different
approaches to create a solution optimized for the needs of neuroscientists. To achieve this goal, we first
developed a new multiphoton photoexcitation method, termed 3D-Scanless Holographic Optogenetics
with Temporal focusing (3D-SHOT), that enables simultaneous photoactivation of arbitrary sets of
neurons in 3D. Our technique uses point-cloud holography to place multiple copies of a temporally focused
disc, matched to the dimensions of a neuron’s cell body, anywhere within the operating volume of the
microscope. However, since improved placement of light, on its own, is not sufficient to allow precise
control of neural firing patterns, we also developed and tested optogenetic actuators ST-ChroME and
ST-eGtACR1 that fully leverage the new experimental capabilities of 3D-SHOT. The synergy of fast opsins
matched with our technology allows reliable, precisely timed control of evoked action potentials and
enables on-demand read-write operations with unprecedented precision. In this chapter, we review the
steps necessary to implement 3D-SHOT and provide a guide to selecting ideal opsins that will work with
it. Such collaborative, interdisciplinary approaches will be essential to develop the experimental capabilities
needed to gain causal insight into the fundamental principles of the neural code underlying perception and
behavior.

Key words Optogenetics, Temporal focusing, 3D holography, ChroME, Brain-machine interfacing,

Multiphoton, 3D-SHOT, Soma-targeted, Opsin

1 Introduction

Although optogenetics have become a mainstay of neuroscience

research, used to probe causal relationships between circuit activity
and behavior [1–6], it is only recently that multiphoton optoge-
netic techniques have been used to modulate neural activity.
Numerous technical advances in optics [7–10] and opsins [11–
15] over the last decade have led to an increase in usage and

Eirini Papagiakoumou (ed.), All-Optical Methods to Study Neuronal Function, Neuromethods, vol. 191,
https://doi.org/10.1007/978-1-0716-2764-8_4, © The Author(s) 2023

101
102 Ian Antón Oldenburg et al.

adoption of multiphoton optogenetic strategies. Multiphoton

optogenetics have been used to examine visual discrimination
[3, 14], coding features of detection [16, 17], ensemble activity
[18], cortical circuitry [19], and more [20, 21].
In this chapter, we will briefly review the state of the field and
introduce 3D-SHOT [7]. We will detail the steps required to build,
align, calibrate, and validate 3D-SHOT as an add-on on the light
path of a 2-photon microscope. We will then discuss how to assess
and select ideal opsin and reporter combinations for use with a
3D-SHOT system.
Optogenetic techniques have been rapidly and widely adopted
in neuroscience research because they enable precise and reversible
external control of neural activity with high temporal precision by
means of minimally invasive optical signals. However, the spatial
precision is generally too poor to manipulate individual neurons
because light does not propagate well through dense brain tissue.
The vast majority of optogenetics studies primarily leverage genetic
specificity rather than spatial control. However, since many neural
computations and behaviors rely on populations of neurons that are
genetically similar but spatially intermixed [18, 22–24], precise
targeting of individual neurons with optical methods is necessary.
Two-photon activation of opsins is an attractive approach for
improving spatial resolution. The longer wavelengths used in
two-photon excitation are less affected by optical scattering [25],
which dramatically improves the axial resolution and the accessible
depth of sculpted illumination patterns [26, 27]. Further,
two-photon absorption is a nonlinear effect which further restricts
opsin excitation to a narrow axial plane [8, 28].
Most biological studies using 2-photon optogenetics have used
scanning-based approaches [3, 14, 18–21]. Similar in principle to
two-photon imaging, a femtosecond-pulsed infrared laser beam is
focused into a single diffraction-limited spot which is scanned in
2D or 3D, with galvo-mirrors [27], acousto-optic modulators
[29, 30], spatial light modulators (SLM) [31], or micro-
electromechanical systems (MEMS) [32]. A raster [8, 13] or spiral
[18, 28, 33] pattern is scanned across the soma to target neurons in
3D. These techniques are power efficient [34] but require opsins
that are either extremely strong or slow to deactivate (ideally both),
so that the photocurrent can accumulate as the spot is scanned
across the neuron soma, usually at the expense of temporal
resolution [11].
To improve temporal precision, whole-cell illumination techni-
ques that forgo scanning have been developed to simultaneously
illuminate the entire cell soma with a larger spot and activate all the
opsin at once. Some approaches achieve whole-cell activation with
low NA objective [13, 20] at the expense of spatial resolution, but
the preferred method relies on computer-generated holography
(CGH) [35–37] with a spatial light modulator (SLM) to synthesize
High-Speed All-Optical Neural Interfaces with 3D Temporally Focused Holography 103

custom illumination patterns that are matched to the shape of

individual neurons (see also Chaps. 3 and 11). Compared to scan-
ning approaches, whole-cell activation with CGH enables faster
responses to optogenetic stimulation, but requires higher peak
powers. With traditional multiphoton CGH [38], and even point
scanning methods [33], spatial resolution along the optical axis is
determined by how rapidly the power density is attenuated as light
propagates into and out of the targeted area. These often result in
significant but undesired photoexcitation above and below the
target. In practice, physiological spatial resolution is highly
power-dependent, and single neuron spatial resolution (e.g., axial
FWHM ~30 μm) [9, 39] is generally impossible across large
volumes, even with high numerical aperture (NA) objectives.
To eliminate the tradeoff between target dimensions in the (x,
y) plane and decreased axial (z) resolution [40, 41], 3D-SHOT
relies on temporal focusing (TF) [40, 41] where a diffraction
grating decomposes femtosecond pulses into separate colors, such
that the different wavelengths components within the original
pulse propagate along separate light paths. Each component of
the decomposed pulse has a narrow spectral bandwidth and is
therefore broadened in time, which dramatically reduces the peak
intensity and prevents two-photon absorption. However,
two-photon absorption can be enabled again when the original
pulse is retrieved by constructive interference of all the chromati-
cally separated components at conjugate images of the diffraction
grating [40, 41]. TF restricts multiphoton absorption to a narrow
(z) depth that depends on the grating’s spatial frequency, not on
the dimensions of the targeted area. TF has been successfully
applied for selective two-photon tomographic fluorescence imag-
ing [42, 43], has been implemented with mechanical scanning
[44], and with random-access volume sampling of functional fluo-
rescence [45]. A detailed presentation of TF is available in Chap. 9.
For two-photon photostimulation applications, TF can activate
opsins over a wide area matching the neuron’s shape in the focal
plane, without compromising depth specificity [39, 46]. TF also
mitigates scattering [47, 48] even through thick layers of brain
tissue [49, 50] (see also Chap. 9). Although multiphoton CGH
with TF can achieve wide-field photostimulation with cellular reso-
lution and high temporal precision, most implementations only
enable excitation within a single 2D plane [20, 39, 46]. Thus,
neurons located above or below the focal plane are not addressable,
a necessary condition for many experiments designed to interface
with neural circuits in vivo, where relevant neurons may be loca-
lized anywhere in the 3D volume of interest. Multi-level temporal
focusing has been implemented with holograms tiled into clusters
on the SLM surface which can be individually defocused in space by
applying digital lens patterns on a second SLM [51]. This strategy is
limited in the number of distinct depth levels used before in-plane
104 Ian Antón Oldenburg et al.

resolution is degraded, constraining the neuronal population that is

simultaneously addressable with optical stimuli.
To overcome this outstanding challenge 3D-SHOT leverages
the advantages of CGH to simultaneously address custom 3D
locations on demand, and TF for enhanced spatial resolution at
the scale of individual neuron soma. 3D-SHOT forgoes the ability
to create custom patterns to make TF and 3D CGH compatible,
instead it replicates multiple identical copies of a temporally focused
excitation pattern matched to the dimensions of a neuron soma at
each target location. The result is a technology that is specifically
tailored to optogenetic photostimulation applications and enables
single-shot in vivo photoactivation of custom neuron ensembles
distributed anywhere in the accessible volume, with single-neuron
spatial resolution.
Understanding the causal relationship between neural activity
and behavior is a fundamental goal of neuroscience. However, this
causal inference requires manipulations that act at the scale of
natural activity, i.e., writing temporally precise patterns of activity
in many cells with single-cell specificity. While optical approaches
such as spiral scanning or 3D-SHOT can confine light to the
dimensions of a single cell, molecular actuators, also known as
opsins, are required to convert light into neural activity. The prop-
erties of these opsins and how they interact with the stimulation
system will determine how well one can drive precise trains of
activity. The fast and potent opsin ChroME [11] was engineered
alongside the development of 3D-SHOT to achieve this goal, but is
only a single example of a class of opsins with appropriate speed and
sensitivity properties that have yet to be discovered. As such, the
fourth section of this chapter outlines the criteria necessary to select
an opsin optimally paired to 3D-SHOT for the purpose of writing
precise trains of activity into groups of neurons.
Fundamental to the goal of writing temporally specific patterns
is to pair extremely fast opsins with “flash”-based optical
approaches, i.e., those that simultaneously illuminate an entire cell
such as 3D-SHOT. In order to drive precisely timed action poten-
tials faithfully and at high rates, the underlying evoked photocur-
rents must be very strong and very fast – both rising and falling very
rapidly. Simultaneous illumination systems ensure that all possible
opsin molecules are activated at the same time, leading to the
shortest response time, while opsins with both fast kinetics and
high conductance ensure that action potentials are faithfully driven,
without the risk of creating doublets. Indeed, opsins with slower
decay kinetics tend to have higher overall conductance but are not
capable of driving pyramidal cells at high rates and often have large
jitter in action potential timing [34, 52, 53].
Finally, multiphoton optogenetic stimulation is rarely per-
formed as a standalone technique, and is almost always paired
with calcium imaging. Special considerations are needed to match
 High-Speed All-Optical Neural Interfaces with 3D Temporally Focused Holography 105

the properties of opsins and reporters to ensure that both multi-

photon optogenetics and imaging are compatible for simultaneous
use. At present, the most successful imaging approaches rely on
“green” calcium indicators (i.e., the GCaMP series) that absorb
~920 nm matched to “red” opsins with longer excitation wave-
lengths. However, even opsins with peak absorption >1000 nm are
sensitive to these imaging wavelengths. We discuss these constraints
and the mitigation approaches that are available to combine multi-
photon optogenetics with imaging under those circumstances.
Precise control of neural activity cannot be achieved by any one
technique alone. New optical developments, new molecular tools,
and new approaches to unite these devices are needed to reach the
next step of precise causal manipulation of neural systems. As
technologies are constantly being developed and improved; new
experimental capabilities provide a path to answer previously intrac-
table neuroscience questions. By combining different skillsets and
expertise, the technological solutions that emerge are better than
what would have been created from the perspective of a single
discipline. Our intent is for this chapter to serve as a guide and
resource for future users who will implement and improve upon
multiphoton optogenetics and usher in a new epoch of neurosci-
ence discovery.

2 Methods

2.1 3D-SHOT Optical Our experimental setup, shown in Fig. 1, is based on the standard
System Design design of a holographic microscope with a spatial light modulator
(SLM) in the Fourier domain (pupil plane). The SLM shapes the
phase of a coherent femtosecond laser light source to synthesize
custom 3D shapes [54, 55] digitally synthesized with Computer
Generated Holography [35–37] (CGH). Unlike scanning
approaches, CGH wide-area holograms matched to the dimensions
of each neuron’s soma enable simultaneous, flash-based, activation
of a large number of opsin molecules, yielding photocurrents with
fast kinetics [39].
In most brain structures, neurons are distributed continuously
in 3D, not in discrete layers. Therefore, the inability of 2D opto-
genetics approaches (i.e., 2D CGH with temporal focusing) to
target neurons at any arbitrary number of axial planes simulta-
neously is a major obstacle for large-scale optogenetic interrogation
of neural circuits. To overcome this outstanding challenge
3D-SHOT leverages the advantages of 3D-CGH, to simulta-
neously address neurons in custom locations. To make 3D CGH
and temporal focusing mutually compatible, 3D-SHOT forgoes
the ability to create custom patterns. Instead, the optical path is
optimized to holographically replicate multiple identical copies of a
temporally focused excitation pattern, termed “custom temporally
106 Ian Antón Oldenburg et al.

Fig. 1 Experimental setup for 3D-SHOT. This is made of two consecutive optical systems. First, a diffraction
grating and a rotating diffuser are imaged onto each other by a f-f optical relay. This assembly shapes
femtosecond laser pulses both spatially and spectrally to create a custom temporally focused pattern (CTFP)
matched to the dimensions of a neuron soma. The resulting engineered point spread function is then spatially
modulated by a second system that enables 3D computer-generated holography (CGH). A spatial light
modulator (SLM) placed in the Fourier domain modulates the phase of the CTFP to target custom 3D locations
with a point-cloud hologram. The resulting sculpted illumination pattern replicates identical copies of the CTFP
at each targeted location. The 3D hologram is further demagnified by a tube lens and a microscope objective.
A zero-order block eliminates any remaining undiffracted light from the hologram. The grating frequency
determines the spectral dispersion, “a”, and the diffuser determines the beam dimension “b” at the surface of
the SLM. Those parameters, along with the focal lengths of lenses L1–L4, are adjusted to match the desired
addressable volume and CTFP dimensions within constraints imposed by the SLM size, the laser source, and
the numerical aperture of the microscope

focused pattern” (CTFP). The CTFP is specifically engineered to

match the dimensions of a neuron’s soma, and to be compatible
with 3D CGH so that identical copies of the CTFP, with individu-
ally specified brightness can be placed at each target neuron any-
where in the accessible 3D volume. The result is a technology that is
tailored for optogenetics applications and enables single shot
in vivo photoactivation of custom neuron ensembles distributed
anywhere in the accessible volume, with single-neuron spatial
resolution.
 High-Speed All-Optical Neural Interfaces with 3D Temporally Focused Holography 107

To implement 3D SHOT in a multiphoton microscope, the

first step is to create a static, temporally focused object matched to
the dimensions of the desired target. For this, we illuminate a
reflective blazed diffraction grating with a femtosecond laser light
source. The incidence angle is adjusted so that the first diffracted
order reflects orthogonally to the surface of the grating. The grove
depth, material, coating, and incoming wave polarization are
adjusted to best match the desired LASER wavelength and optical
power density and to maximize the amount of light in the first
diffracted order. Lenses L1 and L2 are in a 4-f configuration and
create an exact optical relay that place a virtual copy of the diffrac-
tion grating at the surface of the transparent, rotating diffuser. The
rotating diffuser applies an engineered (gaussian) phase pattern to
the temporally focused image that is continuously randomized by
the mechanical rotation. This phase perturbation is necessary to
distribute the energy in the Fourier domain (i.e., to uniformly
illuminate the SLM), a critical step that enables the compatibility
between 3D CGH and temporal focusing and maximizes the dif-
fraction efficiency.
The CTFP is matched to the characteristic dimensions of a
neuron soma by selecting the magnification M ¼ f2/f1, where f2
and f1 designate the focal lengths of lenses L2 and L1 respectively.
The axial confinement (temporal focusing) can be adjusted by
selecting diffraction gratings with a higher (or lower) grating spatial
frequency (in lines per mm). Both properties can be adjusted
independently of the additional phase perturbation induced by
the rotating diffuser.
A second 4-f system made with lens L3 and L4 with focal length
f3, resp. f4 relays the CTFP, first to the SLM in the pupil plane, then
to the volume where the 3D hologram is first synthesized. In this
configuration, the SLM applies a multiplicative phase pattern in the
Fourier domain, which corresponds to a convolution operation in
the real domain. To utilize 3D SHOT for neural stimulation, a
CGH algorithm only needs to compute a hologram made of a 3D
cloud of diffraction-limited points centered on each target neuron,
and the optical system will yield one copy of the CTFP at each
target point. To compensate for spatially dependent diffraction
efficiency, and non-uniform losses through the optical system, the
respective brightness of each target can be adjusted by computing
digitally compensated holograms that redistribute the available
laser intensity across each point of the 3D cloud based on expected
losses, and the total energy can be adjusted globally across all
targets by modulating the power of the laser beam.

2.1.1 3D-SHOT Design 1. The size of the CTFP must be adjusted to match the dimensions
Parameters “d” of a neuron. In a typical implementation of 3D SHOT, the
3D hologram (see Fig. 1) is relayed into a microscope with an
additional tube lens (L5, f ¼ f5) and microscope objective (L6,
108 Ian Antón Oldenburg et al.

f ¼ f6) with magnification M ¼ f5/f6. The dimensions of the

CTFP is M·d in the 3D hologram, Mdf3/f4 at the rotating
diffuser, and correspond to an incoming beam of width
M·d·f3 f1/( f4f2) at the blazed grating.
2. The rotating holographic diffuser is a transparent material with
an engineered surface that deflects incoming light in a Gaussian
pattern angular distribution with a characteristic diffraction
angle, αd. The diffuser spatially stretches the wave in the Four-
ier domain by an amount b (see Fig. 1), optimized to ensure an
even illumination of the SLM active area, and given by.

b ¼ f 3 αd

3. The line spacing of the blazed diffraction grating, l, or spatial

frequency, fg, (l ¼ 1/fg) must be adjusted to match the spectral
bandwidth δλ of the femtosecond laser, and the desired dimen-
sions of the CTFP along the (z) axis. The angular dispersion of
the diffraction grating is given by α ¼ δλ/l, and stretches the
pulse in the Fourier domain. At a distance f1 from lens L1, the
dimension of the stretched pulse satisfies af2/f3 ¼ α f1. Hence,
the spectral dispersion, a, at the SLM, is given by:

a ¼ δλ f 1 f 3 =ð l f 2 Þ:
The numerical aperture NA of the microscope objective is
generally the limiting factor that defines the accessible volume
and CTFP minimal dimensions for 3D-SHOT. The SLM pattern
(see Fig. 1) of width a + 2b is imaged onto the back aperture of the
microscope objective. Hence, to fully capture the light modulated
by the SLM, a suitable design constraint is to ensure (a + 2b)f5/
f4 < NA f6. In this configuration, the characteristic size, δz, of the
CTFP along the (z) axis in the demagnified hologram under the
microscope objective is given by


2
δz ¼ λ f 4 f 6 = a f 5

2.1.2 Implementation 3D-SHOT is implemented as a secondary system on the path of a

Guidelines for 3D-SHOT multiphoton microscope that is also designed for two-photon
imaging, typically with a secondary laser. Imaging and photostimu-
lation are most efficiently merged with a dichroic mirror or a
polarizing beam splitter cube. When using a polarizing cube, the
orientation of the SLM, grating, and path-merging cube must be
adjusted to match polarization constraints, with one path (e.g.,
photostimulation) being reflected and the other one being trans-
mitted so that the merged beams are co-aligned. Any incompatibil-
ities can be resolved by inserting additional half-wave plates along
High-Speed All-Optical Neural Interfaces with 3D Temporally Focused Holography 109

the optical path. However, since no element has perfect transmis-

sion any additional optical element will reduce the overall power
throughput. 3D SHOT is best assembled by first building the laser
beam line at a fixed height on an optical table, then by installing the
additional optical components starting from the beam merging
cube or dichroic mirror, and in the sequential order outlined below.
1. Fully assemble a standard multiphoton imaging microscope
with the addition of a dichroic or polarizing beam splitter
before the tube lens. Our experimental setup was built around
a modified Movable Objective Microscope (MOM, Sutter
Instrument Company), but alternate commercial and custom
microscope designs may be used. We recommend using an
additional 4-f relay of lenses (2-inch diameter, achromatic
IR-coated doublets, 200 mm focal length), if the microscope’s
image plane is not directly accessible at a location that is suit-
able to place the zero-order block.
2. Place lens L4 and the SLM first so that the lens is at a distance f4
from the SLM and the image plane. If a beam expander is
added along the laser path before the SLM, it is possible to
use the experimental setup as a multiphoton holographic
microscope. Several tests such as evaluating the accessible vol-
ume under the microscope objective, as well measuring the
spatial dependence of losses in diffraction efficiency are possible
and applicable in this form. However, in the final calibration
procedure there will be a very fine evaluation of diffraction
efficiency and accessible volume once all parts are included.
By finely adjusting the position of lens L4 along the optical
axis while the SLM displays a uniform phase mask, it is possible
to precisely place the center of the 3D hologram in the center
of the image, and at the same focal depth as the two-photon
imaging plane.
The laser should operate at minimum power levels during
the alignment procedure. Some laser models are fitted with
co-aligned low-power red lasers that may be used to safely
align the entire system with the exception of the diffraction
grating tilt that must be aligned for the desired excitation
wavelength. The SLM surface is one of the most sensitive
areas and light should not be focused on it (this may happen
when placing lens L3). The safest approach is to temporarily
replace the SLM with a flat mirror and to align the SLM at the
end by focusing undiffracted light on the zero-order block.
3. The second design step is to replace the collimated beam
illuminating the SLM with the engineered CTFP. For this,
one must place and align lenses L3, L2, L1, in that order, in
successive 4-f configurations. Pinholes and far-field images of
the infrared beam can be used to check centering and beam
110 Ian Antón Oldenburg et al.

collimation, respectively, to ensure that each newly inserted

lens is properly centered and spaced. The reflective grating is
placed last, at a distance f1 from lens L1 with its reflective
surface orthogonal to the optical axis.
4. The laser beam line must then be readjusted to illuminate the
grating with an oblique incoming path so that the first dif-
fracted order propagates back along the previously established
optical axis. Placing temporary iris apertures on the lenses can
help recentering the optical path. Fine adjustments of the beam
propagation direction can be made by attaching the diffraction
grating to a 2-axis tilt mount and precisely tuning the orienta-
tion of the diffracted beam.
5. The final assembly step is to place the zero-order block at the
focal spot of the default SLM image (uniform phase mask),
where undiffracted light is expected to create a sharp focused
spot, and the rotating diffuser at a distance f2 from lens L2.
Various designs have been proposed to create a zero-order
block and careful consideration should be given to choosing
the adequate design. Most commercial SLMs are expected to
allow a few percent of undiffracted laser light, which represents
a significant amount of power on a small surface area. Thin
metal films are not recommended because they cannot dissipate
heat rapidly enough to avoid overheating. Instead, we suggest
that one depolish the center of a flat optical surface (IR coated
coverslip) with a small diamond-coated grinding tip. The
depolished glass surface will diffuse most undiffracted light in
all directions without accumulating heat. Alternatively, if a
larger than necessary zero-order block is acceptable, a ~1 mm
metal object, such as a neodymium magnet on a coverslip, will
be able to withstand these powers.
During operation, special attention should be given to ensure
that the rotating diffuser is spinning before allowing high laser
power settings to avoid SLM damage.

2.2 Characterization Our primary goal is not to render a visually accurate hologram but
and Performance instead to increase contrast for two-photon excitation at selected
Metrics for 3D-SHOT locations while avoiding inadvertent photoactivation of
non-targeted areas, which relaxes constraints on hologram compu-
tation. Hence, the traditional metrics used to characterize imaging
systems such as resolution, contrast, and speckle are not adequate
to evaluate the capabilities of 3D-SHOT in experimental
applications.
Instead, to evaluate the capabilities of 3D-SHOT and quantify
how two-photon absorption is spatially distributed in 3D, we
placed a thin fluorescent film on a microscope slide under the
excitation objective to record the corresponding two-photon fluo-
rescence image with a fixed sub-stage objective coupled to a camera
 High-Speed All-Optical Neural Interfaces with 3D Temporally Focused Holography 111

Fig. 2 Optical characterization of the spatial resolution of CGH vs 3D-SHOT. (a) We used a fluorescent
calibration slide and an inverted microscope to quantify two-photon excitation in 3D. (b) For conventional
holography, we consider a 10 μm diameter disk target, and show (from top to bottom) projection views of
two-photon absorption in the (x,y), (z,y), and (z,x) planes. (c) With 3D-SHOT, the CTFP was adjusted to a 10 μm
diameter target and the same projection views were recorded. (d) We measured the FWHM of the radial (top)
or axial (bottom) PSF measured through brain slices of varying thickness (* indicates p < 0.05, Kruskal-Wallis
Test with multiple comparison correction. Data represent the mean and standard deviation of n  5
observations for each thickness of brain tissue). (Adapted from Pegard et al. [7])

(Fig. 2a). We recorded tomographic slice images by mechanically

moving the excitation objective along the z axis by 1-μm incre-
ments. The resulting data correspond to a quantitative 3D mea-
surement of two-photon absorption induced by the CTFP. We first
consider the case of conventional 3D holography (Fig. 2b). We
computed a 10 μm disk image target at z ¼ 0 where we imposed a
high-frequency speckle pattern to maximize spatial confinement
along the z axis. Projection views of two-photon absorption along
the y, x, and z axes show how, even in an optimized hologram,
inadvertent photostimulation remains possible above and below a
neuron targeted with this method. With 3D-SHOT however,
experimental results (Fig. 2c) show that temporal focusing signifi-
cantly enhances spatial confinement along the z axis in the CTFP.
112 Ian Antón Oldenburg et al.

Since 3D-SHOT is developed for the primary-use case of

optogenetic stimulation in brain tissue, we also recorded the effect
of propagation through scattering medium on the radial and axial
confinement of 3D-SHOT excitation. We cut acute mouse cortical
brain slices of varying thickness and placed them between the
excitation objective and the fluorescent slide. Recording
two-photon absorption through physiologically relevant amounts
of brain tissue revealed that scattering degraded radial resolution
only after passing through 400 μm of mouse brain (Fig. 2d,
200 μm: p ¼ 0.22, 300 μm: p ¼ 0.21, 400 μm: p ¼ 0.001,
Kruskal-Wallis). Although the axial point spread function exhibited
apparent degradation when imaged through brain tissue, this
decrease was statistically significant only after passing through
300 μm of scattering tissue (Fig. 2d, 200 μm: p ¼ 0.56, 300 μm:
p ¼ 0.04, 400 μm: p ¼ 0.05, Kruskal-Wallis).

2.2.1 Scanless 2P For neurobiological applications, we evaluated the spatial resolu-

Optogenetics Using 3D- tion of 3D-SHOT via quantitative measurements of the photocur-
SHOT rent amplitudes elicited by optogenetic stimulation in neurons. To
do so, we expressed microbial opsins in neurons through in utero
electroporation of mice embryos. Opsin expressing neurons were
then brought under the objective either in acutely prepared cortical
brain slices or in vivo with head-fixed animals.
To measure the spatial resolution of optogenetic excitation
(or “Physiological” Point Spread Function – PPSF) we recorded
the neuronal response to multiphoton photostimulation as a func-
tion of the displacement between the holographic target and the
patched cell (Fig. 3a).
The efficacy of two-photon excitation is not only dependent on
the shape of 3D-SHOT’s CTFP, but also on the precise targeting of
this pattern to the cell soma, the level of opsin expression in the
targeted neuron, and the laser intensity. Computer-generated
holography already offers micron-level spatial resolution for placing
holographic targets onto the desired neurons with a microscope
objective. However, the level of opsin expression varies from neu-
ron to neuron, and consequently so does the required power level
for photostimulation. We experimentally compared (Fig. 3a) the
spatial confinement of 3D-SHOT and 2P-CGH (without temporal
focusing) for photoexcitation as a function of incident laser power
density. Toward this end we obtained voltage-clamp recordings of
neurons, and recorded the PPSF at a variety of different laser
powers (Fig. 3b). With conventional holography, we observed
substantial photocurrents 25–50 μm above and below the disk
image target, indicating that photoactivation of non-targeted neu-
rons is likely to be an issue. Temporal focusing significantly
enhances spatial resolution with 3D-SHOT, and photocurrents
are more significantly attenuated above and below the primary
focus (Fig. 3c). We observed that the axial resolution with
 High-Speed All-Optical Neural Interfaces with 3D Temporally Focused Holography 113

Fig. 3 3D-SHOT generates axially confined photoactivation. (a) A photostimulation pattern generated with CGH
(top) or 3D-SHOT (bottom) was mechanically stepped along the optical axis (z) and passed through a cell
expressing opsin. Photocurrents were recorded in the whole-cell voltage-clamp configuration in neurons. (b)
FWHM of the characteristic response profile for both methods at various power levels. (c) Photocurrent
response profile for CGH (left) and 3D-SHOT (right) with a 10 μm disk target and different power levels. (d)
Spatial profile of two-photon evoked spiking of a L2/3 pyramidal neuron in a mouse brain slice (left) in the
radial dimension. Black: CGH; red: 3D-SHOT, p < 0.56 Mann-Whitney U-test, and (right), along the axial
dimension ( p < 0.006, Mann-Whitney U-test). (e) Quantification of the FWHM comparing CGH and 3D-SHOT.
(f) Full volumetric assessment of photostimulation resolution, points throughout the volume were tested, but
only points that elicited spike probability greater than zero are shown. (Adapted from Pegard et al. [7])

3D-SHOT was significantly improved relative to CGH, even using

several orders of magnitude more laser power. Whereas
two-photon photoexcitation with CGH relies only on defocusing
to confine the excitation light to the desired volume, 3D-SHOT
benefits from simultaneous defocusing and temporal confinement,
as femtosecond pulses are temporally stretched above and below
the desired target which further attenuates the nonlinear response
regardless of the targeted area in the (x,y) plane [41]. 3D-SHOT’s
shallow relationship between laser power and spatial resolution is
critical, in that it allows sufficient excitation light to generate action
potentials without significant loss of spatial confinement, as it nor-
mally occurs with CGH, and gives the user the option to use
additional power to reliably stimulate neurons when the exact
level of opsin expression is unknown without significantly affecting
spatial resolution.

2.2.2 3D-SHOT We next quantified the physiological spatial resolution of CGH and
Photostimulation with 3D-SHOT in neurons by measuring the spiking probability along
Single-Neuron Resolution the radial direction in the imaging (x,y) plane and along the optical
(z) axis. We compared holography and 3D-SHOT by projecting a
single photostimulation target placed at a distance (x,y,z) from a
patched neuron in mouse brain slice, either with single copy of the
114 Ian Antón Oldenburg et al.

Fig. 4 3D-SHOT provides cellular resolution photostimulation in a large volume through digital focusing. (a) To
quantify the spatial resolution of 3D-SHOT as a function of hologram target depth, we recorded the spike
probability in cortical neurons while digitally targeting varying positions along the optical axis (z), and
measuring resolution by mechanically sweeping the objective over the entire (z) range and measuring the
response at each point. (b) Spike probability in cortical neurons while targeting the same cell from different
axial displacements ( p ¼ 0.2, Kruskal-Wallis Test). (c) Spike probability resolution as a function of digital
displacement – shaded green colors denote mechanical sweeps across the optical axis for different digital
displacements. (d) Quantification of the FWHM for the axial fit of spike probability as a function of digital
defocus from the focal plane ( p ¼ 0.17, Kruskal-Wallis Test). (Adapted from Pegard et al. [7])

CTFP with 3D-SHOT or a disk-shaped pattern of equivalent size

using CGH. We measured spike probability as the hologram was
displaced in small increments by mechanically moving the objective
relative to the patched cell. Experimental results (Fig. 3d) show
similar spatial resolution with both methods in the radial direction
in the focal plane, with a FWHM of 10  2 μm for holography, and
9 μm 1.3 for 3D-SHOT ( p ¼ 0.57, Mann-Whitney U-Test)
consistent with the dimensions of the disc and Gaussian patterns
at the focal plane (Fig. 4a).
However, with conventional holography, the spike probability
along the z axis does not permit single-cell resolution
(FWHM ¼ 78  6 μm). In contrast, 3D-SHOT provides far
superior resolution (FWHM ¼ 28  0.7 μm, p ¼ 0.006, Mann-
Whitney U-Test, Fig. 3d) compatible with single-cell resolution in
all three dimensions, in that the FWHM of spike probability is on
par with the typical dimensions of a cortical neuron and their inter-
somatic spacing (Fig. 3e). We recorded from neurons in brain slices
and measured the spiking probability in response to 3D-SHOT
excitation by digitally refocusing a hologram to stimulate positions
throughout a 50  50  100 μm (x,y,z) grid. This experiment
revealed that the neuron was photoactivated only when the disc
image was targeted to the cell body (Fig. 3f).

2.2.3 Spatially Precise A major advantage of holographic optogenetics is the ability to

Remote Control with 3D- photoactivate neurons at different depths that are part of the
SHOT same circuit. Since the major advance of 3D-SHOT is its ability to
target temporally focused patterns arbitrarily in 3D, it is vital that
3D-SHOT maintains its ability to activate neurons with high spatial
resolution even when digitally focusing light far from the zero-
order of the optical system (e.g., the center of the optical axis at
 High-Speed All-Optical Neural Interfaces with 3D Temporally Focused Holography 115

the natural focal depth of the system). Therefore, we next evaluated

the accessible depth within the volume of interest by measuring the
activation and spatial resolution as a function of the distance from
the holographic zero order. Toward this end we measured spike
probability in neurons via current clamp in mouse brain slices
(Fig. 4a). To test if the CTFP can be digitally displaced along the
z axis, we systematically moved the digital focus of the hologram
(with a lens term on the SLM), and accordingly corrected the
mechanical position of the objective by the same distance
(δzDigital ¼ δzMechanical). This test showed that 3D-SHOT effec-
tively photostimulates cells at locations distal to the zero order, as
photocurrent and spike-probability were not affected by digital
offset in z (Fig. 4b, p ¼ 0.2, Kruskal-Wallis).
We next asked if the axial resolution of stimulation was constant
when stimulating away from the natural focal plane at z ¼ 0. For
this we measured the FWHM of the axial PPSF as a function of
digital defocus. As before, we digitally moved the holographic
target along the z axis, but instead of matching the digital and
mechanical offset, we stepped the objective across the entire range
of the z axis and measured the physiological response at each
location. This allowed us to measure the axial spatial resolution of
photostimulation from locations distributed on either side of the
optical axis. Results show that 3D-SHOT effectively confines exci-
tation to the desired depth range throughout the 180 μm range
that we sampled, as the FWHM of stimulation did not change as a
function of digital defocus in z (Fig. 5c, d, p ¼ 0.17, Kruskal-Wallis
Test). These experiments show that 3D-SHOT retains axial con-
finement capabilities that are compatible with single-cell resolution
for photocurrents and spike probability while targeting neurons at
any depth within the accessible volume defined by the SLM and the
microscope assembly.

2.2.4 Volumetric In addition to being able to create multi-target patterns at arbitrary

Optogenetics at High depths with precise power control, the utility of 3D-SHOT for
Spatial Resolution various applications is also determined by the absolute targetable
volume and the number of targets that can be placed in a single
hologram with the appropriate spatial resolution. To test the limits
of the volume that can be simultaneously targeted using 3D-SHOT
in our setup, we randomly selected up to 75 points distributed
throughout the 350 μm  350 μm  280 μm volume of interest,
and generated a point cloud hologram simultaneously targeting all
of these points. We measured 2P absorption using a sub-stage
camera (as in Fig. 2) and we measured the radial and axial confine-
ment of each spot in the multi-target hologram (Fig. 5a). Quanti-
fying the radial and axial FWHM showed that adding additional
targets (up to 75) did not degrade the confinement of light in
multi-spot holograms (Fig. 5b, p ¼ 0.34 Axial FWHM, Kruskal-
Wallis).
116 Ian Antón Oldenburg et al.

Fig. 5 Spatial resolution with simultaneous targets throughout a large volume. (a) 3D-SHOT was tested by
simultaneously targeting 75 randomly distributed targets within the full operating volume defined by the SLM’s
spatial range for the first diffracted order. Projection views of the 3D reconstruction of 2P-induced fluorescence in
a calibration slide are shown along the (y, z), (x, z) axis, with a 3D projection. (b) Similar experiments were
repeated with 20, 30, 50, and 75 targets. The FWHMs of the two-photon response were computed for each target,
and show that spatial resolution and axial confinement are not significantly degraded by increasing the number of
simultaneous targets in any given hologram (axial FWHM: p ¼ 0.34, Kruskal-Wallis; radial FWHM: p < 0.001 for
75 ROIs; p > 0.05 for all other comparisons). (Adapted from Pegard et al. [7])

Overall, our experiments show that with 3D-SHOT as with

CGH, the accessible volume and available optical power under the
objective depends on the diffraction efficiency of the SLM, the laser
power, and on cumulative losses across the optical path. Altogether,
these design parameters determine the number of neurons that can
be simultaneously illuminated with the desired spatial resolution.
Here, with 600*800 pixels on the SLM, we characterized single-
shot photostimulation of up to 75 targets (limited by laser power)
with no degradation of resolution within a 0.034 mm3 volume
(350 μm  350 μm  280 μm, Fig. 5). For comparison, custom
photostimulation patterns have been demonstrated in previous
works within a 0.017 mm3 operating volume with multi-level TF
[51] (240 μm  240 μm  300 μm), ~6.25  104 mm3 with point
scanning methods (250 μm  250 μm  15 μm) [18, 33].

2.3 Calibration of The calibration and alignment of the optical system is critical to the
3D-SHOT with Imaging successful use of any multiphoton stimulation system, this is made
System even more challenging when improving the resolution of such a
system. Furthermore, whereas it is customary to report the best
possible resolution in optics publications (to explain the potential
of the technique), it is also known that the resolution is not con-
stant throughout the entire working volume. However, for
biological experiments, it is necessary to know what the actual
resolution is at any given point in the working volume.
High-Speed All-Optical Neural Interfaces with 3D Temporally Focused Holography 117

Furthermore, even subtle errors introduced by aberrations of lenses

and the SLM can lead to mistargeting problems that will prevent
accurate experiments if they are not accounted for. To that end, we
developed a calibration protocol and numeric tools to map the 3D
holographic stimulation path with the multiplane two-photon
imaging path. This calibration empirically accounts for aberration
and deformities that are introduced both by the SLM and asso-
ciated lenses, but also by the optomechanical defocusing method
(in this example an electro tunable lens; ETL). This strategy pro-
vides both an improved calibration over less thorough procedures,
and even more critically accurate measurements of the size of the
holographic disk throughout the useable volume. While this proce-
dure is quite slow, it is fully automated and can be run overnight (see
Note 2.3.1). Scripts are available at https://github.com/
adesniklab/3D-SHOT/AutoCalib
In addition to the 3D-SHOT and 2P imaging system described
above, the calibration requires a substage camera (Fig. 6a). While
many camera objective pairings are theoretically useable, we used a
Basler camera (acA1300-200um) with a 5 Olympus air objective
(Olympus MPLFLN) and a thin fluorescent slide (see Note 2.3.2).
Care should be made to match the substage camera’s field of view
to be at least as large as the imaging field of view.

Calibration Procedure
(a) Manually position the substage camera such that the slide is in
focus, and the illuminated area is in the center of the substage
camera’s field of view. Tip: It may help to zoom in the 2P image
and/or reduce the line count to get more visible fluorescence on
the substage camera. However, care should be made not to
photobleach the slide, and it will be necessary to return the
imaging conditions to their normal settings before the rest of
the calibration.
(b) Compute 500–1000 test holograms containing a single target
randomly placed throughout the imaging accessible volume.
Tips: Random spots work slightly better than regularly placed
spots to avoid overfitting.
(c) Coarse Alignment. Sequentially illuminate each hologram
with the same power and record the fluorescence on the
substage camera (Fig. 6a). Move the objective mechanically
in 25-μm steps increments throughout the useable volume to
get a stack of images for each hologram. From this stack you
can get the expected XY location of each hologram, and the
peak fluorescence depth. Tips: Make sure that the power level
you use here is neither saturating the substage camera, nor too
dim to be visible. As holograms near the center of the optical axis
will be brighter (better diffraction efficiency) we recommend
118 Ian Antón Oldenburg et al.

Fig. 6 Calibration protocol for 3D-SHOT. (a) Substage camera assembly for calibration with a uniform
fluorescent thin film on a microscope slide. (b) A single hologram is imaged at 13 different planes by moving
the hologram with respect to the thin fluorescent slide. The full range is 40 μm from the estimated center of
the hologram. (c) We fit a Gaussian curve to the measured fluorescence at each plane for each hologram
recorded in (b). Relevant resolution characterization values (peak intensity, FWHM, and depth) are extracted
for each hologram. (d) We first identify the relationship between the predicted SLM defocus and the detected
depth of the corresponding holograms. (e) Mapped relationship between hologram FWHM and the hologram
depth (left) is measured in the entire volume accessible by the SLM (right). (f) Hologram diffraction efficiency is
measured throughout the field of view. (g) True depth of the two-photon imaging planes, as detected by the
substage camera. We note that the planes are neither flat, evenly spaced, nor parallel to the axis of the
camera, but that the calibration will account for all those discrepancies. (h) The final hole pattern in SLM space
accounts for aberrations and curvature from both the SLM/stimulation path and imaging path. (i) Images of the
holes ablated in the first plane, and for a subsequent plane. The hole pattern is asymmetric, so that
subsequent planes will not burn in the same location. (j) Simulated targeting error over the full calibrated
volume
High-Speed All-Optical Neural Interfaces with 3D Temporally Focused Holography 119

using a test hologram near the zero order block and set the power
such that it is just below saturating the camera pixels.
(d) Fine Alignment. Take a z stack at 5–6 μm spacing for the
40 μm around the expected Z location of each test holo-
gram (Fig. 6b). Extract the fluorescence at the center of the
hologram across the measured depths and fit this value to a
Gaussian. The width of this Gaussian is the axial FWHM for
the measured XYZ depth, and the peak of the Gaussian will be
used to determine the diffraction efficiency for this point
(Fig. 6c). The radial FWHM is determined by the fluores-
cence profile of the holographic spot at the best depth. It is
best measured by fitting the observed fluorescence to a
Gaussian, but as it is measured at a high spatial resolution
already, this is not critical. Tip: while there may be aberrations
or curvature from the SLM or other optical elements, they don’t
need to be explicitly corrected as the general mapping strategy
will account for all smooth distortions.
(e) Fit SLM locations to substage cameras. For every SLM XYZ
coordinate we now have a corresponding XYZ location
measured by the substage camera with the depth determined
from the z stack. Some holograms may need to be excluded if
they were not properly detected (see Note 2.3.3). Use a
polynomial fit to map SLMXYZ to CameraXYZ (Fig. 6d).
Tip: When fitting make sure to scale both the camera pixels and
the z depth to similar size units (such as μm) to prevent over-
weighting one axis. It is best practices to use ‘hold out’ data to
test the fidelity of your fit. This will allow you to detect if there are
systematic problems in your procedure.
(f) At this point you will have collected all the necessary infor-
mation to detect any variation in the shape of the holograms
throughout the useable volume. Axial FWHM is often
degraded when reaching the limits of your optical system
(both radially near the edges, and axially at far defocus levels).
It is important to restrict your experiments to locations where
this FWHM is acceptable for your application (Fig. 6e).
(g) To determine the diffraction efficiency as a function of target
location in the accessible volume, you may divide the
observed peak fluorescence from the fine calibration by the
best fluorescence observed in the experiment. Here again, we
recommend a polynomial interpolation to map the scalar
“diffraction efficiency as a function of the SLMXYZ coordi-
nates” (Fig. 6f). Tips: The diffraction efficiency accounts for
many spatially dependent losses of observed fluorescence beyond
holographic diffraction efficiency itself. This however works out
to the more useful measure when running biological experi-
ments. Furthermore, it would be more correct to get the best
120 Ian Antón Oldenburg et al.

possible measurement of fluorescence with the zero-order block

removed and a zero-order hologram. However, in practice this is
unnecessary, the disruptions from removing and replacing the
zero-order block are non-negligible, and the absolute value of the
measured diffraction efficiency is less important than the exact
profile along which it falls off.
(h) Record the XYZ shape of each imaging plane. Due to aberra-
tions induced by the ETL and the other lenses in the imaging
path (and the substage camera itself), each imaging plane will
have some three-dimensional shape, and they will be different
for all defocusing levels. Image the slide at a single ETL-based
depth and record the fluorescence on the substage camera.
Then move the objective axially to create an orthoscopic
image of its 3D profile. Repeat for many ETL defocus levels
corresponding to the range you want to calibrate (Fig. 6g).
Tips: Make sure to use enough power to be visible on the substage
camera but not bleach. Increase the camera acquisition time, or
frames averaged to get sufficient signal to noise.
(i) Using a polynomial fit, map the measured Z depth to each
ETL depth value as a function of XY position. Tips: you should
be able to see the shape of each of your imaging planes, curvature
and ripple in these planes is common and usually not detrimen-
tal to imaging quality or stimulation success.
(j) To perform the final step in the alignment relating camera
space to imaging space we calculate the SLM coordinates that
are necessary to place holographic spots on each of the ETL
planes that will be calculated. We use a grid of at least 16 tar-
gets per plane, each target offset laterally so that the projec-
tion of all targets onto a single plane will not be overlapping.
Calculate single target holograms for each of these targets
(Fig. 6h, i).
(k) Ablate or bleach a hole in the fluorescent slide for each of this
second set of holograms. Take a two-photon image of the
slide at the appropriate ETL depth before and after each hole.
The difference between this before and after image will reveal
the XY position of each hole and the ETL depth can be used
as the detected depth to create a 2PXYZ map for each of the
second set of holograms. Tips: setting the ablation power can
be tricky, higher peak energy, lower wattage pulses appear to
bleach the slide more than they create cavitation and thus
provide a more representative “hole”. We use 5x the standard
imaging power for 500 ms to ablate a hole.
(l) Fit the picked CameraXYZ locations to the detected 2PXYZ
locations using a polynomial fit as above. The final calibration
is 2PXY + ETLZ -> CameraXYZ -> SLMXYZ (Fig. 6j). Tips:
All of these fits are symmetric so it can be self-checked by
High-Speed All-Optical Neural Interfaces with 3D Temporally Focused Holography 121

measuring the error by attempting to map coordinates onto

themselves with a “round trip” interpolation between any two
coordinate systems. Additionally, since the last step ablates tar-
gets for which both SLMXYZ and CameraXYZ coordinates are
known, a second mapping can directly compare SLMXYZ to
2PXY + ETLZ coordinates, but since this approach does not
include a true measurement of Z it is assumed to be less accu-
rate. Typically, the mean interpolation error for a calibration is
<2 μm throughout the entire useable area.
(m) Troubleshooting. There are two very common sources of mis-
calibration. First, imaging conditions can change. This can
occur due to evaporation of the immersion liquid, instability
in lasers, or other sources. Sometimes the fluorescent inten-
sity of holograms decreases over time. To detect this, plot the
peak hologram intensity in order in which it was measured, as
the locations are randomized, if any trend is visible it indicates
some instability. Imprecise points can be manually excluded
or the whole procedure can be repeated with steps taken to
ensure that this problem will not occur again.
Second, the calibration slide may move. Since the calibra-
tion can take many hours, even subtle movement of the slide
will disturb the calibration. This can be an insidious problem
as depending on when this movement happens it can manifest
in different ways. Prevention is the best remedy, and firmly
securing the slide and the substage camera will mitigate this
issue. A wise step at the end of a calibration is to conduct a
hole test by ablating targets above and below a test location,
with offsets of a few microns, to test the XZY accuracy (even
2 μm offset target will burn less efficiently than a properly
targeted hologram). Typically, slide movements will only
result in a XYZ offset and a digital offset can be applied
without the need for a full recalibration. This approach can
also help fix small post-calibration misalignments that may
occur if there is any drift of the laser beam, when a full
recalibration is not desired.
Notes

2.3.1: Overnight calibrations: While the described calibration is

designed to run autonomously, several problems can arise that
will disrupt it. First, the immersion liquid between the objective
and the slide can dry up. To avoid this, we use a 1:10 dilution of
Ultrasound Gel (NA 1.0, Parker Aquasonic 100), and create a
well to hold excess liquid. Second, if the setup is much more
susceptible to movements during calibration than it will be in
its final form. We also recommend signage to ensure nobody
disturbs the procedure.
122 Ian Antón Oldenburg et al.

2.3.2: Thin Fluorescent Slide: The detected resolution will be

dependent on the thickness of the thin fluorescent slide. We
manually spray Fluorescent Red (Tamiya TS-36) spray paint on
standard microscope slides, and then screen each to be of
uniform, minimal, and comparable thickness using the
two-photon imaging system. As the diffraction-limited spot
(DLS) of the imaging system is smaller than the 3D-SHOT
spot, it is a useful benchmark for slide quality. For this step, we
take a z-stack of the stationary imaging DLS using various
slides. Variation of the same spot in the same location is caused
by the slide thickness, and thus slides that report the smallest
axial FWHM will be the thinnest. It is very important to pick a
slide and a field of view that is even and flat. Small and sparse
blemishes will be tolerated by the redundancy of the calibra-
tion, but large uneven fluorescence, or frequent scratches or
holes will prevent accurate power calibrations.
2.3.3: There are many reasons why a given hologram may be
poorly detected (e.g., out of range of the camera, insufficient
diffraction efficiency, high noise), but these poorly read data-
points will increase the overall error of the calibration and
decrease its usefulness. It is therefore better to have a smaller
high-quality calibration than a larger one that contains inaccu-
rate data. To ensure data quality, we set a brightness threshold
for each hologram to be included in the calibration. The peak
fluorescence should be at least 2 the Standard deviation of the
imaging noise, points that do not satisfy this criterium are
removed from the calibration data.

2.4 Comparison of Precisely timed neuronal activation cannot be achieved through

Opsins for Precise technological progress in optical instrumentation alone. The
Activation of Activity molecular actuator, i.e., the opsin, is also a critical element for
proper control of neural activity. The opsin must be selected delib-
erately, such that it synergizes with your chosen optical stimulation
technique. In this section, we specifically discuss the selection of
opsins that best leverage new capabilities introduced by 3D-SHOT
to simultaneously illuminate the entire cell with strong photocur-
rents. Simultaneous illumination approaches as opposed to “spiral
scanning” favor opsins with very fast kinetics and high peak ampli-
tudes, enabling precise timing of action potentials with minimal
jitter. Oppositely, asynchronous scanning-based approaches may
benefit from slower opsins allowing for greater integration times,
and can be activated with less laser power, but at the expense of
temporal precision. Both approaches require opsins that are well-
activated by 1030-nm light to best match commercially available
high-power stimulation lasers.
The requirements for opsin constructs for 2-photon optoge-
netics differ considerably from those used for one-photon activa-
tion. Most notably, multiphoton approaches benefit from “soma-
High-Speed All-Optical Neural Interfaces with 3D Temporally Focused Holography 123

targeted opsin constructs”, i.e., those that are only expressed in the
soma and proximal dendrites. Without soma targeting, the spatial
resolution is compromised [11, 19, 56]. Furthermore, other opsin
properties, such as photocurrent amplitude, absorption spectrum,
and photocurrent kinetics, will strongly affect the experimental
abilities of a 3D-SHOT system. These biophysical properties will
interact with both the imaging and 3D-SHOT system and will alter
the resolution, precision, and scale of neural control that is possible.
We will briefly summarize how these properties interact, before
describing a protocol for assessing opsin properties with regard to
how to best activate or suppress a neuron. While many techniques
are employed in this evaluation process, we will focus on those steps
that are germane to opsin evaluation and two-photon optogenetics
while directing the reader elsewhere for some technical procedures.
While we will focus on selection of activating opsins, we will briefly
discuss the additional concerns surrounding selection of a suppres-
sing opsin.
We will also include, where possible and relevant, data on
existing opsins. While many studies focus on individual features of
opsin behavior, proper evaluation of an opsin requires a holistic
understanding of many properties of those opsins. There are rela-
tively few commonly used activating opsins used with multiphoton
optogenetics the most common variants are ChR2 [28], C1V1
[8, 13], ChrimsonR, Chronos [15], ChroME [11], ReaChR
[57], CoChR [56], and ChRmine [14] each with their own advan-
tages and disadvantages. Opsins for two-photon suppression are
less well characterized with only Arch [13], NpHR3, PsuACR, iC+
+, GtACR1 [11], and GtACR2 [58] being described.
We will select for opsins that:
l Are well-trafficked to the somatic membrane with little toxicity.
l Have large photocurrents.
l Have fast kinetics.
l Are well activated by 1030-nm light.
l Are compatible with the all-optical approach of choice.
l Can reliably drive spiking in vivo.
Procedure
1. Subcellular Targeting.
When expressed in neurons, the opsin must be properly traf-
ficked to the cell membrane but restricted to whatever extent
possible away from the distal dendrites and axons (Fig. 7a). A
sequence from the Kir2.0 channel [59] can be very helpful to
export from endoplasmic reticulum, while a portion of the Kv2.1
channel [60] has become the standard (but not the only [56])
“soma targeting” motif, facilitating both membrane expression
and restriction to the soma and proximal dendrites. It is advisable
to use all subcellular targeting motifs even when testing opsins in
124 Ian Antón Oldenburg et al.

Fig. 7 Characterizing opsin characteristics for use with 3D-SHOT. (a) Comparison of non-soma-targeted opsin
localization (left) to soma-targeted opsin localization (right). (Adapted from Mardinly et al. [11]). (b) Compari-
son of photocurrent FWHM (right) at different points on the opsin response function (left). Closer to saturation
(dark blue), the actual FWHM of the photocurrent is larger than the theoretical opsin FWHM. (c) Comparison of
photocurrent amplitude and kinetics for several commonly used opsins expressed in CHO cells. (Adapted from
Sridharan et al. [61]). (d) Schematic of three common opsin kinetic metrics: left, time to peak used to measure
opening kinetic. Center, desensitization. Right, tau off, a metric of decay kinetic. (e) Schematized absorption
spectra for three opsin types compared with GCaMP absorption spectrum (dashed green). (f) Schematic of
how, with fast imaging and slow opsins (top left) scan-induced photocurrents can accumulate to produce
unwanted spiking, while in other conditions they decay and do not produce spiking. (g) Schematic displaying
how under different stimulation conditions, short laser light pulses (left) can produce more or less reliable
spike trains depending on opsin characteristics
High-Speed All-Optical Neural Interfaces with 3D Temporally Focused Holography 125

reduced preparations such as CHO cells, as membrane targeting

can affect photocurrents substantially. To begin opsin characteriza-
tion, it is advisable to examine the trafficking of your opsin by
creating a construct with the opsin fused to a fluorophore, even if
ultimately a different fluorophore configuration is preferable. This
way, one can ensure that the construct is well-trafficked to the
membrane, while still restricted as much as possible to the soma.
Furthermore, internal protein aggregation may be an indicator of
toxicity. For more detailed discussion of toxicity assessment at
multiple stages, see Note 2.4.1. In our experience, with the excep-
tion of incidences of overexpression, the gene delivery technique
(e.g., AAV, IUE, transgenic) does not dramatically change intracel-
lular trafficking patterns. Still, the most rigorous approach is to
examine your particular opsin with the delivery mechanism you
will use (see Note 2.4.2 for a discussion of delivery approaches).
2. Photocurrents.
Photocurrent is perhaps the most obviously important measure
of potency for an opsin. In many cases the opsin that fluxes the most
current will be the most useful, as more potent opsins can drive
more cells with less energy. While it may be convenient to compare
the one-photon response to light, 3D-SHOT relies on the multi-
photon process and thus one-photon responses are not an accept-
able substitute (see Note 2.4.3).
There are two main criteria to consider when evaluating pho-
tocurrent. First, it is important to probe a large range of light
intensities. Different opsins will saturate (i.e., reach maximal pho-
tocurrent) at different illumination powers and at different photo-
current levels. While it is necessary to reach a certain threshold to
spike a cell (1 nA is a good approximation to spike a L2/3 pyrami-
dal cell with a 5-ms pulse), the shape of this curve will impact your
resolution. The minimal power needed to spike a cell will dictate
the total number of cells that can be activated with a given micro-
scope, and the total heat added for a given number of activated cells
(see Note 2.4.4). In addition, the further this current is from the
saturating current the better the effective resolution will be
(Fig. 7b).
While peak current is often reported, the average current over a
given pulse duration, or the total charge fluxed, is what ultimately
drives cell activation and thus is a more relevant measure for photo-
activation. This is especially important considering that many
opsins show desensitization (see detailed discussion in step 3,
Opsin Kinetics).
Available opsins differ greatly in photocurrent magnitude
(Fig. 7c). While direct comparisons of all commonly used opsins
is not available (though see Sridharan et al. [61] for comparison of
many), only ChroME family [11, 61] and ChRmine family [14, 62]
opsins appear to reliably reach the 1 nA benchmark. Cells
126 Ian Antón Oldenburg et al.

expressing either CoChR [56], and ReaChR [57] occasionally

reached this threshold, but not reliably. It is possible that further
improvement of targeting and expression will help these opsins
reach this benchmark.
3. Opsin Kinetics.
While selecting the opsin with the maximum photocurrent
makes sense in many cases, it may come at the expense of speed.
Fast opsins are necessary to take full advantage of the temporal
control offered by 3D-SHOT and to write specific spike trains,
with low spike jitter and high fidelity.
A fast opsin must both open and close its ion channel very
quickly. If the closing kinetic is too slow, two or more action
potentials may result from a single stimulation [34, 52, 53], and
it may be impossible to drive cells to fire at high rates, disrupting
the ability to write a known train. Similarly if the opsin opening
kinetic is too slow, longer stimulation periods will be needed to
drive the cell to fire and the uncertainty (jitter) in action potential
timing [52] will increase.
Moreover, the opening (but not closing) kinetics of both acti-
vating and suppressing opsins [11, 46, 63] are often dependent on
laser stimulation power, adding additional complexity [11]. As the
precise mechanics that lead the opsin to transition between con-
ducting states is not yet fully understood [64, 65], and even less is
known about how these transitions may be affected by the
two-photon process, there is often no way to model or infer kinetics
based on the structure of an opsin alone. Instead, the only solution
is to measure these kinetics with each new prospective opsin.
The opening kinetic is often measured using the time to peak or
90% current, but τ derived from an exponential fit can also be
reported [8, 11, 15, 56] (Fig. 7d). During prolonged pulses,
many opsins’ photocurrents reduce with time, a phenomenon
known as desensitization (Fig. 7d). This may be either through
inactivation of a population of channels and/or by individual chan-
nels entering states with different conductance [53, 65]. At the
cessation of a pulse, the opsin current decays gradually. This closing
kinetic is typically reported by fitting an exponential fit and calcu-
lating the τ (Fig. 7d) [56]. At times, a double exponential may
better fit the data than a single one [11].
Several groups have endeavored to identify or engineer very fast
opsins for one- or two-photon use [11, 15, 66–69]; these promise
to be valuable tools for optogenetic control. While mutations that
speed up an opsin often come at the expense of total photocurrent,
this does not appear to be an absolute rule (note the success of
ChroME and ChETA [11, 68]). Furthermore, it is hard to know
how fast is “fast enough”? Chronos and the mutant forms,
ChroME opsins, are the fastest opsins used in 2p-activation to
date, and can be used in vivo to drive spike trains with jitter much
High-Speed All-Optical Neural Interfaces with 3D Temporally Focused Holography 127

less than one millisecond [11, 61, 70]. Nevertheless, in the correct
conditions even much slower opsins such as ChrimsonR [52],
CoChR [56], or ChRmine [14] can reach jitter of about 1 ms.
However, cells expressing these last three opsins struggle to follow
rates over 20 Hz [14, 52], probably due to their slower off kinetics.
4. Two-Photon Spectra.
The two-photon absorption spectrum of an opsin affects its
compatibility with imaging approaches and suitability for use with
the high-power lasers typically used for 3D-SHOT. A chief concern
with simultaneous imaging and holographic activation is the phe-
nomenon of crosstalk, in which the scanning diffraction-limited
spot used to excite GCaMP fluorescence also activates opsin mole-
cules (the reader may also refer to Chaps. 2, 3, 5, 6, and 11 of this
book for an extended overview of this phenomenon). Opsins with
low absorption in the range of wavelengths used to image GCaMP
(typically 910–930 nm) reduce crosstalk. Unfortunately, many tra-
ditional opsins are highly activated by blue light, so this has
required the development of many new opsins. Alternate calcium
indicators that absorb in other wavelengths are also available but are
much dimmer than available GCaMPs [71–73]. In addition, most
commercially available high-energy lasers emit around 1030 nm
[63]. Thus, the optimal opsin would have a peak photocurrent
around 1030 nm with a comparative minimum at the wavelengths
to image GCaMP (~930 nm) (Fig. 7e, see Note 2.4.5 for further
discussion of alternate strategies).
It is well known that fluorophores have two-photon absorption
spectra that are considerably different from their one-photon coun-
terparts [74]. To assess the sensitivity at various 2 photon wave-
lengths, the simplest approach is to use a 2p imaging microscope
with femtosecond laser (e.g., Ti: Sapphire oscillator) that is tunable
across a large range of wavelengths. Since spectral response is not
known to be affected by the light delivery method, using a scanning
imaging system to photoactivate opsins is a suitable approach to
compare the relative activation at different wavelengths and mea-
sure the spectral response profile. Recording photocurrents from
CHO cells at different wavelengths while scanning will provide a
two-photon spectrum for the opsin. Emission power varies with
wavelength, so be sure to test power out of the objective at all
testing wavelengths.
Of the common excitatory opsins, ChR2 [28] and CoChR
[56] are blue-shifted making them suboptimal for pairing with
GCaMP imaging (Fig. 7e). Several 2p-optimized opsins, including
Chronos, ChroME [11], and ReachR [57] peak around 1000 nm,
and more red-shifted opsins such as C1V1 [13], ChrimsonR [11],
and ChRmine [14] peak beyond 1040 nm.
128 Ian Antón Oldenburg et al.

5. Characterizing Crosstalk in Imaging Conditions.

In most experiments, multiphoton optogenetics will be paired
with multiphoton imaging. It is important to expressly consider the
relevant ways that these two systems interact. While the stimulation
laser can create an artifact on the imaging system (see Note 2.4.6),
the more insidious form of crosstalk is where the imaging laser can
activate the opsin. This crosstalk results from opsins that are some-
what sensitive to imaging wavelengths, as discussed above. Even
when these depolarizations (or hyperpolarization in the case of
inhibitory opsin) are not enough to overtly change the spiking
rate of cells they can cause significant currents which may alter the
timing of action potentials.
Opsin kinetics and strength also influence compatibility with
2p imaging. A diffraction-limited spot is used for imaging scans
across a cell in a very different way than 3D-SHOT. Here, fast
opsins are again advantageous because activation will decay to
baseline between imaging frames, thus not driving the cell to
spike (Fig. 7f). Strong, slow opsins may be most vulnerable to the
effects of crosstalk, but this can be addressed at least in part by
interleaving several frames in different areas to extend the time
between repeated stimulation. In contrast, as activation is propor-
tional to dwell time of the imaging laser on a cell, approaches that
increase this dwell time, such as having a smaller field of view, will
suffer worse crosstalk [11].
Due to the many variables affecting crosstalk currents, includ-
ing imaging speed, power, wavelength, opsin kinetics, and more, it
is important to test the actual currents induced in your typical
imaging preparation [11]. Empirical measurements with your
opsin of interest and in the typical imaging conditions are essential
to understand the level of crosstalk that you will experience. Whole-
cell recordings, even in ex vivo slice, under analogous imaging
conditions will give the resolution needed to observe how large
the imaging-induced photocurrents are.
6. Characterizing In Vivo Spike Fidelity.
Ultimately the end goal of selecting an opsin is to cause neu-
rons to fire action potentials. While many opsin/stimulation com-
binations can drive cell spiking [11, 34, 52] it is important to
quantify the fidelity of this spiking. One should quantify to the
fraction of cells spikeable, the fidelity of response (i.e., the fraction
of pulses in which the cell spikes), the reproducibility of a response
(i.e., the fraction of pulses resulting in one and only one spike, also
known as no “doublets”), and the jitter of the resulting action
potentials (to understand the limits of your timing control).
Furthermore, these evaluations should be performed at a vari-
ety of frequencies, and in conditions most similar to your biological
experiments possible. Under different stimulation patterns and
High-Speed All-Optical Neural Interfaces with 3D Temporally Focused Holography 129

strengths, opsins may be more or less faithful (Fig. 7g), such as

strong, slow opsins producing doublets at high stimulation powers.
We recommend cell-attached or whole-cell recordings from the
anesthetized animal. Of these in vivo criteria, being able to reliably
evoke one and only one action potential per pulse is one of the most
challenging and useful features. While this analysis has not been
performed on all opsins both ChroME [11] and ChrimsonR [52]
can be driven in the regime where one and only one spike is
possible.
7. Considerations for Multiphoton Suppression.
Multiphoton suppression involves using an inhibitory or sup-
pressive opsin (one that hyperpolarizes a cell) instead of an activat-
ing one to prevent a cell from firing. Multiphoton suppression
employs many of the same concerns as multiphoton activation.
Concerns around spectrum, photocurrents, imaging compatibility,
and toxicity requirements are all very similar between activating and
suppressing opsins.
The primary difference is in the requirements for kinetics,
whereas activation requires a fast off kinetic to prevent doublets;
suppressing opsins have no such constraints. In fact, slower off
kinetics can be helpful, as they allow discontinuous (aka duty
cycled) light to achieve continuous inhibition. Similarly, as it is
rare to know the precise timing of a naturally occurring spike,
there is a diminished need for a fast on kinetic, but this comes at
the cost of needing to suppress activity for a long duration. With
increased photostimulation durations come increased hazards of
heat buildup, and corruption from optical artifacts. Furthermore,
opsins that desensitize to two-photon stimulation, i.e., have a high
peak current but a lower sustained current, such as iC++ [11], are
disadvantaged as the peak current contributes less to the overall
suppression of activity.
Finally, whereas photoactivation is a nearly binary process, a
spike is either evoked or it is not, suppression is graded. Even if a
given opsin stimulation can suppress spontaneous activity, a partic-
ularly large endogenous stimulus might overwhelm this inhibition.
As such many aspects of benchmarking inhibitory opsins are harder.
Nonetheless, GtACR2 [58], GtACR1, and Arch [11] have all been
used to successfully suppress activity in vivo. Although of these
Arch and GtACR2 only suppressed ~50% of spontaneous activity
[11, 58], whereas GtACR1 was more effective [11].
130 Ian Antón Oldenburg et al.

3 Conclusion

When selecting an activating opsin for very precise manipulation of

spike trains there are a variety of factors that need to be weighed.
Thus, a holistic approach that evaluates all qualities of the opsin is
required to pick the optimal tool. Of the opsins that have been
tested for their compatibility with two-photon optogenetics, only
ChroME, ChRmine, CoChR, and ReaChR reach the photocurrent
benchmark of 1 nA. Of these only ChroME, and ChRmine can be
used to drive spikes with sub-millisecond jitter, and only ChroME
responded reliably at rates above 40 Hz. However, as more opsins
are developed for multiphoton use, this set of useable opsins will
continue to grow. In the near future, there will be a generation of
new opsins that are discovered or mutated from existing opsins that
will advance our ability to control the firing of cells. As the field
grows out of its infancy it is likely that more criteria will present
themselves as essential for selecting the best opsin. Until they do,
we hope this guide will aid in the benchmarking of future opsins for
the precise recreation of neuronal activity patterns.

Notes
2.4.1: While observing aggregates in structural imaging is a clear
indicator of possible toxicity, many exogenous proteins can
have deleterious effects on the health of cells without necessar-
ily forming aggregates. Thus, we recommend further assess-
ment of cell health for all preparations used for experiments.
Viral overexpression and/or combination with other stressors,
including calcium buffering from GCaMP, may further lead to
cells no longer responding physiologically. It is important to
benchmark the health of cells with your chosen opsin and
expression strategy. There are two broad categories to examine:
First the intrinsic properties of cells, and second their physio-
logical responses to stimuli. Intrinsic properties, such as a cell’s
input resistance, resting membrane potential, membrane time
constant, action potential threshold, and shape of an action
potential should all remain unchanged, between opsin expres-
sing and opsin negative cells, and are easily measurable in
ex vivo whole-cell recordings. Similarly, the resting firing rate
of cells in vivo should not be altered by the presence of an
opsin, nor when imaging with standard GCaMP imaging con-
ditions. Physiological assessment of cell health is more chal-
lenging and is often overlooked. Ideally, one would measure
the in vivo responses to given sensory stimuli and show that
they do not change with presence or activation of the opsin.
2.4.2: Opsin expression. While AAV delivery of opsin is a popular
and effective strategy for expression in neurons, it can
High-Speed All-Optical Neural Interfaces with 3D Temporally Focused Holography 131

introduce variability, especially dependent on differences in the

viral preparation [75]. Especially if new constructs are being
created, the time to develop new viral delivery systems may be
preventative. For this reason, we recommend using transfected
CHO cells for opsin biophysics such as kinetics, and in utero
electroporated cortical neurons for experiments assessing neu-
ronal responses. For photocurrent assessment, while CHO cells
will provide some information, we recommend using neurons.
Subcellular targeting and the interactions between endogenous
channels and opsins could potentially alter the effectiveness of
different opsin constructs. For this reason, we recommend
whole-cell voltage-clamp recordings in neurons in utero elec-
troporated with the opsin construct of interest.
2.4.3: The two-photon response of opsins: while relatively little
work has been devoted to comparing the two-photon
responses of opsins, several lines of evidence support that the
activation we see is indeed a two-photon process. First, others
[13, 46] have fit very low power activation of C1V1 or ChR2
and concluded that it better followed a quadratic fit rather than
a linear one. More recent work has shown [16] that a dispersed
laser pulse, with lower peak energy, is unable to activate Chrim-
sonR while a compressed pulse is.
2.4.4: The risk of brain heating is greatly expanded with multipho-
ton imaging and optogenetics. It has been shown that even
modest temperature increases used in one photon optogenetics
can sometimes affect firing rates of cells [76, 77]. Thus, the
much higher energies used in multiphoton optogenetics, and
to a lesser extent imaging, could in theory lead to aberrant
behavior. This concern is mitigated somewhat by a 2–3  C
decrease in the overall temperature of the brain simply through
the cranial window and water immersion objective that is used
in the microscope [78, 79]. Nonetheless, brain heating espe-
cially with multiphoton optogenetics remains a risk [80, 81].
2.4.5: While, by far the majority of groups doing all-optical multi-
photon optogenetics use red-shifted opsins [11, 14, 16, 34,
82], others have focused on blue-shifted opsins with red indi-
cators [58]. In theory, 3D-SHOT should be fully compatible
with a lower wavelength laser. However, 3D-SHOT requires a
high-energy laser to stimulate many cells. As currently there are
few available lasers that have very high peak power at 930 nm, it
is difficult to build a system that can control many cells. None-
theless, this approach offers several advantages that should not
be overlooked. All opsins recorded to date have substantial
“blue shoulders”, i.e., current fluxed at lower wavelengths
than their peak. As such with a red-shifted opsin there will be
some activation by the imaging laser. This “scan activation”
132 Ian Antón Oldenburg et al.

presents a potential confound that if not accounted for could

change the results of a biological experiment.
2.4.6: Stimulation laser-induced artifacts in imaging. Just as the
imaging laser can somewhat activate the opsin, so too can the
1030 nm stimulation laser drive GCaMP fluorescence, albeit
sub-optimally. This creates an artifact whereby pixels recorded
during stimulation will be contaminated and will appear
brighter, in severe cases (such as when stimulating many tar-
gets) this artifact can be much brighter than the GCaMP
fluorescence. Some groups have opted to exclude frames or
pixels containing artifact [14, 18] but this can be preventative
when stimulating at high rates, or for long durations, as is
required during optogenetic suppression. Instead, we recom-
mend syncing the firing of the stimulation laser to the phase of
the fast-resonant mirror. By only allowing the stimulation laser
to fire during the edge or flyback of the imaging field of view we
ensure that very few neurons are obscured by the stimulation
artifact. This can be achieved via a fast analog or digital circuit
that controls a sufficiently fast electro-optic modulator
controlling the stimulation laser’s power. As the rate of the
resonant mirror is ~8 kHz, this modulated cycle will be approx-
imately ~16 kHz much faster than any known opsins on or off
rate. This very fast duty cycle ends up being a significant advan-
tage. While this “gating” of the laser might exclude up to 50%
of the total energy hitting the sample, we see photocurrents
that are only reduced by 15–20%.

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 Chapter 5

An All-Optical Physiology Pipeline Toward Highly Specific

and Artifact-Free Circuit Mapping
Hendrik Backhaus, Nicolas Ruffini, Anna Wierczeiko, and Albrecht Stroh

Abstract
All-optical physiology of neuronal microcircuits requires the integration of optogenetic perturbation and
optical imaging, efficient opsin and indicator co-expression, and tailored illumination schemes. It further-
more demands concepts for system integration and a dedicated analysis pipeline for calcium transients in an
event-related manner. Here, firstly, we put forward a framework for the specific requirements for technical
system integration particularly focusing on temporal precision. Secondly, we devise a step-by-step guide for
the image analysis in the context of an all-optical physiology experiment. Starting with the raw image, we
present concepts for artifact avoidance, the extraction of fluorescence intensity traces on single-neuron
basis, the identification and binarization of putatively action-potential-related calcium transients, and finally
ensemble activity analysis.

Key words All-optical physiology, Functional calcium imaging, System integration, Optogenetics,
Event-related analysis of functional fluorescence traces, Stimulation artifact avoidance, Spectral
independency

1 Introduction

The advent of optogenetics, pioneered already more than a decade

ago [1, 2], has revolutionized our understanding of the contribu-
tion of individual, genetically defined neurons to circuit function.
For the first time, we could start to unravel the causal relation of a
distinct genetically defined compartment of the network to whole-
brain circuit dynamics and ultimately behavior. Optogenetics
impinged upon all fields of neuroscience. For each of those fields,
individual roadblocks needed to be overcome based on the princi-
ple of optogenetics, i.e., the illumination of a defined brain volume
with rather high light intensity exceeding 1 mW mm
2. In the field
of single-cell electrophysiology, the problem of the Becquerel effect
had to be solved by the design of specific electrodes, in the field of
behavior the problem of flexible and non-tethered light delivery
had to be solved. These obstacles could be removed already in the

Eirini Papagiakoumou (ed.), All-Optical Methods to Study Neuronal Function, Neuromethods, vol. 191,
https://doi.org/10.1007/978-1-0716-2764-8_5, © The Author(s) 2023

137
138 Hendrik Backhaus et al.

first years upon the advent of optogenetics [3]. Yet, there is one
field of neuroscience, which maybe poses the ultimate challenge for
the integration of optogenetics: optical functional imaging. The
field of optical imaging, particularly of individual neuron function
in the intact tissue, relies on detecting subtle changes in light
intensity [4]. The implementation of two-photon microscopy in
combination with calcium indicators in the early 2000 allowed for
the first time a functional readout of neuronal circuits comprising of
several hundred neurons in the rodent cortex [4–6]. This leads to a
tremendous advance in our understanding on how complex circuit
dysfunction arises, particularly also in the early stages of neurologi-
cal disorders [7–11]. The ability to identify a rather small fraction of
dysregulated neurons in the circuitry makes the true difference in
comparison to single-cell or population readouts. And of course,
also here, the implementation of optogenetics holds tremendous
potential, as a causal manipulation of, e.g., single dysregulated
neurons and the simultaneous readout of circuit function would
truly advance our understanding on cause and effect in network
disorders. And indeed, in 2012 [12, 13] the efficient two-photon
excitation of opsins became a reality, followed by combined
two-photon imaging and two-photon optogenetics [14–17]. In
these early proof of concept studies, it already became apparent,
that all-optical approaches pose unique challenges, both in terms of
system integration, experimental design, and not the least, analysis.
First, conventional line-scanning excitation schemes have proven to
be rather less efficient. Second, only a few opsins seem to be
two-photon excitable, and there is yet no molecular or structural
predictor, which could guide molecular engineering of opsins to
modify their two-photon cross section. Thirdly, as mentioned
above, the light intensity used for two-photon optogenetics ranges
orders of magnitude higher compared to the light intensities used
for the excitation of fluorophores, creating problems in terms of
indicator bleaching and tissue heating. Lastly, also the analysis poses
problems in terms of artifact removal and synchronization. All of
this prevented up to now the broad implementation of all-optical
approaches, despite their promises. Here we will focus on the entire
workflow for an all-optical experiment in circuit neuroscience,
reporting on recent advances, and giving guidance for the unique
requirements of all-optical physiology.

2 Experimental Framework

Circuit neuroimaging historically originated in invertebrate species,

and, alongside with the technique maturation and development,
has been introduced in the application of various model organisms,
such as Zebrafish, allowing, e.g., for the whole-body imaging of
transparent zebrafish larvae [18]. In recent years, optical imaging of
 All-Optical Physiology Pipeline 139

local circuits has been predominantly applied in mouse models.

This is on the one hand due to the availability of mouse models of
human disorders, and also due to the rather preserved cortical
cytoarchitecture in comparison to humans. The limitation of pene-
tration depth and of course the invasiveness of the method cur-
rently seems not to allow the implementation in human preclinical
research. Here, we focus on the implementation of all-optical
approaches in mouse cortex. Cortical networks are critically
involved in fundamental tasks of the brain, starting from sensory
processing [19–21], to decision making [22–24] and mechanisms
involved in consciousness [25, 26]. Optical neuroimaging using
genetically encoded calcium indicators [27] can resolve the activity
of local neuronal population even with single-action potential
(AP) resolution of sparsely firing neurons. Recent advances
[28, 29] even allow for the detection of cortex-wide activity of
thousands of neurons in real time. Certainly, despite these
advances, genetically encoded calcium indicators are inherently
slow, so single APs can only be resolved if the inter-AP interval is
sufficiently long, preventing the resolution of individual spikes,
e.g., of fast-spiking interneurons, such as PV interneurons
[27]. What is more, the frame rate of current commercially available
two-photon microscopes ranges at 30 Hz for full-field imaging, at
least for the majority of microscopes equipped with resonant scan-
ners [30]. Using random-access scanning, the temporal resolution
can be increased, but at the expense of a limited ability to do post-
hoc operations for movement correction. While inertia-free AOM
systems allow for a much higher framerate [31] (see also Chap. 3),
the current standard in the field is single-plane full-field imaging at
30 Hz, a good trade-off between speed, signal-to-noise ratio
(SNR), and field of view size. This has important implications for
the integration of optogenetics in an optical microcircuit imaging
framework. With an effective temporal resolution of 30 Hz, the
window of coincidence which can be resolved equals 33.3 ms. It is
therefore imperative to design a framework which avoids the loss of
even a single imaging frame. Here, with these limitations in mind,
we put forward concepts focusing on artifact avoidance or removal,
to retain the utmost temporal resolution. Even more so, for the
relation of a per se unspecific signal, i.e., the increase of fluorescence
intensity to an underlying AP, specific criteria need to be met in
terms of the temporal dynamics of an AP-related event. The entire
workflow of an optical functional imaging approach has to be
tailored, starting from the design of the hardware, to the experi-
ment itself, and the subsequent analysis. This chapter does not
strive for giving an overview of all possible solutions of the integra-
tion of optogenetics for all-optical experiments, but rather focuses
on a few tried-and-tested pipelines.
140 Hendrik Backhaus et al.

3 Technical Framework for Functional Neuronal Circuit Mapping

An optimal system integration needs to be tailored toward the

specific requirements for addressing the neurophysiological
research question. Investigating action-potential-related neuro-
physiological events mandates a precise temporal synchronization
of all acquired signals and interventions, in our case opsin excita-
tion, to ultimately gain evidence of cause and effect in the investi-
gated neural circuit.
Multichannel-data-acquisition (DAQ) interfaces can be
employed to collect signals from all involved subsystems. We
would strongly suggest to design a hierarchical configuration, in
which all subsystems, including the microscope, report the critical
signals, such as the frame trigger to this unifying and synchronizing
device. Commonly used manufacturers are National Instruments
(Austin, Texas, USA) or Cambridge Electronic Design (Cam-
bridge, UK) [32–34]. Integrating multichannel-data-acquisition
hardware in the microscope itself is also a viable solution offered
by many major microscope companies. Connecting this device with
a suitable integration software generates a master file for each
experiment, containing individual traces for all relevant signals,
such as breathing rate, temperature, frame trigger, and certainly
optogenetic or sensory stimulation pulses. Referring to the men-
tioned manufacturers, LABView is used for National Instruments
interfaces, whereas DAQ boards from Cambridge Electronic
Design use the software package Spike2. Particular care has to be
exerted in terms of gathering all relevant information important for
the current experiment in this master file. For example, the frame
trigger signal of a microscope can give insights at which absolute
time point images were acquired. For tactile stimulation, a
TTL-pulse can be generated by the DAQ boards itself, avoiding
varying time delays as occurring with using PC-based USB control.
Thereby, the experimenter gains full control and a complete report
of all relevant systems parameters without temporal jitter.

4 Somatic Calcium Influx as Correlate of Suprathreshold Neuronal Activity

4.1 Two-photon Functional calcium imaging utilizes the action-potential-related

Raster Scanning calcium influx into a cell as a correlate of neuronal activity [35–
37]. A genetically encoded calcium indicator (GECI) is undergoing
conformational changes upon binding of calcium ions, causing a
change in its fluorescence properties [38]. Currently, the most
commonly employed GECI is GCaMP6. It has to be mentioned
that there are different subtypes of GCaMP6: GCaMP6s (the “s”
stands for “slow”), exhibits a high SNR but slow off kinetics,
GCaMP6m (“intermediate”) exhibits medium, and GCaMP6f
 All-Optical Physiology Pipeline 141

(“fast”) fast kinetics [27] (see Note 1). We recommend using

GCaMP6f whenever possible, particularly for event-related analyses
with the aim of extracting action-potential-related calcium tran-
sient with utmost temporal resolution. The excitation spectrum of
GCaMP6f has its maximum at 497 nm [27] under a one-photon
excitation regime. However, the penetration depth using the prin-
ciple of one-photon excitation is limited, inter alia caused by light
scattering in the brain which requires a pinhole to avoid detection
of scattered light that does not derive from the focal point
[39, 40]. Furthermore, the deposited energy in the brain by
photons of this wavelength can lead to phototoxicity and bleaching
of the indicator [41]. The development of microscopes using the
principle of two-photon excitation combined with raster scanning
approaches [42] overcame these limitations and therefore revolu-
tionized functional neural circuit imaging [5]. Furthermore, pin-
hole solutions became dispensable since all emitted photons
necessarily originated from the fluorophores excited at the focal
point.
Fluorescence excitation by two-photon excitation requires two
photons being absorbed by the fluorophore within a time window
of less than 10
16 s [40], resulting in a tremendously low probabil-
ity for a two-photon excitation. Even though two-photon excita-
tion can be achieved by continuous-wave lasers [43, 44], the
development of mode-locked femtosecond pulsed lasers, like the
Titanium-Sapphire (TiSa) laser (Fig. 1a), enabled focusing light at
high-intensity spots and thus efficient two-photon excitation at the
focal point at moderate laser power in vivo [42]. Two-photon
excited fluorescence images can be acquired by employing raster
scanning of the focused laser beam (Fig. 1a), where the beam is
deflected by a galvo-resonant mirror system to the specimen cover-
ing the field of view (FOV) [5]. The dimensions of the FOV
depend on the chosen objective and its numerical aperture (NA).

A Source Delivery Image generation Detection

Two-photon Femtosecond Microscope Raster scanning, PMTs &
Microscope Laser objective galvo-resonant Bandpass filter

B
One-photon Gradient-index
LED & Gradient-index Full-field
Miniature lens &
Bandpass filter lens excitation
Microscope CCD Chip

Fig. 1 Principles of functional calcium imaging. (a) Scheme of two-photon-based readout using a femtosecond
laser and raster scanning principle. The laser is focused by the objective and the focal point is guided by a
galvo-resonant scanhead over the field of view. Emitted fluorescence is collected and detected by a PMT. (b)
One-photon Miniature Microscopes used for freely moving animals require a miniaturization of light sources,
achieved by bandpass filtered LEDs. A full-field illumination via a GRIN lens is carried out and a CCD-Chip
collects emitted fluorescence
142 Hendrik Backhaus et al.

The duration that the laser beam remains focused on a pixel is

defined as the pixel dwell time. For fluorescence excitation, a pixel
dwell time of 1 μs is commonly applied, resulting in frame rates of
30 Hz for galvo-resonant systems [9]. Note that this temporal
resolution is critical for the subsequent identification of
AP-related calcium transients based on their temporal dynamics.
The emitted fluorescence light is collected by the objective and
is deflected to a photo-multiplier tube (PMT). In front of the PMT,
a spectral bandpass filter ensures that only light in the bandwidth of
the fluorophore’s emission band is detected.

4.2 One-photon In the field of in vivo calcium imaging, a recently emerging method
Miniature Microscope are miniature microscopes, mounted on an implanted baseplate on
Full-Field Imaging via the head of the animal. In this chapter, we solely refer to
GRIN Lens one-photon miniature microscopes, whereas Chap. 7 gives a
detailed introduction to two-photon miniature microscopes.
Recently, the first three-photon miniature microscope was pro-
posed by the group of Jason Kerr [45]. Miniature microscopes
paved the way for the combination of functional imaging with
behavioral assays in which the animal moves freely. What is more,
brain regions not accessible by two-photon microscopy due to its
limited penetration depth can be targeted. First developed by the
group of Mark Schnitzer [46], nowadays several manufacturers
offer ready-to-use systems (INSCOPIX, Palo Alto, CA, USA
[47]) but also open-source systems are being provided by the
community, e.g., the UCLA Miniscope [48] or the
FinchScope [49].
Retaining the image information also in deeper brain regions is
afforded by a gradient-index optic (GRIN lens, Fig. 1b). GRIN
lenses have a cylindrical shape with a radially decreasing refraction
index, and planar surfaces for optimizing optic interfacing. These
two factors, a gradient of refractory index and the planar surface,
retain, at least to some extent, the image information throughout
the passage of light through the GRIN lens. It is important to note
that there is a deterioration of image quality with increasing the
length of the GRIN lens. As a light source, a LED in combination
with spectral bandpass filters is used [47], and the image is typically
recorded by a CCD chip. The sampling rates for functional calcium
imaging differ depending on the chosen model, ranging from
15 Hz with a field of view of 1440 by 1080 pixels (nVoke, Inscopix,
Palo Alto, California) up to 60 Hz at a field of view of 752 by
480 pixels (Miniscope-v4, UCLA, Los Angeles, California). The
following issue needs to be considered: the reliable recording of
neuronal layers depends on the location of the GRIN lenses tip,
neurons located 100–300 μm below the tip can be resolved by
electronically adjusting a focusing lens [47]. However, changing
the x-y-position of the recorded field of view is not possible and is
predefined by the implantation site of the GRIN lens.
 All-Optical Physiology Pipeline 143

5 The Principles of Optogenetic Manipulation Methods in a Nutshell

There is a vivid research in the field of molecular biophysics on the

development of new opsin classes paralleled by the improvement of
existing opsins by molecular engineering. The researcher needs to
decide which opsin is best suited for the question at hand, i.e.,
should a specific component of the network be silenced or stimu-
lated. Importantly, at this time, there are no a priori predictions on
the suitability of a given opsin for two-photon excitation. Here, we
cannot give a comprehensive guide on the specific requirements for
any specific opsin. We suggest caution on the use of newly devel-
oped opsins for non-experts in the field of optogenetics: Achieving
strong and stable expression of an opsin requires dedicated and
careful titration steps [33, 50, 51]. Also, it so happened more than
once, that a new opsin, while promising at first, showed rather
negative cytotoxic effects, requiring the development of new ver-
sions [52]. Consequently, if there are no critical requirements such
as kinetics or specific ion conductance, we would strongly suggest
to use tried-and-tested opsins, even if they might not be tailored to
the specific neuronal class. For example, if a neuronal subpopula-
tion is probed which exhibits an intrinsic firing rate exceeding the
optimal kinetics of a given opsin, it might still be advantageous to
choose this opsin. Remember, the opening time of an opsin is not
determined by the duration of the excitation pulse, but by the
opsins’ intrinsic dynamics, described by the parameter τoff, i.e.,
the time at which the probability of an open state is reduced to
1/e [50]. While this can result in not achieving to optogenetically
mimic the intrinsic firing frequency, it might increase the overall
probability of a successful all-optical experimental approach. Criti-
cally evaluate, which experimental aims are mandatory, and which
parameters of the experimental design can be adapted. This is of
particular importance for an all-optical experiment, requiring the
optimal expression of indicator and opsin, and all the other techni-
cal requirements stated above and below. Spending too much time
on individual components of this chain might reduce the chance of
completing the overall aims of the given study in the inherently
limited time frame. It is already quite complex to set up an
all-optical experimental framework, and we suggest to limit the
complexity whenever possible.

5.1 One-photon For one-photon optogenetic interrogation, typically a solid-state

Raster Scan Opsin laser at the optimal excitation wavelength for the respective opsin
Excitation should be employed [50, 53]. Opsins successfully used for the
manipulation of neurons in a one-photon regime include the vet-
eran ChR2 (134) for AP initiation [2, 54] and ArchT for inhibition
[2, 51]. For these tried-and-tested opsin pairs, two solid-state lasers
can be employed, with 488 nm for ChR2 and 552 nm for ArchT,
144 Hendrik Backhaus et al.

A Delivery Pattern generation

Microscope Raster scanning,

objective galvo-galvo
(one-photon) scanhead

Microscope Raster scanning,

Two-photon
objective galvo-galvo
Microscope
(two-photon) scanhead

Microscope Computer Generated

objective Holography,
(two-photon) SLM

B
One-photon Gradient-index
Full-field
Miniature lens
excitation
Microscope (one-photon)

Fig. 2 Schematic overview of system combinations for opsin excitation. (a) Two-photon functional calcium
imaging can be combined with one-photon or two-photon raster scan methods to target opsin-expressing
neurons. The method of Computer Generated Holography is a scanless approach and enables the experi-
menter to excite several neurons at the same time under a two-photon regime using spatial light modulators
(SLMs). (b) Miniature microscopes equipped with a second LED for opsin excitation are based on one-photon
full-field illumination delivered via an implanted GRIN lens

provided that the light intensity at the excitation site is

1 mW mm
2 [55]. The one-photon light source is coupled to
the microscopes beam path via optic fibers. The region of interest is
raster-scanned typically by a galvo-galvo scanning head (Fig. 2a).
To avoid the detection of the laser pulse for opsin excitation, a
notch filter is blocking the excitation wavelength from entering the
PMTs. Note that there might be a problem in terms of the drastic
increase of autofluorescence during the one-photon excitation
pulse, in the wavelength bandwidth of the signal of interest, here
of GCaMP, leading to a stimulation artifact. Alternatively, if neces-
sary, the PMTs can be blanked by a shutter during opsin stimula-
tion, which results in a sacrifice of signal detection during
stimulation. This might pose problems for subsequent trace analy-
sis, as particularly detecting the stereotypical onset dynamics of an
AP-related calcium transients is vital for ensuring specificity. Apart
from focusing on a single neuron, this approach can also be used to
illuminate a section of or the whole field of view to excite a larger
subset of opsin-expressing neurons. However, the downside of this
approach is that not only the layer recorded in the field of view gets
excited, but also neurons located above or below the recorded layer
can get excited as long as they are exposed to a light intensity that
exceeds 1 mW mm
2, as depicted in Fig. 3a.
 All-Optical Physiology Pipeline 145

Fig. 3 Mechanisms for opsin excitation. (a) One-photon excitation by a focused beam is carried out by raster
scanning the target neurons. Neurons located above or below the imaging plane are exposed to the excitation
beam cone as well. (b) One-photon full-field paradigms are used in miniature microscopes and do not allow
for neuron-specific excitation but illuminate the entire FOV at once, eventually exciting neurons below the
recorded layers. (c) Two-photon raster scanning approaches overcome this disadvantage due to the physical
principle of two-photon excitation, reaching the necessary photon density only in the focal spot. (d) The
scanless paradigm also enables experimenters to simultaneously excite several target neurons under a
two-photon regime

5.2 Two-photon Utilizing the same physical mechanism like in functional

Raster Scan Opsin two-photon calcium imaging, two photons of roughly the double
Excitation wavelength of the optimal one-photon excitation can evoke an
activation, i.e., conformational change of an opsin. It has to be
noted that there cannot be an a priori assessment on the suitability
146 Hendrik Backhaus et al.

of a given opsin for two-photon excitation regimes (see Note 2).

One of the first opsins probed for two-photon excitation represents
C1V1, typically excited at around 1080 nm [12, 13]. Yet, excitation
beyond the standard range between 1030 and 1100 nm of Ytter-
bium lasers [56] is possible. We explored wavelengths of up to
1250 nm using an Optical Parametric Oscillator (OPO) as light
source, being able to decrease the spectral cross-talk [34]. There is a
wide range of possible light sources for two-photon optogenetic
raster scan-based excitation: A tunable Ti:Sa laser is well suited for
wavelengths up to 1080 nm. However, the femtosecond pulse
dynamics of a two-photon imaging laser is not as critical for opsin
activation as it is critical for imaging, since it is not necessary to
obtain the shortest pixel dwell time possible. Therefore, fixed
wavelength Ytterbium lasers with lower pulse repetition rates
became the most widely applied light source [57]. Since opsin
activation is based on conformational changes of the protein
initiated by an isomerization from all-trans to 13-cis retinal upon
photon absorption, the physical principle differs from functional
calcium imaging with fluorophores, i.e., GECIs. Therefore, the
pixel dwell time is, besides the chosen objectives point-spread
function (PSF) and laser intensity, a crucial factor. Studies
suggest a pixel dwell time of at least 3.2 μs for efficient excitation
[13, 33, 58].
The raster scan method enables the experimenter to define
several target neurons that are scanned successively in the same
plane (Figs. 2a and 3c). Depending on the chosen line spacing,
this can lead to rather long excitation times of up to 100 ms. Please
note that there is an ideal line spacing value, to cover the entire
neuronal membrane. The minimal excitation volume of a micro-
scope depends on the theoretically reachable optical resolution,
defined by the full width at half maximum (FWHM) of the PSF,
and can be estimated by the magnification of the objective and the
chosen wavelength. The reachable optical resolution can be calcu-
lated with Δr  2∙pffiffi2λ∙NA [59]. With a given wavelength of
λ ¼ 1100 nm and a NA ¼ 1.0, an average neuron surface of
20  20 μm and a pixel dwell time of 6 μs, it takes 16 ms to cover
the entire neuron. For spiral excitation patterns, a similar, yet
slightly shorter stimulation time is needed, ranging at 12.5 ms per
neuron. If the experimenter aims to excite successively 6 neurons,
the excitation process itself takes 96 ms even without consideration
of the time required to move from target to target. These technical
roadblocks limit the effective use of a raster scanning-based
approach, at least if the entire membrane of the neuron needs to
be excited for AP-inducing depolarization or AP-suppressing
hyperpolarization, respectively. The development of opsins with
either a higher ion conductance or more efficient two-photon
excitability might reduce the membrane area which needs to be
excited, and consequently decrease the stimulation time.
 All-Optical Physiology Pipeline 147

5.3 Two-photon As described in Subsection 5.2, if more than two neurons need to
Scanless Opsin be efficiently activated within the time window of a typical imaging
Excitation via CGH frame, a two-photon raster scanning approach is reaching its limits.
Parallel excitation methods in principle allow for an infinite number
of simultaneously activated regions of interest (ROIs), limited only
by the laser power and the spatial resolution afforded by the spatial
light modulator used [59]. Parallel methods most commonly use
Computer Generated Holography (CGH) for the generation of the
excitation pattern [60] (Fig. 2a). In CGH, liquid crystal spatial
light modulators (LC-SLMs) are integrated into the beam path to
modulate the phase of the electric field of the laser beam, and
typically a low-repetition rate, high-energy Ytterbium laser is used
as a light source [57]. A precise control of the spatial specificity of
the calculated phase hologram is achieved by applying a Fourier
transform-based iterative algorithm on previously acquired fluores-
cence images of the region of interest [61]. A detailed description
of the technical aspects of this method can be found in Chaps. 4
and 11.
With this approach, an effective, simultaneous excitation of
several neurons is amenable. The illumination targets do not need
to be located in the same z plane, neurons above or below the
imaging plane can be interrogated as well, provided that the loca-
tion of all targets is known (Fig. 3d). Depending on the respective
microscope and illumination concept, neurons can be excited
which are located several hundreds of μm distant from the current
z plane. Yet, upgrading an existing microscope setup with this
technique can be cumbersome and expensive, as major changes in
software and hardware need to be done. However, it might be an
interesting option for scientists experienced in two-photon micros-
copy to upgrade a microscope with this technology. A further
interesting option represent hybrid solutions, combining CGH
and scanning [12]. These hybrid solutions guide the illumination
patterns generated by the SLM on a galvanometric mirror. These
patterns comprise multiple typically rather small focal points, but
with high light intensity. This cloud of focal points is then scanned
by the galvanometric mirror, resulting in each focal point to cover
an entire given neuron either using line or spiral scanning
approaches. Thereby, a limitation of CGH-only approaches in
terms of the ever-decreasing light intensity when increasing the
number of neuron-sized patterns is circumvented. For a constant
laser power, the number of neurons which can be effectively acti-
vated by a two-photon regime, as discussed in the previous section,
and in [12] is consequently higher with hybrid solutions (see also
Chap. 11).
148 Hendrik Backhaus et al.

5.4 One-photon Recent one-photon miniature microscopes are capable of opsin

Miniature Microscopes excitation via an implanted GRIN lens. Typically, for optogenetics,
with GRIN Lenses a second LED light source equipped with corresponding bandpass
filters is used in these systems. The excitation paradigm is based on
one-photon excitation (Fig. 2b).
A clear advantage of using GRIN-lens-based solutions is the
ability to specifically target deep structure for optogenetic interro-
gation, without undesired activation, e.g., of passing opsin-
expressing neurons or neurites dorsal of the target region. The
ventral regions below the GRIN lens are illuminated by the light
emitted by the lens, in the geometrical shape of a frustum (Fig. 3b).
An estimation of the effective penetration depth, i.e., exceeding the
threshold for opsin excitation, can be achieved using the Kubelka-
Munk model of light transmission through diffuse scattering media
[55, 62, 63]. The light intensity is decreasing by the distance to the
lens surface, estimated by:
I ðz Þ ¼ I 0 T ðz ÞG ðz Þ,
where I0 is the light intensity at the tip of the lens, T(z) the
transmission factor according to the Kubelka-Munk model for
diffuse scattering media, and G(z) is a geometry factor. The trans-
mission factor is defined as:
1
T ðz Þ ¼ ,
Sz þ 1
with S ¼ 11.2 mm
1, the damping constant in mice [55]. As light is
spreading in a conical shape from the tip of the  lens, the angle of
divergence is calculated by θdiv ¼ sin
1 NA n , where NA is the
numerical aperture of the GRIN lens and n ¼ 1.36, the refraction
index of gray matter in the brain [64]. Using the definition ρ ¼
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
 n 2 ffi
r NA
1 , the geometric component, the conical light spread
can be calculated as:
ρ2
G ðz Þ ¼ :
ðz þ ρÞ2
Therefore, the intensity depending on the depth is defined as:
I 0 ρ2
I ðz Þ ¼ :
ðSz þ 1Þðz þ ρÞ2
For a GRIN lens with a diameter of 200 μm, a numerical
aperture (NA) of 0.5, a light intensity of 35 mW mm
2 at the tip
of the lens, and an excitation wavelength of 630 nm, the effective
penetration depth for opsin excitation assuming a minimal thresh-
old for excitation of 1 mW mm
2 is 650 μm with a geometric loss of
0.08, resulting in a total volume of 0.118 mm3 with the Kubelka-
 All-Optical Physiology Pipeline 149

Munk model. Note that the lateral spread close to the tip of the
fiber may be underestimated by the Kubelka-Munk model [54, 62],
at least for highly scattering shorter wavelengths.

6 Everything You Always Wanted to Know About All-Optical Data Processing But
Were Afraid to Ask

Independently of the chosen technical implementation, the goal of

any pipeline for data processing is to extract a subset of features
from the raw data, either based on a priori hypotheses, or by data-
driven unsupervised methods. Here, we present a pipeline based on
the idea of an event-related analysis: We put forward the notion that
any functional circuit imaging of a neuronal ensemble at the end
only serves as a tool to extract the underlying action potential train
of each individual neuron. Consequently, we provide a step-by-step
guide to extract putatively AP-related calcium transients from the
raw images. Once this optically derived matrix of AP-related events
is generated, e.g., by transforming an extracted intensity trace to a
binarized train of zeros and ones, already well-established correla-
tion or interaction concepts can be employed on these
dimensionality-reduced datasets.
All-optical experiments require a tailored pipeline for each
technical implementation and mandate the avoidance of false-
positive identification of putatively AP-related calcium transients.

6.1 Roadmap for Prior to image data acquisition, the synchronicity of all necessary
Processing All-Optical subsystems needs to be guaranteed (Fig. 4a). This can be achieved
Data by copying all relevant trigger and stimulation pulses to a multidata
acquisition interface. These signals include the frame trigger of the
6.1.1 System Integration microscope, the logic level controlling the light source for opsin
excitation, and biomonitoring such as the breathing rate and body
temperature. Upon digitalization, the signals are being centrally
displayed by a control software, and a master file for each experi-
ment is generated. Reading out the master files allows for a post hoc
exact assignment of each raw image to a given time and the status
of, e.g., the stimulation regime.

6.1.2 Image Data The initial step for analyzing the data is to reduce the impact of
Acquisition movement artifacts (Fig. 4b). We differentiate between two types of
artifacts: displacements induced by movement in the x-y plane, and
in z plane. While changes in the x-y plane can be corrected retro-
spectively by algorithms based on Hidden-Markov-Model [65] or
discrete Fourier transformation-based image alignment [66],
provided that the field of view is sufficiently large, movements in
the z plane are more complicated to correct. Note that the possibil-
ity for x-y movement correction represents the main reason why
random-access scanning is not recommended, and the field is
150 Hendrik Backhaus et al.

Fig. 4 Workflow of a pipeline for an event-related analysis of all-optical data. Raw data is segmented into ROIs
and intensity traces are extracted. A binarization of transients is carried out and a temporal and spatial
classification of the recorded microcircuit is applied

currently mainly conducting single-plane resonant imaging. Simply

put, if a soma of a given neuron is displaced below the current x-y
imaging plane, this information cannot be regained. A potential
solution for this problem is to record a 3D volume. However, this
results inevitably in a reduction of temporal resolution for any given
neuron recorded. If working with currently most used resonant
systems with a frame rate of 30 Hz, we would strongly discourage
recording 3D volumes, as the temporal resolution is critical for
subsequent identification of putatively AP-related calcium transi-
ents. This is a prime example on how analysis needs should inform
the previous technical implementation steps: Take all efforts to
minimize movement by devising mechanical stability, this cannot
be stressed enough (see Note 3).
 All-Optical Physiology Pipeline 151

6.1.3 Segmentation The raw images comprising typically 512  512 pixels contain
several biological compartments: neuronal somata, axons, den-
drites, blood vessels, and certainly a multitude of non-neuronal
cells, such as astrocytes. While the one critical advantage of micros-
copy using fluorescent indicators represents the reduction of image
complexity, i.e., ideally only the features of interest express the
fluorophore, still, only a fraction of the image contains the signal
of interest. What is more, in the context of neuronal microcircuit
imaging, it is of advantage to integrate several pixels which reflect
the same functional compartment: when the experimenter aims for
identification of the suprathreshold activity of individual neurons in
the microcircuit, each neuron can be identified as functional unit.
Consequently, each neuronal soma is defined and segmented as
ROI (Fig. 4c). In recent years, the range of applications that sup-
port the experimenter in ROI segmentation grew rapidly: mathe-
matical models to perform automatic segmentation based on deep
learning algorithms drastically shorten the time-consuming step to
manually identify neurons in the recorded images [67]. While there
are deep learning-based methods that process the average of all
images, other techniques integrate the temporal information of
neuronal activity by using subsets of all images for the segmentation
of active ROIs [67, 68]. The approach proposed by Soltanian-
Zadeh et al. is based on a 3D technique: subsets of the acquired
images are created and used to predict 2D probability maps for
active neurons utilizing a neural network. Upon applying a thresh-
old to exclude low-probability regions from each probability map,
individual somata are extracted from high-probability regions
[67]. Ultimately, all somata positions from each image from the
image sequence are combined to acquire a final output of active
somata areas. However, manual segmentation of functional calcium
imaging data by marking neuronal somata with polygon shapes still
prevails due to the applicability on datasets of varying quality, e.g.,
datasets of a particularly low signal-to-noise ratio, when deep
learning algorithms reach their limits. Depending on the strength
of GCaMP6 expression and the overall SNR ratio, if targeting
somatic changes in calcium concentrations, a decontamination of
the ROIs containing the neuronal soma from neuropil signal might
be useful. The decontamination is carried out by expanding a given
ROI in cardinal and diagonal directions, beyond the region of the
soma, which are then separated into several neuropil regions sur-
rounding the initial ROI. The size of each neuropil subregion
should equal the size of the initial ROI. Under the assumption
that the intensity trace of a given ROI is generated by a mixture of
different underlying signal components, but certainly one of the
components exhibits the somatic signal of interest, non-negative
matrix factorization or independent component analysis are
employed to perform a blind source separation, resulting in a
separation into the underlying signal components. By weighting
152 Hendrik Backhaus et al.

the presence of each component in the central ROI, under the

assumption that the somatic signal is the prevailing component
contributing to the ROIs intensity trace, the strongest signal com-
ponent is defined as the somatic signal [69].

6.1.4 Trace Extraction After the locations of somata constituting a ROI have been identi-
fied within the image sequence, the signal over time of each ROI is
extracted (Fig. 4d). The intensity values of all pixels in a given ROI
are averaged for every image of the temporal sequence, resulting in
an intensity trace for every ROI, with a temporal resolution deter-
mined by the frame rate. It has to be noted that the absolute level of
intensity is based on multiple factors such as autofluorescence, the
expression levels of the GECI, or stray light entering the objective.
Therefore, it is recommended to calculate the relative change of
fluorescence, as these dynamic changes, depending on their tem-
poral dynamics, are most likely due to changes in intracellular
calcium levels, and can therefore be termed calcium transients.
Note that there is inevitably a drift of the baseline levels due to
bleaching. These drifts can be compensated for, as long as linear
operations are being used (see Note 4). There is no convention on
how to perform baseline correction. An option used by us and
others represents the definition of a sufficiently long period of
quiescence, i.e., stable baseline non-interrupted by any transients,
and defines this period as baseline (F0), separately for each neuron
[7, 9]. The relative change of fluorescence is then calculated by
relating the intensity of each time point (F) to the baseline fluores-
cence ΔF ¼ F
FF 0 [70].
0

6.1.5 Artifact Removal Prior to binarizing the extracted intensity traces, potential photo-
stimulation artifacts superimposing the signal need to be identified
and, if possible, corrected (Fig. 4e). Characteristic properties of
photostimulation artifacts can serve as a basis for reliable identifica-
tion and subsequent correction. Firstly, looking only at individual
responses to an optogenetic stimulus might lead to the notion of a
physiological signal. Yet maybe the most decisive difference
between an artifact and a physiological response is the inherent
variability of the physiological signal. Artifacts, with rare excep-
tions, are rather consistent. Overlaying and averaging the individual
responses therefore gives important cues on the probability of a
physiological origin. For that, the temporal section of a trace
upon the photostimulation is assessed by a nm matrix M, with
n ¼ stimulus intervall, m ¼ total frames/n. Figure 5b shows an
overlay of the intensity traces of all photostimulation periods of
Fig. 5a (gray lines).
Photostimulation artifacts exhibit several typical features:
Firstly, a photostimulation artifact will show both a sharp onset,
and a sharp offset. Functional calcium transients of physiological
 All-Optical Physiology Pipeline 153

Fig. 5 Evaluation of photostimulation artifacts. (a) Artifacts caused by photostimulation do not represent a
physiological signal. Onsets of photostimulation are indicated by black triangles. (b) By overlaying (gray) and
averaging (red) periods of photostimulation, an estimation on the impact of the artifact can be made. Here,
100 sample points prior to and 200 sample points after the photostimulation onset, indicated by the black
triangle, are considered. The averaged signal waveform can be used in an algorithm to minimize the
photostimulation artifact. (c) Subtracting the averaged waveform depicted in (b) at each stimulation onset
can reduce the intensity of the photostimulation artifact. Note that signal components containing the
physiological signal of interest can be altered by the algorithm as well and, in the worst-case scenario, will
be completely eliminated

origin might also display a rather sharp onset, due to the high
affinity of calcium to the indicator represented by a small dissocia-
tion constant KD and the high temporal gradient of calcium influx.
But it will be characterized by a slow decay, an inevitable conse-
quence of the high affinity of the indicator to calcium. The off
kinetics does not mirror the true time course of the decrease of
somatic calcium concentration. If a researcher would be interested
in assessing the re-uptake of calcium, an indicator with a high KD/
low affinity would be advantageous, but these indicators typically
display a lower SNR [71]. Any trace deflection with a sharp decay
when using a low KD indicator is therefore almost certainly not
associated with an AP-related somatic calcium response. Secondly,
if the duration of the intensity deflection equals the duration of
light administration, a physiological origin is highly unlikely. As
depicted in Fig. 5b, the intensity traces during photostimulation
exhibit both of the mentioned features.
What is more, but this very much depends on the imaging
setup used, the latency between the onset of the stimulation pulse
and the putative response is critical: If the latency ranges <3 ms, it
cannot be considered as a physiological response, due to the time
needed for the AP initiation and the influx of calcium into the
cytosol. Please note that this criterion can only be used if the
sampling rate of the GCaMP emission channel is high enough to
resolve durations less than 3 ms. Lastly, a physiological response
might be subject to adaptation, and often times does not occur
upon every stimulus, e.g., due to changes in local inhibition
[54]. Consequently, while a signal deflection might surely be the
result of a physiological response if it occurs upon each stimulus,
nonetheless, if a response is drastically changing its amplitude, or
sometimes is not present, it is likely to be of physiological origin. To
154 Hendrik Backhaus et al.

test for that, it might be useful to modulate the light intensity of the
optogenetic stimulus: if a decrease of stimulation leads to the
sudden disappearance of a transient, it is again likely that an
AP-related origin can be assumed, as a technical artifact would
simply scale with the light intensity, even though also non-linear
correlations can occur. Certainly, a control experiment comprising
of indicator-only expressing cells should be conducted in any case.
The subsequent correction of photostimulation artifacts in the
intensity trace is a delicate task that needs to be conducted carefully.
A possible approach is to employ non-negative matrix factorization
[72] to identify the background noise of a given ROI containing
the stimulation artifact. In a second step, the identified noise com-
ponent is subtracted from the ROIs signal [73]. Here, for illustra-
tion purposes we obtained the averaged intensity trace of all time
periods of photostimulation (Fig. 5b) and subtracted the given
value from the raw intensity trace (Fig. 5a). This results in an
intensity trace devoid of at least the majority of the
non-physiological signal sources (Fig. 5c). Please note that the
processed intensity trace has to be treated with caution: we can
still observe fluctuations in the signal that could be misinterpreted
as functional calcium transients. Furthermore, using methods
based on complex mathematical models often act as a black-box,
making it difficult to grasp the underlying methods for
non-experts. We strongly emphasize to design the experimental
paradigm in a manner to avoid any stimulation artifact during the
measurement and to minimize the need for post-processing of raw
data as much as possible. Characteristic temporal dynamics and
latencies of the used GECI, as put forward before, can serve as a
sanity check, and, following up with an event-based identification
of AP-related calcium transients is mandatory.

6.1.6 Event-Related Action potential-related calcium transients exhibit typical signal

Binarization dynamics and waveforms. Methodologically, the rather stereotypi-
cal dynamics of the somatic calcium influx and re-uptake respec-
tively efflux is convolved with the binding dynamics of the
fluorescent calcium indicator [74]. Therefore, each binarization
approach critically has to be tailored to the calcium indicator
used. The resulting AP-related calcium transient can be described
based on parameters such as rise time, decay, and duration, or,
alternatively, an idealized transient is used for performing deconvo-
lution approaches [75–77]. A rather straightforward but yet effec-
tive method, with rather low false positives [7, 9, 34] represents a
threshold-based algorithm. For that, first, the standard deviation of
the noise band of the trace is determined, ideally in a section of the
trace not containing the signal of interest. In the case of an
all-optical experiment, time periods outside of stimulation events
and with low spontaneous activity should be used. Once the signal
 All-Optical Physiology Pipeline 155

exceeds this threshold, and if additional criteria such as minimal

duration are met, a putative event is detected. Furthermore, as the
decay of an AP-related calcium transient typically follows a roughly
exponential decay, at least for commonly used indicators such as
GCaMP6 and OGB-1, a regression of the trace with an exponential
function can be conducted. The goodness of fit for this regression
gives additional evidence for the physiological nature of the given
transient. By identifying onset, peak, and offset for each detected
transient, we can represent the fluorescence signal as a binarized
event array (see Notes 5 and 6). Note that also deconvolution-
based methods provide such a binarized array. All following ana-
lyses are carried out on these reduced datasets (Fig. 4f).

6.1.7 Connectivity The coherent activity of neurons of a given microcircuit or ensem-

Analysis ble in the field of view is calculated and visualized based on two
variables for each neuron: first, the window of co-incidence, in
which two events are considered to occur simultaneously, has to
be defined (Fig. 4g).
Certainly, for achieving information on Granger causality [78],
this coincidence window should be as short as possible, ranging at
only a few milliseconds. Yet, in the field of imaging, the minimal
coincidence window cannot fall below the imaging frame rate, for
resonant scanning approach this ranges at 33.3 ms, and it might
even be necessary to further lengthen this co-incidence window,
particularly in scan-based imaging methods. Otherwise, coinci-
dence would be biased by the individual location of a given neuron
in the scan field. Within this given coincidence interval, at least one
other neuron needs to exhibit an event. Second, the average num-
ber of simultaneously active neurons is determined. The binarized
calcium transients of each neuron i are used to identify the number
of active neurons per frame by introducing a counter ci, registering
the coincident active time points, and an array Ai, storing the
number of coincident active neurons for each time point. Now,
we ask for each active time point j if the sum of active neurons is nact,
i > 1. If this is the case, the counter ci is incremented by 1 and Ai is
appended by nact, i
1. Afterward, ci is divided by the total number
of active time points defined by Xi, representing the relative num-
ber of coincident active time points of each neuron i and the mean
of Ai gives insights into the average number of coincident active
neurons.
The number of simultaneously active time points ci relative to
the number of all active frames per neuron ci/Xi are plotted against
the overall number of active frames of the respective neuron Xi.
Additionally, a linear regression analysis is applied to the data.
The average number of simultaneously active neurons can be
visualized using box plots. The distribution of the average number
of coherent active neurons are compared by a statistical test, Stu-
dents t-test, or Wilcoxon rank sum test.
156 Hendrik Backhaus et al.

To gain insights into the connectivity pattern of the given

microcircuit, the information about the position of a ROI, its
activity, and the correlation between each pair of ROIs, a network
graph can be constructed. Each ROI is represented by one vertex.
The position of each vertex is given by the ROIs x and y position.
The color is based on the ROIs activity. In the example given in
Fig. 4, the color scale is constructed as a non-linear gradient
between green, yellow, and red in order to show single ROIs as
hypoactive (green), normal (yellow), or hyperactive (red). The
pairwise Pearson’s correlation between each pair of ROIs is repre-
sented by the width of the edges.
Together, such a connectivity analysis takes advantage of the
spatial information obtained by an imaging experiment, which at
least partially overcomes the inherent drastic limitations in terms of
temporal resolution compared to methods such as single-cell patch-
clamp recordings. Only both methods together, high temporal
resolution of electrophysiological methods, directly acquiring the
signal of interest, and functional imaging obtaining the local func-
tional architecture, can provide a holistic understanding of neuro-
nal network (dys)function.

6.2 Toward Cross- While the strong light intensities needed for an effective excitation
Talk-Free of an opsin pose the aforementioned problems for simultaneous
Experimental Designs functional calcium imaging, we need to also consider a putative
impact on the constant excitation of the calcium indicator in terms
6.2.1 Assessing the of unwanted activation of opsins (see also Chap. 2). The activation
Impact of Continuous of an opsin requires a distinct quantal energy; in case of the ChR2-
Illumination for Calcium
based opsins or the cis-trans isomerization of retinal, this threshold
Imaging on Opsin ranges from 0.1 to 1 mW mm
2. All-optical one-photon experi-
Excitation ments, even using the same wavelength for imaging and opsin
activation, is therefore possible, as long as the excitation intensity
ranges below this threshold [32, 47, 54]. However, for two-photon
line scanning conditions, the effective light intensity per pixel can
well exceed this barrier. Nevertheless, fortunately, there is also a
temporal barrier. It so seems that any pixel dwell times below 3.2 μs
may not suffice for efficient opsin excitation at least for opsins such
as C1V1 [12]. Pixel dwell times of regular resonant scanning range
well below that number. However, novel generations of opsins
designed for more efficient two-photon excitation might allow
shorter pixel dwell times.

6.2.2 Increasing the Avoiding cross-talk in the GECI emission channel is an important
Spectral Separation criterion in the design of all-optical experiments. Both for
Between Opsin and one-photon and two-photon regimes, the excitation light can be
Indicator to Minimize rather easily prevented from entering the PMTs, e.g., by notch
Optogenetic Stimulation filters. But nonetheless, the strong light pulse might lead to a
Artifacts on the Imaging broadband increase in autofluorescence, also in the emission band
Data of the fluorescence calcium indicator, leading to a decrease in SNR.
 All-Optical Physiology Pipeline 157

While in principle, the utmost spectral separation of opsin and

indicator excitation wavelengths is advisable, there might be good
reasons to choose an opsin indicator pair with similar or even
identical excitation wavelengths, if, e.g., both indicator and opsin
are best suited for the scientific question at hand, as mentioned
above. Post-hoc identification of photostimulation artifacts is man-
ifold and should be conducted with utmost care. Common
approaches compare the dynamics of the signal’s intensity during
photostimulation to the expected dynamics of calcium transients
[17] to make an assumption of the presence of a physiological
response (see also Subheading 6.1.5). As a consequence, the speci-
ficity of the analysis toward the detection of AP-related calcium
transients is reduced. Other approaches sacrifice pixels that are
considered to contain signal components that represent an artifact
[79, 80] at the cost of a reduction of the field of view. Another
option, if a spectral separation of excitation and emission wave-
lengths cannot be implemented, is to perform gating of light
sources, inevitably leading to a loss of data [81]. Yet, it is possible
to avoid any stimulation artifact altogether, which has to be tailored
for each opsin/GECI pair (see Note 7). For the pair GCaMP6/
C1V1, artifact-free all-optical physiology experiments are possible
if using excitation wavelengths at or exceeding 1100 nm while
simultaneously imaging GCaMP6 fluorescence at 920 nm
[34]. Employing a blue-shifted opsin and a red-shifted calcium
indicator resents another viable option [58].

7 Outlook

All-optical physiology in neuroscience, i.e., simultaneously record-

ing and manipulating individual functional components of a given
microcircuit, opens up truly new experimental designs [80]. Partic-
ularly in the field of research on preclinical rodent models of neu-
rological disorders, these “dream” experiments can provide
evidence for the impact of the (dys)function of individual neurons,
and neuronal ensemble activity to network performance and behav-
ior. Yet, all-optical physiology did not at all exploit its full potential
yet. The reasons are manifold: First, the ideal opsin for efficient
induction of action potentials, mimicking the temporal dynamics of
physiological inputs, while tremendous progress has been made, is
not available yet. While efficient two-photon excitation seems to
have almost achieved this goal, particularly the efficient two-
photon-based inhibition is still in its infancy [82]. Nevertheless,
the opsin and the stimulation method is only a part of the entire
workflow. We need to move beyond rather synthetic,
non-physiological stimulation patterns, which often evoke
non-physiological responses, e.g., due to hypersynchronization of
158 Hendrik Backhaus et al.

the network caused by the simultaneous activation of multiple

neurons. For that, we not only need to record the neuronal activity
but also analyze the activity pattern in real time. Only then, we will
be able to re-play and meaningfully modify the endogenous ensem-
ble activity, closing the loop. In this chapter, we covered key aspects
of system integration and the basic concept of analysis of all-optical
functional data. Indeed, in our view, the entire workflow, from the
acquisition of raw images to the identification and binarization of
events, and the design of the stimulation pattern, has to be trans-
formed toward real-time closed-loop performance [83], as put
forward in the context of global functional brain state changes
[84]. This requires first of all dedicated fast hardware. Each com-
ponent of the system: microscope, optogenetic pattern generator,
signal acquisition, and signal computation, has to operate on real
time. This is in reach for the microscope, optogenetic pattern
generator, and signal acquisition, as put forward in this chapter,
but, is still far from achievable for an event-based signal processing
and signal analysis in real time. Achieving real-time capability of the
critical analysis step requires the close collaboration between neu-
roscientists, mathematicians, and (bio-)informaticians. First, the
neurophysiological event of interest has to be identified and
described in painstaking detail. For instance, in our own research
interest involving state transitions of local populations, these events
needed to be discerned from the large parameter space of neuro-
physiological events, and described in terms of their spatio-
temporal dynamics and its variance. Only then, the mathematicians
can either take advantage of statistical methods, or of new unsuper-
vised machine learning algorithms. Indeed, the advent of artificial
intelligence may enable to accelerate at least key aspects of analysis
routines which traditionally take weeks and months, to seconds or
even milliseconds [67, 85, 86].
The fast-evolving field of all-optical physiology of neuronal
microcircuits can only thrive in a multi-disciplinary environment
and is critically dependent on each component to be optimized and
ideally integrated. Yet, now is the time, now all individual advances
can come together to make real-time closed-loop all-optical physi-
ology a reality.

8 Notes

1. Choice of indicators: Adapt the kinetics of your indicator to the

respective firing properties of the neuronal populations of
interest: i.e., in rather fast-spiking neurons, indicators with a
slow off kinetics such as GCaMP6s would not be advisable,
but, in cases of deeper structures with low SNR, slow indicators
might be the better choice.
 All-Optical Physiology Pipeline 159

2. Testing expression levels for novel experiments: We suggest testing

the functionality of opsins in in vivo experiments by initially
exciting the microcircuit by one-photon stimulation. Since the
success rate of opsin excitation is generally higher under
one-photon regimes, conclusions regarding the level of the
expression can be drawn easily before a more error-prone
approach based on two-photon excitation is used. If no activa-
tion upon opsin excitation is detected, a sanity check if the cell
is expressing the opsin can be carried out by combining the
opsin with a fluorescent marker with different spectral proper-
ties than the used GECI. For example, mCherry or tDtomato
can be used in combination with an opsin while functional
calcium imaging is carried out with GCamP6. If a sanity
check using optical methods is not possible, electrophysiologi-
cal recordings can be used as an alternative.
3. Mechanical Stability: Since movement-induced distortions in
z-direction cannot be corrected post hoc, it is of utmost impor-
tance to ensure mechanical stability of the integrated and
mechanically connected unit comprising of the brain as target
structure, the cranium, and the holder. The head holder pro-
vides the mechanical connection to the stage and represents the
most critical component. Here, one-point fixations on only one
hemisphere of the skull, often designed as a metal bar
connected to the skull by dental cement or UV-glue, are not
providing sufficient stability in our view, at least a two-point
fixation is considered as mandatory. We strongly suggest to
maximize the adhesive surface between the fixation device
and skull and to attach it to both cranial hemispheres.
4. Avoid non-linear bleaching: Typically, in the first few seconds of
data acquisition, the intensity trace displays a rather non-linear,
exponential decrease which cannot be compensated for. Any
non-linear manipulations are highly problematic, as they affect
the dynamics of the calcium transients and therefore strictly
speaking manipulate the data. However, linear bleaching arti-
facts can be addressed by baseline correction [9, 34, 87].
5. Implement event-related analysis to identify AP-related transi-
ents: Temporal classifications of noisy transients, caused by the
physical principle of the recording as well as the source of the
physiological signal (burst firing), are often carried out by
correlation approaches which are suitable for highly fluctuating
signals. However, we propose the application of event-related
analysis pipelines whenever possible to ensure the specificity of
neuronal responses with respect to the underlying signal of
interest, i.e., the neuronal action potential.
160 Hendrik Backhaus et al.

6. Perform manual inspections: Every algorithm has its strengths

and weaknesses, and, due to the highly variable quality of
all-optical experiments, e.g., in terms of SNR, might lead to
false-positive results. Therefore, no matter which method is
used to identify AP-related calcium transients in an intensity
trace, a manual inspection of the binarized event array needs to
be conducted to eliminate all potential false-positive results.
7. Avoid cross-talk at any time: Capturing the onset of a calcium
transient can be impossible due to cross-talk between emis-
sion/excitation wavelengths: Artifacts can be ruled out by
blanking the PMTs at the cost of sacrificing information
about fluorescence during opsin excitation, while on the
other hand applying algorithms to minimize photostimulation
artifacts may result in a loss of the physiological signals of
interest. We strongly suggest to design experimental paradigms
in all-optical physiology to avoid cross-talk at any time by
choosing spectral independent opsins and calcium indicators.

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 Chapter 6

Spatial and Temporal Considerations of Optogenetic Tools

in an All-Optical Single-Beam Experiment

Damaris Holder and Matthias Prigge

Abstract
All-optical experiments promise neuroscientists an unprecedented possibility to manipulate and measure
neuronal circuits with single-cell resolution. They rely on highly fine-tuned microscopes with complex
optical designs. Of similar importance are genetically encoded optical actuators and indicators that also have
to be optimized for such experiments. A particular challenge in these experiments is the detection of natural
firing patterns via genetically encoded indicators while avoiding optical cross-activation of neurons that are
photon-sensitized to allow optical replay of these patterns. Most optogenetic tools are sensitive in a broad
spectral range within the visible spectrum, which impedes artifact-free read-and-write access to neuronal
circuits. Nonetheless, carefully matching biophysical properties of actuators and indicators can permit
unambiguous excitation with a single wavelength in a so-called single-beam all-optical experiment.
In this chapter, we evaluate the current understanding of these biological probes and describe the
possibilities and limitations of those tools in the context of the all-optical single-beam experiment.
Furthermore, we review new insights into the photophysical properties of actuators, and propose a new
strategy for a single-beam two-photon excitation experiment to monitor activity minimizing cross-
activation with the actuators. Finally, we will highlight aspects for future developments of these tools.

Key words Channelrhodopsin, Two-photon excitation, Photobiophysics, Optogenetics, Cross-

activation

1 Introduction

Over the last few years, the development of advanced optical stim-
ulation technologies in combination with novel optogenetic probes
foretold a bright future for the interrogation of brain function in
behaving animals. In particular, optically imprinting naturalistic
firing patterns onto neuronal circuits while simultaneously obtain-
ing activity readouts at a single-cell level is considered the holy grail
of a holistic understanding of neuronal circuits [1–3]. In this chap-
ter, we refer to such an experiment as an all-optical experiment and
offer a review on the possibilities and pitfalls of two-photon stimu-
lation of photon-sensitized single neurons while optically

Eirini Papagiakoumou (ed.), All-Optical Methods to Study Neuronal Function, Neuromethods, vol. 191,
https://doi.org/10.1007/978-1-0716-2764-8_6, © The Author(s) 2023

165
166 Damaris Holder and Matthias Prigge

monitoring activity in the entire neuronal circuit. These days such

experiments have mostly been performed in specialized laboratories
with a long-evolved expertise in advanced two-photon microscopy
or optogenetic tool development groups. They are, however, more
difficult to implement in common neuroscience laboratories. To
allow the execution of these all-optical experiments, both optical
technologies and optogenetics probes need improvements in terms
of allowing high signal-to-noise ratio (SNR) imaging and obtaining
decreasingly flawed readouts. Furthermore, obstacles for most
laboratories include the enormous costs of optical systems includ-
ing two beam lines and optical modulators. Nevertheless, the over-
all idea for such an approach is straightforward: optical stimulation
of single neurons expressing a sensitive blue-absorbing actuator in
combination with a highly sensitive red- or IR-light absorbing
indicator to report activity of single neurons in a large field of
view (FOV) [4–6].
For a more extensive introduction into the optical design of
these kinds of systems, several implementations have been
employed and are discussed within this book (Chaps. 1, 2, 3, 4,
and 5) or reviewed elsewhere [7]. Briefly, kilohertz resonance mir-
rors are commonly implemented for fast imaging, resulting in line
scan speeds between 50 and 100 μs. Alternatively, acousto-optic
deflectors (AODs) are of an order of magnitude faster and can yield
line scan speeds of 10 μs and lower. With electrically tunable or
ultrasound lenses and piezo- and acousto-optic elements scanning
speeds can be drastically increased and 3D imaging and random-
access scanning can be realized [8–10]. Furthermore, liquid-
crystal-based spatial light modulators (SLMs) are used to extend
imaging into the third dimension via computer-generated hologra-
phy (CGH) [11].
CGH with liquid crystal SLMs is most often used to optically
stimulate dozens of target cells in a three-dimensional brain volume
on a single-cell level [5, 12–14]. Therefore, two strategies for
single-cell optical stimulation have emerged in the last decade.
The first strategy involves spiraling of the two-photon laser beam
on the somata of neurons expressing an actuator. An increasing
number of actuators are then activated along the spiral beam path
on the surface of the membrane, and their single-channel photo-
currents are integrated toward the depolarization of the entire cell,
eventually leading to spiking of the neuron [15]. By combining
SLM and galvanometric mirrors, such a spiral approach can easily
be multiplexed [2]. Yet, latency and spike jitter are rather ill-defined
and can vary around 20 ms and from 5 to 20 ms, respectively due to
biological factors such as cell morphology, cell excitability, or
expression level. Additionally, the temporal spike accuracy also
depends on an exact technical implementation in terms of spiraling
speed, determination of light powers, and axial resolution for the
excitation of the actuator. Furthermore, how efficiently the
 Spatial and Temporal Considerations of Optogenetic Tools 167

photocurrent of a single-channel translates into neuronal depolari-

zation depends on the off-kinetics of the actuator in use: slow-
closing actuators integrate photocurrent over a longer time and
therefore drive spiking more reliably in neurons. Nonetheless,
slow-closing actuators increase the probability of multiple spikes
during a single spiral scan.
The second approach for single-cell stimulation is the so-called
scanless stimulation approach, which can be implemented using
holographic stimulations [16, 17]. Here, an SLM is conjugated to
the rear focal plane of the objective, and can generate multiple
(circular) patterns to stimulate several entire somata simulta-
neously. Sub-millisecond jitter can be achieved with holographic
stimulation [18, 19], and actuators with fast off-kinetics can be
used [20, 21]. Such technologies for the precise control of spike
timing in many neurons in parallel will be pivotal to probe compu-
tational principles of neuronal circuits or even to artificially drive
animal behavior without sensory inputs [1, 22].
Thus in summary, a rapidly increasing number of optical tech-
nologies have been developed for neuroscientific applications.
However, imaging and optical manipulation still face challenges
on both technological and tool fronts as the channels for imaging
and manipulation are spectrally distinct and therefore require two
independent optical pathways with heavy weight and sensitive as
well as expensive optical equipment.

1.1 Optogenetic Several advances are being made in tool development as new and
Tools for All-Optical better indicators and actuators are constantly being designed.
Experiments However, the most commonly used green-absorbing calcium indi-
cators GCaMP6 through 8 are still superior to their red-absorbing
counterparts jRGECO or K-GECO1 [23, 24]. It is believed that
only the pairing of red indicators and blue-absorbing actuators will
completely prevent cross-interference between imaging and stimu-
lation. Yet, for the more commonly used combination of a blue-
light absorbing indicator in combination with a red-absorbing
opsin the actuator is always cross-activated during imaging, since
retinal isomerization is triggered via the hypsochromic shifted exci-
tation light (shorter wavelength relative to maximum absorption
see Fig. 1a). This can elicit electronic transitions to higher elec-
tronic states (e.g., S0 ! S2), which are less efficient than those to a
lower energy state (S0 ! S1) [25, 26]. To some extent, lower
imaging light powers can mitigate such effects, but deeper imaging
and in vivo applications are often antagonistic to low power
imaging.
Despite ongoing developments toward better red-light absorb-
ing indicators, artifacts from photophysical processes induced by
blue light imaging excitation in these red absorbing indicators still
constitute a major obstacle for their widespread use in all-optical
experiments.
168 Damaris Holder and Matthias Prigge

A hypsochromic imaging stimulation B stimulation bathochromic imaging

(spectral overlap) (no spectral overlap)
p (minimal spectral overlap) (no spectral overlap)

GCaMP Chrimson ChR2 RCaMP

absorption absorption absorption absorption
Activation

Activation
lowest
lowest energy
higher energy transition
electronic transition
state transitions

Wavelength Wavelength

Fig. 1 Spectral combinations of indicators and actuators in an all-optical experiment, and their respective
cross-activation. (a) illustrates the overlap between the absorption spectra of GCaMP with the red-light
absorbing opsin ChRimson. Here, during hypsochromic imaging of the indicator, higher electronic state
transitions in the all-trans retinal can evoke photoactivation of ChRimson. While in (b) the absorption spectra
of RCaMP and the action spectra of ChR2 are shown. Here, both spectra are sufficiently separated to avoid
cross-activation of the blue-light absorbing opsin via bathochromic imaging, while contamination of RCaMP
emission during opsin photoactivation is minimal due to low absorption of the red indicator for shorter
wavelengths

For example, promising red-light absorbing calcium indicators

such as jRGECO1a and RCAMP2, which originate from mApple,
exhibit a blue-light sensitive protonation state giving rise to
increased red fluorescence emission with similar kinetics as
calcium-induced changes [23, 24]. Similarly, far-red calcium indi-
cators, excited at approximately 590 nm, such as R-GECO and
K-GECO also exhibit blue-light artifacts, albeit with faster kinetics
than calcium-induced changes [27].
In the last decade, the rise of voltage indicators has additionally
augmented the sensor landscape, however, the aforementioned
bottleneck is only further enhanced by commonly used genetically
encoded voltage indicators with a high SNR such as ASAP or
Marina that also absorb in the blue/green spectral range
[28, 29]. Additionally, a somewhat baffling finding is that
promising candidates of opsin-based voltage indicators are not
accessible via two-photon excitation due to their poorly understood
photophysical processes related to the lifespan of the excited state
[30]. Moreover, opsin-based indicators have inherited the afore-
mentioned S2 hypsochromic excitation. Thus, although the collec-
tion of genetically encoded tools is constantly expanding and
improving, an artifact-free and robust combination of indicators
and actuators has yet to be established.
 Spatial and Temporal Considerations of Optogenetic Tools 169

2 All-Optical Single-Beam Experiments

A single-beam experiment refers to an approach in which a single

laser beam at a given wavelength is multiplexed to simultaneously
image and stimulate neuronal circuits. The most straightforward
approach to this problem is to target two distinct cell populations
with an actuator and indicator. A frequently used approach is to
restrict the expression of actuators and indicators to different brain
regions (anatomy-defined approach Fig. 2a). Here, cross-activation
of the axonal projections in one target region, in which neurons
express an indicator, is reduced, since photon-sensitized axons
require higher light power to trigger an action potential than
photon-sensitized somata [31]. However, such an approach

A Anatomy-defined approach B Cell-type-defined approach

soma
activation axon
activation ROI
1
reg ROI
ion 2
1

reg
ion
2

C Path-defined approach D Dual-colour experiment

Fig. 2 Fundamental approaches for all-optical experiments. In (a) the expression of actuator and indicator are
separated through space. In this anatomical approach, full-field one-photon illumination of axons still triggers
the activation of actuators and hinders simultaneous imaging of indicators, but low power imaging of the
indicator can reduce cross-activation of the axon drastically. Moreover, if expression of the actuator is
restricted to the soma, photo-stimulation can be entirely spatially separated, rendering an artifact-free
simultaneous all-optical experiment possible. (b) Illustration of a cell-type-defined approach in which different
cell types express either an indicator or actuator, so that imaging and optogenetic stimulation are segmented
into small subregions containing only specific cell types. The approach in (c) is a variation of (b): while using a
distinct expression of indicators and actuators, here, technology to define arbitrary paths allows for the
selection of neurons with indicator expression in the entire field of view. In (d), a typical dual-color all-optical
experiment is illustrated. Here, both indicator and actuator are expressed in the same cell. In this case, the
choice of indicator/actuator combination is crucial. Usually green-absorbing indicators are employed along-
side red actuators to minimize optical crosstalk
170 Damaris Holder and Matthias Prigge

necessitates identification of the light power regime for artifact-free

imaging of indicators and carefully performed control experiments.
The light power used to evoke the release of a neurotransmitter
from photon-sensitized axons depends on several factors such as
myelination, directionality of the axons relative to the plane of light,
and axonal caliber [32].
A related anatomy-defined strategy is often used for in vivo
all-optical experiments. Here, a brain region harboring photon-
sensitized somata is stimulated to elicit action potentials that prop-
agate orthodromically to a projection region in which neuronal
activity is monitored optically [32]. While such an approach con-
siders the natural latencies and branching of axonal collaterals, it
also requires restriction of actuator expression to the soma to avoid
unwanted activation of axons. Additionally, for an in vivo applica-
tion in a single beam experiment, the laser beam would have to be
split and the resulting beamlets guided independently to two sepa-
rate brain regions. Overall, both anatomy-defined strategies are
limited to the study of axonal innervation in a specific brain region
and are highly dependent on anatomical architecture.
For an all-optical experiment within the same region, actuator
and indicator can be expressed in distinct cell populations, e.g.,
excitatory versus inhibitory neurons. In this case, a small imaging
region of interest (ROI) containing few cells (e.g., excitatory neu-
rons) devoid of actuator expression can be selected, while a subset
of neurons expressing the actuator (e.g., interneurons) are holo-
graphically stimulated outside these ROIs (Fig. 2b).
Similar to the aforementioned anatomy-defined approach, a
cell-type defined approach also requires strict soma-targeting of
both actuator and indicator. Instead of limiting imaging ROIs to
two small subsets, fast scanning approaches using AODs or
resonant-galvo-galvo configurations allow an imaging path to be
defined in which only neurons expressing indicators are scanned
(see Fig. 2c). In particular, AOD technology allows one to traverse
such an arbitrary pathway in the kHz range, making it ideal for use
with fast voltage indicators as well as for calcium imaging [10, 28].
Importantly, anatomically and genetically defined approaches
do not enable both activation and imaging from the same set of
neurons. Ultimately, only simultaneous expression of both indica-
tor and actuator can grant all read-and-write access to neurons but
it is still particularly challenging to prevent cross-activation with a
single laser beam.
Dual-color experiments are classically considered to be a bul-
letproof concept for an all-optical experiment. Yet, compromising
on indicator SNRs due to less sensitive red-absorbing indicators
make such an approach challenging in terms of detection (Fig. 2d).
Even though dual-color experiments usually rely on two different
laser beams, they can serve as an interesting framework and intro-
duce valuable parameters to consider in a single-beam experiment.
 Spatial and Temporal Considerations of Optogenetic Tools 171

Ay x B 1st 2nd 3rd ...

FRAME Tframe 32 ms
T
frame

Ton_cell LINE Tline 63 μs

Tline

Tline don_cell d off_cell

don_cell 30 μm
r
Tframe xy

d d 1.4 μm

Ton_cell < 3 μs
rxy = 275 nm =Tline Toff_cell
- = 63 μs - 59.82 μs
Toff_cell Ton_cell

rz = 1.4 μm Tpulse 140 fs

Tpulse f repetition 80 MHz

Fig. 3 Illustrations of scanning parameters and actuator stimulation patterns in an all-optical dual-color
configuration experiment. (a) For a given x-y plane, the scanning mirrors move a laser spot across the field of
view (FOV) with a given lateral rxy and axial resolution rz [33, 34]. (b) As an example, we assume a system with
a 16x objective with NA ¼ 0.8, yielding a FOV of 700  700 μm (dFOV); an 8-kHz resonant scanner, scanning
with a resolution of 512  512 lines; and a laser with frepetition ¼ 80 MHz, and a single pulse width of
Tpulse ¼ 140 fs, where we scan a single neuron with a dimension Dsoma ¼ 30 μm (in teal an idealized neuron,
which was approximated to a rectangular scan area (gray shaded area) of 30  30 μm for estimating
parameters). Scanning the entire FOV takes Tframe with a single line scan of the duration Tline. We define the
distance don-cell as the distance scanned by the laser on the cell and doff-cell as the distance the laser scans
over non-opsin expressing parts of the FOV. The distance d is defined in y-direction as the distance between
two scan lines. Depending on the resolution, the laser spot runs over a neuron expressing an actuator several
times per frame. Ton_cell is the time the laser spends scanning the cell for a given line. Depending on the
position of the neuron within the FOV, the subsequent line can give rise to another illumination period, i.e., cell
is located on the border of the FOV so that doff-cell is very small on one end of FOV. Within a single given frame,
a neuron in our example will be exposed to Nsoma ¼ 22 lines with each of them having Ton_cell < 3 μs

As a practical example shown in Fig. 3, the imaging beam

(longer wavelength than that of actuator absorption) passes over
neurons expressing the actuator and indicator with a specific lateral
(rxy) and axial (rz) dimension. The spacing between consecutive
scan lines (d) depends on the resolution of the acquired image
(Rscan), and defines how many times (Nsoma) the laser passes an
idealized soma (quadratic dimension given as Dsoma) during a single
frame. Based on typical values used in all-optical experiments
(shown in Fig. 3), the following relations apply:
d FOV
d¼ ¼ 1:4 μm ð1Þ
Rscan
Here, the spacing d between two consecutive lines is 1.4 μm
and FOV denotes the field of view with respect to the entire laser
scanning range, which is 700  700 μm in our example. Consider-
ing a 16x objective (0.8 NA) with a scanner speed (νscan) of 8 kHz
172 Damaris Holder and Matthias Prigge

and a resolution of 512  512, a laser scan line (dimension rxy)

passes over a 30-μm (rectangular) neuron approximately 22 times:
D soma D soma Rscan
N soma ¼ ¼ ¼ 22 ð2Þ
d d FOV
During these 22 passes of the beam area of rxy over the cell
surface, only 20% of the total membrane surface will be illuminated
(we assume the idealized neuron to be a square of an area of
900 μm2):

A illuminated ¼ N soma r xy D soma ¼ 182 μm2 ð3Þ

The scanning frequency defines the dwell time (Ton_cell), which
denotes the time the laser beam spends scanning a cell during each
scan line of the bidirectional movement of the resonant scanner
(example):

T oncell ¼ D soma ðνscan 2 d FOV Þ1 < 3 μs ð4Þ

In our example, the average dwell time per cell and line is
roughly 3 μs. However, for a single opsin molecule, dwell times
are even shorter (cross-sectional diameter approximately 2 nm). In
our example, an opsin that diffuses within the plasma membrane
with 0.5 μm/s can be regarded as immobile during the dwell time
of 3 μs [35]. Each opsin will be exposed to several light pulses
during one scan line, but once activated, an opsin molecule with its
millisecond off-kinetic can only be triggered once during a single
line, and depending on rxy and the FOV only once within a single
frame acquisition. This is in drastic contrast to indicators that have
fluorescent lifetimes of 2–10 ns, and can be excited and emit a
single photon with every light pulse (approximately 10 light pulses
when sweeping a beam across a single molecule). Therefore, the
number of emitted photons indicative for the state an indicator is in
can be further increased through prolonging dwell times in the
microsecond range, while avoiding increased photocurrent
through double-activation of an opsin with its milliseconds
photocycle.
To better understand two-photon activation of actuators and
indicators, two-photon cross-section (unit: Goeppert-Mayer; GM)
data are invaluable to estimate cross-activation. Yet, GM values are
rare for opsin-based indicators, though retinal-based actuators gen-
erally tend to have higher values than those of fluorescence pro-
teins. As mentioned above, the light sensitivity and dynamic range
of genetically encoded indicators are being periodically improved.
Current versions allow two-photon imaging at low excitation
powers. A recent report demonstrated that GCaMP6 can be
imaged using two-photon light powers as low as 50–80 mW
(using an 80 MHz Ti:Sapphire laser) and that this did not lead to
any significant change in firing rates of neurons expressing a
red-shifted opsin (λmax ¼ 540 nm) [22, 36]. Nevertheless,
 Spatial and Temporal Considerations of Optogenetic Tools 173

subthreshold depolarizations were not monitored, but likely

induced and potentially biased ongoing basal excitability levels,
thereby causing measurement artifacts in the neuronal circuit
under investigation. Ultimately, further two-photon optimized
indicators together with carefully selected opsins, as discussed
below, can potentially enable single-beam all-optical experiments
with little to no cross-activation. In particular, this includes a well-
controlled expression level of opsins that is just sufficient to reliably
trigger spikes holographically, while minimizing cross-activation
when line-scanned.

3 Temporal Considerations of Actuator Kinetics in Single-Beam All-Optical

Experiments

The possibility to optically probe neuronal systems is tightly linked

to the discovery and exploitation of light-gated ion channels [37–
39]. Channelrhodopsins are the main actuators used in neurosci-
ence. The success of opsin-based actuators triggered an avalanche
of protein engineering studies as well as metagenomic exploitation
to design opsins for specific experimental settings [40–42]. This
new hype around opsin research was accelerated by the ground-
work laid by earlier studies on light-driven ion pumps (reviewed in
[43]). Based on their established spectroscopic, crystallographic,
and electrophysiological paradigms, in less than 10 years, causal
relationships between channelrhodopsin structure and
ion-conducting pathways could be drawn on an atomic level [41].
The sequence of conformational changes and ion movements is
described in a photocycle (see Fig. 4). We now know that an

A all-trans 15-anti 13-cis 15-syn B Δt

...
double two-photon absorption cycle
single two-photon absorption cycle

DA
N
H
R
τ
rec
N
R DA
LA
DA LA
H
O1 + O2 τ off
O1 + O2

anti - syn - I steady state

P*
P
cycle τ
off1 τ off2
cycle
τ on O1
O11 O12 τ
Current

H
des
τ rec
N
H
N R
O1 Time
R
all-trans 15-syn
I peak
13-cis 15-anti

Fig. 4 Unified photocycle for Channelrhodopsin2 in relation to its electrophysiological parameters [44, 45]. (a)
is a schematic depiction of the unifying photocycle where a single two-photon process can either trigger an
anti-photocycle from the dark-adapted (DA) ground state or the transition to the light-adapted (LA) state. The
LA state thermally relaxes back to the DA state or a second two-photon process can trigger a syn-photocycle.
After relaxation to the open state(s), O1 or O2 the molecule transitions back to the closed ground state of the
respective cycle. (b) Illustration of a two-pulse experiment in which two light pulses are given with increasing
delay times (Δt) while monitoring the recovery of peak photocurrents during the second light pulse. The
recovery of the peak photocurrent Trec after varying Δt obeys a monoexponential increase referring to the
transition of LA to DA (dotted red line)
174 Damaris Holder and Matthias Prigge

aqueous pore is formed between helix 1, 2, 3 and 7, and is guarded

by two main gates. The pore opening is initiated via a so-called
central gate, which is in close proximity to the retinal chromophore,
and pre-opens the conduction pathway and allows influx of water
molecules into the pore without conducting ions or protons. Only
upon breaching the inner gate, ions and protons can be conducted
along their electrochemical gradients.
The open time of ion channel pores, which has been a subject of
mutational studies that led to many ChR variants, can range from
milliseconds to seconds [42, 46]. After closing, the opsin returns to
its ground state for a new photon excitation. Despite our increas-
ingly detailed understanding of these processes, it is surprising how
photocycles can differ among various ChR variants.
A unified photocycle derived from spectroscopic data also inte-
grates a multitude of electrophysiological parameters such as pho-
tocurrent profile, on- and off-kinetics, inactivation and recovery, as
well as light sensitivity (Fig. 4) [44]. Photocycles can exist in
various forms, from a single photocycle consisting of one dark
(D), an open (O), and a non-conducting state (P), to a dual
photocycle consisting of two conducting states up to highly com-
plex and branched photocycles [47–49].
Indispensable for our understanding of branched photocycles
has been Raman spectroscopy. This technique can provide a finger-
print of molecules based on their vibrational or rotational states,
revealing an additional photo-induced rotation of the retinal chro-
mophore in ChR between two conformational isomers: syn and
anti [50]. These conformational isomers refer to the –C15¼NH–
bond and are distinct from the photon-isomerization of the –
C13¼C14– cis/trans configurational isomers.
Such a second “light-switch” in an actuator has potentially
interesting consequences for all-optical experiments in neuroscien-
tific applications; in the complete dark-adapted state (DA), where
all rhodopsin molecules harbor an all-trans/-C15¼N-anti isomer,
absorption can either cause the molecule to enter into an anti-
photocycle (all-trans/-C15¼N-anti ! 13-cis/-C15¼N-anti) or a
syn-photocycle (13-cis/-C15¼N-syn ! all-trans/-C15¼N-syn)
ending in the light-adapted ground state
(LA) [44, 51]. Molecular dynamics simulation supports that the
ground state in the syn-cycle is indeed a pre-opened central gate,
which can only completely open the ion-conducting pathway
through a second absorption process. The LA ground state can
thermally convert back to DA in the range of seconds (seen as
recovery kinetic τrec), a process that can be monitored with a
two-pulse experiment (see Fig. 4b).
Despite the debate on the contribution of this syn-photocycle
to the overall photocurrent [52], it is evident that such photocycles
exist and can be subjected to further design efforts. Potentially, an
opsin could be engineered to exploit the dual photon absorption
 Spatial and Temporal Considerations of Optogenetic Tools 175

process to initiate an efficient syn photocycle only after dual-photon

processes. Such a dual absorption would strongly depend on Ton_cell
and the likelihood of a second absorption process from LA being
triggered to start the syn-photocycle. The probability of such an
effect will be very low within the time window of Ton_cell. Addition-
ally, the probability of a two-photon absorption process within
Ton_cell would quadratically decrease with light power. Engineering
efforts would also have to be directed toward reducing the conduc-
tance in the anti-photocycle, that is, minimizing photocurrent and
enhancing conductance in the syn-photocycle to reliably trigger
action potentials during holographic stimulation. Holographic
stimulation in the order of a millisecond would efficiently trigger
a dual absorption process, and therefore trigger the syn-photocycle.
After a single absorption event, the opsin molecule then thermally
reverts from LA to DA with τRec.
Yet another interesting photophysical light switch is the
so-called photoinduced closing of an opsin. As spectroscopists
visualized different photo-intermediates through their different
absorption bands, neuroscientists exploited these different states
by illuminating light at the corresponding wavelength of a particu-
lar state during the photocycle, and therefore short-circuiting
photo-intermediates directly back to their ground state. The effi-
ciency to photo-induce an off-switch is highest when certain photo-
intermediates are stabilized and have long dwell times. This has
been shown for bacteriorhodopsin, the prototype of a light-driven
pump, here single-point mutations can result in the accumulation
of specific M and N photo-intermediates which can be photo-
converted to the ground-state [53, 54]. Similarly for ChR2, muta-
tions in the residues C128 or D156 in ChR2 generate a set of
mutants in which opsins accumulate in an open state. These
so-called step-function opsins can be turned on for hundreds of
seconds with a short light pulse (in the millisecond range), thereby
initiating the transition from the dark to stabilized open state.
Because the absorption band of the open states is red-shifted,
they can then quickly be turned off with red light illumination
above 550 nm [46]. Therefore, a one-photon full-field background
illumination with red light could potentially mitigate cross-
activating the opsin during a two-photon all-optical experiment
with a single laser imaging beam around 980 nm.
However, such photo-induced back reactions still remain
poorly understood, particularly during two-photon absorption. It
would be interesting to explore the possibility to excite deep-blue
blue-absorbing ChRs such as PsChR or CheRiFF (λmax ca. 440 nm)
with a 980 nm two-photon beam on the bathochromic (longer
wavelength excitation relative to maximum absorption) side of their
activation spectra. Eventually, cross-activated opsin molecules
reaching O would then be back transferred with 980 nm excitation
light.
176 Damaris Holder and Matthias Prigge

Therefore, the matching of temporal properties of opsin mole-

cules to imaging parameters can be utilized to minimize cross-
activation. Here, off-kinetics that only allow a single activation of
an opsin molecule during an imaging iteration in combination with
indicators with short fluorescent lifetimes are favorable. In particu-
lar, Ton, Toff, and Trec of opsin molecules have been heavily engi-
neered and are ranging from few milliseconds to seconds [6, 55]. In
contrast, photo-induced back reactions are not well understood,
but could potentially act as an optical master switch that can render
opsin molecules photocurrent-effective or ineffective.

4 Spatial Consideration of Optogenetic Tools in a Single-Beam All-Optical

Experiment

For an all-optical experiment with a single laser line, a high SNR

and single-cell resolution are essential. For stimulation of single-
neurons within a large field of view with hundreds of neurons
expressing photon-sensitive opsins, undesired off-target activation
via their closely passing axons (and dendrites) along the soma of the
targeted neuron becomes a challenge. To decrease background
fluorescence as well as confine excitation to selected neurons, it is
advantageous to localize the expression of actuators to specific
compartments. Therefore, restricting the expression of opsins to
the soma and dendrites has been a robust strategy.
Figure 5 gives an overview of different genetic targeting stra-
tegies along the neuronal cell body. Genetically fusing the opsin to

NaV1.2 (II - III TM loop) Ankyrin-G KV2.1

axon initial segment and somatodendritic
Axon initial segment (221aa) C-terminal soma and proximal dendrites
(745aa) N- or C-terminal
(65aa) C-terminal
Greenberg et al., 2011
Grubb and Burrone, 2010
Pégard et al., 2017 and Wu et al., 2013

NaV1.6 (II - III TM loop)

axon hill (27aa) C-terminal KA2
somatodendritic (150aa) C-terminal
Wu et al., 2011 and Zhang et al., 2015
Shemesh et al., 2017

EE-RR
Soma (88aa) C-terminal fusion

AnkTail Shemesh et al., 2020

axon initial segment
segmen
nt and somatodendritic
(400aa) N-terminal
Shemesh et al., 2020

Fig. 5 Overview of cellular localization of different target sequences. The neuron is divided into four segments:
axon initial segment, soma, somatodendritic and soma and proximal dendrites. Here, an overview is given
over the different molecular targeting strategies which are employed depending on which of these segments
are supposed to be expressing the opsin
 Spatial and Temporal Considerations of Optogenetic Tools 177

ankyrin-G protein, couples opsin expression to spectrin-actin net-

work, and consequently restricts expression to the somatodendritic
region and axon hill. Despite the size of its targeting motif of more
than 700 amino acids, the ankyrin-G sequence also targets the
dendritic region and hence still gives rise to off-target activation
[67]. However, targeting opsins to more defined and small subcel-
lular regions such as the axon initial segment (AIS) has failed so far
[56]. Early attempts to deploy targeting motifs found in voltage-
gated ion channels to anchored actuators in AIS such as NaV1.2
have been successful in terms of localizing the transgene to the AIS,
but the number of opsins molecules was too small to optically
induce action potentials [57]. A similar strategy utilizes a shorter
targeting motif derived from NaV1.6 and localizes opsins suffi-
ciently to the AIS, but also changes the intrinsic excitability within
the host cell itself [58, 59]. Based on these insights, short targeting
motifs to prevent expression within the axons are most promising.
For example, fusing a targeting motif from kainate receptor subunit
2 to the N-terminus CoChR yielded a soma-oriented actuator
(soCoChR) allowing for holographically triggered action potentials
with a 1 ms resolution and minimal off-target activation [19]. Simi-
larly, the cytoplasmic C-terminal from the voltage-gated potassium
channel Kv2.1 yielded specific targeting of opsins to somata as well
as proximal dendrites (stChronos, stCoChR, or stGtaCR2)
[20, 21]. Both targeting motifs, NaV1.2 and Kv2.1, have been
successfully used for two-photon connectivity mapping; however,
direct comparison between motifs remains to be elucidated.
Clearly, restricting the expression of optical actuators to a frac-
tion of the entire cell membrane leads to smaller photocurrent. Yet,
the soma-targeted opsins exhibit larger photocurrents than do
wildtype versions, indicating higher membrane expression of opsins
when fused to the Kv2.1 motif [20, 60]. Furthermore, apparent
off-kinetics of soma-targeted versions in neurons are faster than
unmodified versions, since delayed axonal and distal dendritic cur-
rent contributions are removed from the overall kinetics.
On a similar basis, targeting cytoplasmic indicators to the soma
or nucleus can be beneficial. In particular, nucleus-targeted calcium
indicators help to segment calcium transients to individual cells
[61]. However, onset latencies are prolonged due to slower calcium
rise in the nucleus. For single spike events, calcium transients might
not propagate into the nucleus and the overall response sensitivity
of calcium indicators is reduced in the nucleus.
Figure 5 gives an overview of different targeting sequences and
the resulting expression regions. In a recent screening, novel and
optimized targeting motifs have been reported [62]: a shorter
ankyrin-based motif combined with an ER trafficking signal from
Kir2.1 fused to the N-terminus restricts calcium indicators to the
somatodendritic region (Fig. 5). In addition, a de novo synthesized
178 Damaris Holder and Matthias Prigge

coil-coiled peptide that self-assembles into a complex slows down

transport out of the soma.
As cellular targeting helps to avoid cross-activation, a foresee-
able future breakthrough will be the exact control of the expression
level in relation to the membrane/volume ratio. Here, expression
systems that encode an auto-feedback that restricts expression to
the necessary amount will greatly reduce the effect of cross-
activation with indicators [63].

Note 1: Beyond Temporal and Spatial Constraints: Ion

Permeability
Not only spectral properties within a photocycle can be
exploited for neuroscientific applications. Ion selectivity is a
key feature to adapt ChR to specific neuroscientific experi-
ments. As ChRs are intrinsically non-selective cation channels
permeable to protons, sodium, potassium, and even calcium,
they are not designed per se for the sensitive electrochemical
ionic gradient in neurons. With an atomic structure of differ-
ent ChRs at hand [56, 57], several protein engineering
attempts yielded ChR variants with improved ion selectivity
(Permeability, P). Particularly interesting for a neuroscientific
application are PNa+/PH+ ratios. For example, variants such as
ChRomeT (A71S/E90A/H114G), Chronos-D139H, or the
naturally occurring opsin PsChR exhibit shifted ratios of ten
to hundredfold to higher sodium permeability. Further,
PCa2+/PH+ ratios have been modified to improve calcium
conductance in ChR2 mutants such as ChR2-L132C,
ChR2-S63D, or ChR2-L132C-T159C [6, 58, 59]. An
increased sodium selective opsin translates photocurrent
more directly into membrane depolarization in neurons.
In a branched photocycle, the high-conductance O1 state
exhibits lower proton selectivity than does the
low-conductance state O2, and therefore the initial peak pho-
tocurrent carries a larger fraction of sodium in its photocur-
rent. So far, attempts to modify ion selectivity of distinct open
states remain unsuccessful. The structural changes that lead to
different ion selectivities in the respective open state are not
well understood. As both open states share the same
ion-conducting pore, mutational analysis introducing small
structural changes will likely influence the ion selectivity of
both open states.
 Spatial and Temporal Considerations of Optogenetic Tools 179

Note 2: Beyond Temporal and Spatial Constraints: Spectral

Consideration
In an ideal all-optical experiment, actuators and indicators are
sufficiently separated without any cross-activation and optical
setups are equipped with two independent spectral laser lines.
However, in-depth understanding of photophysical processes
underlying wavelength-dependent absorption can help design
better all-optical single-beam experiments.
So far, only phenomenologically understood are the find-
ings that stationary photocurrents current saturate at lower
light powers and can exhibit slightly shifted absorption spectra
[64, 65]. Typically, for one-photon imaging peak photocur-
rents saturate at light powers higher than 15 mW/mm2,
whereas stationary photocurrents saturate already at less
than 5 mW/mm2 [55]. However, for optimized opsins with
large photocurrents, light intensities can be orders of magni-
tude lower [40]. Photocurrent profiles at low light powers
exhibit a slow increase in amplitude that sometimes
completely lacks any peak photocurrent due to lower absorp-
tion probability per time. This feature can be utilized in
two-photon imaging, where indicators are monitored at low
intensities and high scanning frequencies inducing small and
skewed transient currents carried by a mix of O1 and O2 (see
Fig. 3). For 2P optogenetic activation of neurons, high light
powers will evoke large peak photocurrents and efficiently
trigger action potentials. Therefore, for utilizing such a strat-
egy, ChRs with strong and fast inactivation and strong expres-
sion are preferred. Similarly, activation of opsins outside their
maximal absorption range can also lead to photocurrent pro-
files with reduced peak photocurrents and slow
on-kinetics [66].
In summary, low power imaging and excitation wave-
lengths outside the maximal absorption band reduce transient
peak photocurrents and therefore minimize cross-activation
of opsin-based actuators during two-photon resonance
imaging.

5 Summary

To gain cellularly resolved read-and-write access to an entire neu-

ronal circuit, photo-sensitizing proteins, indicators (read), and
actuators (write) need to be expressed within a single neuron.
Ideally, neuronal activity is monitored in a large field of view while
multiple neurons are stimulated in parallel with sub-millisecond
latencies. Here, scanless stimulation technologies for multicell
180 Damaris Holder and Matthias Prigge

A Spiral stimulation B Holographic stimulation

Spiral stimulation Imaging Holographic stimulation Imaging

stChR indicator stChR indicators

Actuators Indicators Actuators Indicators

moderate kinetics high SNR at low light power fast kinetics high SNR at low light power
fast τdesensitization localisation towards soma fast τrecovery localisation towards soma
high expression moderate expression
strong desensitisation strong desensitisation
bathochromic stimulation

Fig. 6 Comparison of different stimulation approaches for a single-beam experiment. (a) outlines the spiral
stimulation approach, while (b) elucidates the holographic approach, both combined with fast scanning
indicator imaging. The grey inserts outline the methods’ respective advantages and drawbacks

excitation, such as holographically multiplexed spiraling or sole

holographic stimulation with spots fitting the size of a neuron, are
used to excite actuators, whereas indicators are imaged using fast
scanning approaches (Fig. 6). Since an ideal combination of spec-
trally distinct and high-efficiency indicator/actuator pairs remains
unavailable and would require expensive setups with multiple laser
lines and a highly complex optical design, in this chapter, we review
unexplored spatial and temporal photophysical features of spec-
trally comparable indicator and actuator pairs permitting an
all-optical single-beam experiment with minimal cross-activation.
Indicators are constantly being improved in terms of higher
SNR and better imaging properties. However, mutational screen-
ing for a new generation of indicators with improved two-photon
cross-sections is rarely performed. Within our introduced theoreti-
cal framework, we demonstrate that each actuator opsin molecule is
only activated once during a laser beam sweep. In contrast, indica-
tors can emit hundreds of photons during the same time window.
Therefore, next to improving the two-photon cross-section of
indicators to allow for efficient activation, decreasing their fluores-
cence lifetimes to below 5 ns can help distinguish between indicator
and actuator activities. Spatial restriction of indicator expression
within the soma can further help prevent background fluorescence
arising from the neuropil, thereby decreasing the imaging contrast.
With regard to actuators, we have highlighted the benefits of
using moderate or slow off-kinetic opsins for spiraling approaches
because they more efficiently integrate single-channel ion
 Spatial and Temporal Considerations of Optogenetic Tools 181

conductances towards crossing the spiking threshold. Fast and

complete desensitization from peak to stationary photocurrents
reduces the probability of multiple action potentials being triggered
during a single spiral scan. In this case, high membrane occupancy
of actuators is advantageous since only a fraction of the entire
membrane is illuminated during a spiral scan.
In contrast, holographic stimulation concurrently activates the
entire opsin-packed neuronal membrane with a millisecond illumi-
nation. Such an activation eliminates the need to sum over single-
channel conductances and favors the use of actuators with fast
off-kinetics. To avoid cross-activation via the imaging beam, we
suggest choosing blue-shifted opsins relative to the imaging laser
beam. Thus, the laser light will only cross-activate actuators with a
decreasing red spectral flank, reducing the overall probability of
activation. However, holographic stimulation can still efficiently
trigger action potentials via such a bathochromic excitation, given
high enough light powers.
As the basic photophysical processes of light-gated ion channels
are already well understood, we mainly focused on photophysical
processes in the context of two-photon excitation. Further, we
discussed photo-induced processes beyond the classical all-trans
! 13-cis isomerization in channelrhodopsins. Particularly, we
describe a second two-photon absorption process based on an
anti/syn conformation.
Harnessing any double two-photon absorption to activate
opsin molecules in an all-optical experiment would render the
light power dependency quadratic for actuators rather than for
indicators. Furthermore, just like in the case of indicators, high-
throughput mutational analysis for two-photon optimized opsin
variants is virtually absent with this being particularly true for
opsin-based actuators. Here, screenings toward high two-photon
cross-sections or the facilitation of double two-photon absorption
processes could enable artifact-free imaging of indicators and actua-
tors with a single-beam line.
As previous years have demonstrated how prolific and diverse
opsin evolution has been through natural selection, future years will
reveal whether artificial screening toward two-photon optimization
will produce a plethora of actuators for all-optical experiments.

Acknowledgments

The study was supported by the German Leibniz Association “Best

Minds” program, and the Center for Behavioral Brain Sciences.
The authors thank Nicole D’Souza and Eirini Papagiakoumou for
their help during the preparation of the manuscript.
182 Damaris Holder and Matthias Prigge

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 Chapter 7

Miniature Multiphoton Microscopes for Recording Neural

Activity in Freely Moving Animals
Baris N. Ozbay, Gregory L. Futia, Ming Ma, Connor McCullough,
Michael D. Young, Diego Restrepo, and Emily A. Gibson

Abstract
Miniaturized head-mounted microscopes for in vivo recording of neural activity have gained much recog-
nition within the past decade of neuroscience research. In combination with fluorescent reporters, these
miniature microscopes allow researchers to record the neural activity that underlies behavior, cognition, and
perception in freely moving animals. Single-photon miniature microscopes are convenient for widefield
recording but lack the increased penetration depth and optical sectioning capabilities of multiphoton
imaging. Here we discuss the current state of head-mounted multiphoton miniature microscopes and
introduce a miniature head-mounted two-photon fiber-coupled microscope (2P-FCM) for neuronal
imaging with active axial focusing enabled using a miniature electrowetting lens. The 2P-FCM enables
three-dimensional two-photon optical recording of structure and activity at multiple focal planes in a freely
moving mouse. Detailed methods are provided in this chapter on the 2P-FCM design, operation, and
software for data analysis.

Key words Two-photon microscopy, Fluorescence microscopy, Lens system design, Fiber optics,
Ultrafast pulse propagation, Neural imaging, Image processing

1 Introduction

Two-photon laser scanning microscopy (2P-LSM) has opened up

opportunities for in vivo biological imaging at the sub-cellular level
[1, 2]. In the neuroscience field, 2P-LSM combined with fluores-
cent indicators can now capture brain activity at high resolution and
in real time. Although ingenious methods to study behavior and
perception in a head-fixed animal have been developed, there are
some behaviors that simply cannot be studied. Examples include
navigation and social behaviors, while additionally there is evidence
that head fixation can alter behavior and underlying neural pro-
cesses [3]. Due to these constraints, head-mounted microscopes

Eirini Papagiakoumou (ed.), All-Optical Methods to Study Neuronal Function, Neuromethods, vol. 191,
https://doi.org/10.1007/978-1-0716-2764-8_7, © The Author(s) 2023

187
188 Baris N. Ozbay et al.

are now being developed that can allow the animal to be unre-
stricted with more naturalistic behavior. The first miniature micro-
scopes to be developed for this purpose were one-photon widefield
miniscopes [4–7]. These systems rely on innovations in compact
CMOS imaging sensors and efficient visible wavelength light emit-
ting diodes for the excitation source. Despite the success of the
miniscopes with thousands deployed in laboratories internationally,
there are limitations. One-photon widefield excitation in tissue
results in high levels of light scattering, reducing signal to noise as
the out-of-focus fluorescence is also collected on the imaging
detector. Computational methods are typically used to select out
transient fluorescent signals from the background from the image
data. Detailed structural information of the brain region being
imaged is sometimes not possible to obtain. Recently, modified
miniscopes have been developed that place a phase plate [8] or
microlens array [9] in the optical detection path which provides
additional information for computational reconstruction in three
dimensions (3D). However, light scattering, particularly in densely
labelled samples, ultimately limits the imaging depth in these 3D
miniscopes.
In contrast to one-photon widefield, two-photon microscopy
removes the out-of-focus fluorescence and provides cross-sectional
imaging only at the focus. 2P imaging provides excellent signal to
noise and detailed structural information allowing high-resolution
images of cells and processes. Previous work demonstrated minia-
ture laser-scanning multiphoton fiber-coupled microscopes for
head-mounted neuronal imaging in freely moving rodents using
different methods of laser scanning including miniaturized micro-
electromechanical systems (MEMS) actuated mirror [10, 11] or
piezoelectric scanner [12] within the head mount. However, none
of these designs leverages the optical sectioning capabilities for full
volumetric 3D imaging without introducing cumbersome com-
plexity and weight. Recently, Zong et al. integrated an optical
design using a MEMS mirror for lateral scanning and a tunable
lens for axial scanning in a head-attached miniature microscope
[13, 14]. However, this miniature microscope is complex to align
and involves custom lenses and tunable optics. In contrast, the
design reported here incorporates a miniature, compact electrowet-
ting tunable lens (EWTL) for increasing the versatility of
two-photon capabilities by adding active axial focusing, while
using a coherent imaging fiber bundle for lateral scanning. The
design uses only commercially available components and can be
implemented on any standard bench-top laser scanning
microscope.
 Miniature Multiphoton Microscopes 189

2 Background

2.1 Optical Design The objectives used for two-photon excitation microscopy in deep
Considerations for tissues typically have a numerical aperture (NA) greater than 0.8
Miniature 2P [2]. Imaging with high NA objectives reduces the spot size of the
Microscopes laser, thereby increasing the two-photon fluorescence signal for a
given excitation power. However, large NA objectives are a prob-
lem for the manufacture of small imaging systems, because the
system aperture is constrained by the diameter of the physical
optics, which in turn forces the optical design to compromise on
other important parameters, such as working distance and field-of-
view [15]. Assuming a fixed aperture size, larger NA lenses require
a reduction in the working distance. Additionally, the complexity of
a lens design can be viewed as a function of its optical invariant, i.e.,
ability to collect light over a large angle and field-of-view (FOV).
Increasing the NA has the effect of making it more challenging to
design for a large FOV as well. When considering the fluorescence
signal detection path, the fluorescent photons are only emitted at
the focus and detected on a photo-multiplier tube (PMT) such that
the background noise of the image is not degraded by scattered
photon trajectories. Therefore, the excitation NA and the collec-
tion NA, more commonly referred to as Etendue, must be consid-
ered separately. The time-averaged intensity of fluorescence
collected geometrically is:



If ,col ffi If ,gen Ωf ð1Þ
where Ωf is the fractional collection efficiency of an imaging system,
summarized as:
rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
 
2
11 1 NAcol
n1
Ωf ¼ ð2Þ
2
When imaging into scattering tissue, the excitation NA is not as
important as the collection NA. This is because high-angle rays
have longer optical paths, with higher probability of scattering,
and are affected by aberrations. Additionally, there are diminishing
returns as the focal volume decreases with higher NA. Therefore, a
good efficiency two-photon imaging system can be designed that
has a relatively low NA (~0.4–0.5) excitation path if the collection
NA path can be increased independently. Such systems are often
employed by using light collection paths that involve large-area
detectors, which can be replicated with large effective-area optical
fibers in miniaturized microscopes [10, 16].
190 Baris N. Ozbay et al.

2.2 Optical Design Unlike methods of axial scanning that physically move the sample
Considerations for or the objective lens, the use of a tunable lens to adjust the axial
Axial Scanning with a focus changes the way light propagates through the optical system.
Tunable Lens More specifically, the curvature at the back focal plane of the
objective lens is modified. When propagated through a lens, this
curvature of the wavefront gets transformed into an axial displace-
ment of the paraxial focus spot. This is analogous to how a tilted
wavefront becomes transformed by a lens into a transverse displace-
ment of the focus.
One important consideration when designing such a system is
the magnitude of curvature that is needed to achieve a desired axial
scan range. An EWTL placed in the back focal plane of a lens
directly shapes the wavefront entering the lens.
The wavefront phase term ϕ can be written as a function of the
pupil radius ρ [17]:
NA2 2
ϕðρÞ ¼ kZc ρ ð3Þ
2M2
where M is the objective magnification and Zc is the distance at
which the focus is formed with a given quadratic phase. Rearran-
ging this in terms of the amount of axial change for a given change
in wavefront phase curvature gives:
ϕ ρ2
¼ kNA 2 ð4Þ
Zc 2M2
This last expression shows that the ability of a tunable lens to
change the axial focus of a lens system is dependent inversely on the
square of the objective magnification. This means that designing
for high magnification with the purpose of increasing resolution or
NA will result in a decrease in axial scan range for a given EWTL.
Another consideration is that any imaging system that produces
magnification cannot perfectly represent both the axial and trans-
verse focus transformations simultaneously. This effect is illustrated
in Fig. 1. Botcherby et al. describe the consequences of this on
imaging properties [18]. In lens design, optical engineers typically
optimize for either transverse or axial focusing. Most commonly
transverse imaging is preserved because imaging lenses are expected
to have uniform performance throughout the field-of-view (FOV).
The consequence is an accumulation of spherical aberrations along
the axial range of the lens, shown in Fig. 1c. One can also design for
the Herschel condition, which instead forms perfect focus spots
along the optical axis but accumulates aberrations in the transverse
dimension.
The FCM imaging system designs presented here are opti-
mized primarily for the Herschel condition, by constraining the
optimization parameters in Zemax optical design software to
 Miniature Multiphoton Microscopes 191

Fig. 1 Axial focusing by wavefront curvature shaping at the objective back focal plane. (a) An input converging
or diverging wavefront is transformed into an axial translation away from the designed focal length of the lens.
A lens producing an ideal diffraction limited focus at the design working distance (b) will not produce a
diffraction limited focus when the input wavefront is divergent (c) due to spherical aberrations as a result of
the Abbe sine condition

maintain consistent imaging properties throughout the full focal

range of the imaging system. One important characteristic that is
maintained is telecentricity, which ensures that there is nearly no
magnification change as the focal length is tuned. However, this
choice sacrifices some imaging performance in the transverse
dimension, and so imaging performance is maintained only up to
the expected FOV. Further, it is necessary to balance high magnifi-
cation, which is required to achieve high resolution, with axial
scanning range. In the end, the relationships between aperture
size, focal length, transverse and axial consistency, and magnifica-
tion become a careful interplay of compromises to reach a useful
solution for miniaturized imaging. In this work, the actual optical
designs are sought empirically based on initial designs and guiding
principles. The designs are restricted by the parameters for size,
resolution, FOV, and axial scan range and additionally restricting
the designs to commercially available optics to allow for easier
replication and affordability.
192 Baris N. Ozbay et al.

2.3 Cranial Windows Cranial windows provide optical access to surface-level brain struc-
tures with traditional microscope objectives. Typically, two-photon
laser scanning microscopy (2P-LSM) is performed on a head-fixed
mouse with a coverslip implanted in a small craniotomy above the
brain region of interest, normally 3 mm2 or less in size. There are
several variations on windows that show the flexibility of this tech-
nique [19–21]. A similar procedure is skull-thinning and polishing,
which can be healthier and stable for the brain tissue, but is time-
consuming and may not be as optically clear [22]. Recently, Wang
et al. demonstrated that application of index-matching epoxy to the
bone can reduce scattering and enhance optical access [23]. Remov-
able windows and permeable windows have also been
described [24].
Some examples of common regions accessed by cranial win-
dows are olfactory bulb [25], barrel cortex [26], and visual cortex
[27]. These regions display high levels of behavior-dependent neu-
ronal activity relatively close to the surface. The extended depth-
limit of 2P-LSM, compared with widefield microscopy or confocal
laser scanning microscopy, allows access to neurons about 500 μm
below the brain surface. In terms of cortical layers in mouse,
500 μm is approximately the depth of the cell bodies found in layers
4/5 of structures such as the motor and somatosensory cortex.
Three-photon excitation microscopy has been recently demon-
strated to reach structures such as the hippocampus, located around
1 mm below the surface in the mouse brain. However, for deeper
structures, it is necessary to excavate the tissue above the target
structure or to implant relay optics, such as GRIN lenses [28].

2.4 Gradient- GRIN lenses have been classically used for simple and space-
Refractive Index efficient collimation of the divergent light exiting a single fiber
(GRIN) Lenses core. In the past decade, GRIN lenses have been re-purposed for
miniaturized imaging applications [29, 30]. The radial profile of
the refractive index, ng, in a GRIN lens varies according to [31]:
 
g 2r 2
ng ðr Þ ¼ ng:0 1  ð5Þ
2
where g is the gradient constant, r is the radial distance from the
optical axis, and ng.0 is the index of refraction at r ¼ 0. Rays
launched into one end of the GRIN lens will be focused based on
the axial length of the lens, defined by the pitch length, pL:
2π
pL ¼ ð6Þ
g
 Miniature Multiphoton Microscopes 193

Fig. 2 GRIN lens illustrations. Top: Single GRIN lens with pitch ~0.5 with a
magnification of 1. Bottom: Two-element GRIN lens with low NA relay at pitch
~0.75 and high NA objective with pitch ~0.25

A pitch of 1 is a full sinusoidal period at a given wavelength.

GRIN lenses are also available in a variety of NA values, which are
defined by the focus angle in the external medium with index n1.
This value can be approximated as [31]:
ng:0 gd
NAgrin ffi ð7Þ
n1 cscðgLÞ
where d is the GRIN lens diameter. These expressions show that
GRIN lenses with smaller diameter, d, need a shorter pitch length
to achieve the same NA. To achieve a long and thin GRIN lens for
deep-brain implants at high NA, it may require a very high pitch.
To get around this constraint, a relay lens of lower NA is used in
front of a high NA lens. Figure 2 shows an illustration of two kinds
of GRIN lens assemblies used for imaging. A singlet lens is shown
compared to a two-element lens with a low NA relay. By increasing
the pitch number of the low NA relay, GRIN lenses of various
lengths can be constructed for reaching deep-brain structures.
GRIN lenses are engineered to be highly space-efficient,
achieving relatively high NA (up to 0.5) for the small diameters
(0.35–1.8 mm). The disadvantages are that they are highly
wavelength-dependent and have rapidly deteriorating off-axis per-
formance for imaging. This is mainly due to the small aperture,
which vignettes the off-axis rays, effectively reducing the NA
toward the edges of the lens. Longer GRIN lenses also suffer
from accumulated geometric aberrations. Methods to extend the
imaging field and resolution include using active adaptive optics
[32] and custom fabricated microlenses [33].
Even with the disadvantages, GRIN lenses offer a tool for
providing optical access to otherwise deep-brain structures. Deep-
brain implants of GRIN lenses have been used with both widefield
fluorescence microscopy and 2P-LSM in structures such as the
hippocampus [34]. Animals may be head-restrained with an appro-
priate objective used to image near the top surface of the GRIN
lens. Usually a baseplate is implanted on the skull to provide stabil-
ity to the fragile GRIN lens assembly.
194 Baris N. Ozbay et al.

2.5 Fiber-Coupling of For 2P-FCM imaging a laser-source and point-scanning system are
Excitation Laser required. For the laser source, high-intensity laser light must be
brought into the miniature microscope enclosure using an optical
fiber. Fibers can be selected for the most efficient propagation of
the laser light, generally such that the fiber-core supports only the
fundamental transverse electromagnetic (TEM00) mode, in which
case it is known as a single-mode fiber. In the special case of
ultrashort laser pulses required for 2P-LSM, the maintenance of
the peak pulse power is dependent on chromatic dispersion, modal
dispersion, polarization dispersion, and non-linear effects
[35]. Pre-conditioning the pulses can help to cancel these different
effects of propagation through the fiber to maintain high peak
powers [36–38]. Alternatively, it is possible to use hollow-core
photonic bandgap fibers [39], in which the electric field exists
mostly in an air-filled core, minimizing chromatic dispersion and
nonlinearities. The choice of fiber should also depend on its weight,
flexibility, and cost for the practical purposes of mouse imaging.
Regardless of the choice, a single fiber core must be focused onto
the sample and actively scanned to form an image.
Fiber-coupled scanning microscopy is a relatively well-
developed field, owed to the progress in endoscopic surgical and
diagnostic clinical tools that use miniaturized imaging heads cou-
pled with fiber-optics to a detection system [40]. There are many
techniques for accomplishing this, two of the most common being
piezoelectric resonance scanning of the fiber-tip [12, 41–44] and
integrated microelectromechanical systems (MEMS) actuated mir-
ror scanning [10, 11, 45].
An extra consideration for these single-fiber delivery systems is
that single-mode fiber-cores are inefficient for the collection of
fluorescence emission. Solutions include the use of large mode-area
fibers flanking the excitation core [16], miniature single-element
detectors mounted on the microscope head [46], and a separate
emission path for large-area collection fibers [10, 11, 47, 48].
Overall, these single-fiber-delivery techniques allow access to
high-efficiency laser-transmission, which is especially useful for
2P-LSM because of the difficulty required in maintaining ultrashort
pulse integrity at the focus. The cost of these techniques is the
added mechanical complexity required to implement miniaturized
laser-scanning. These may result in challenges in the optical design
that limit imaging performance. Frequently, it is difficult to achieve
large scan ranges or maintain large beam apertures using small
mirrors. So far, these systems have been discussed only in the
context of transverse scanning. Miniature fiber-scanning micro-
scopes that implement a third axial scanning dimension can become
overly cumbersome in their complexity.
 Miniature Multiphoton Microscopes 195

Fig. 3 Properties of coherent imaging fiber bundles. Left: Illustration of proximal-to-distal image coherence of
CIFB, as well as the pixelation of an image formed through the bundle because of the discrete fiber cores.
Right: Fujikura CIFB fluorescence image of a uniform target showing core distribution. Image was acquired
using a laser scanning microscope focused on the proximal surface of the CIFB and placing a uniform
fluorescent sample at the distal end

2.6 Coherent A CIFB is made from a longitudinally ordered bundle of optical

Imaging Fiber Bundles fiber preforms that are drawn into dense canes with only a small
(CIFB) amount of cladding between the cores [49]. The resulting CIFB is
spatially coherent on both ends, allowing a pattern of light on one
end to be represented by the cores on the other end, illustrated in
Fig. 3.
CIFBs can be coupled with a variety of simple imaging lenses to
allow for full-field imaging. One of the simplest methods is to use
optical epoxy to adhere an imaging GRIN lens (pitch ~0.5) to the
distal end of the fiber bundle for use as a handheld imaging probe
[50]. An image of the Fujikura CIFB is shown in Fig. 3 with one
region expanded to better visualize the core distribution. Lateral-
laser scanning microscopy using a CIFB can be accomplished by
simply scanning the excitation laser focus across the proximal sur-
face of the fiber. The light is sequentially coupled into each core and
transmitted to the distal surface. The distal surface can then be
imaged onto a sample to create targeted excitation of fluorophores.
The emission light can be collected through the same optical path.
This setup is convenient because all the filters, detectors, and
scanning optics can be located proximal to the fiber bundle, and
can potentially be those of a standard bench-top microscope.
Even with the gained simplicity, there are some issues with
using a CIFB for miniature laser-scanning microscopy:
l The CIFB introduces pixelation to the image due to the
necessary cladding between the fiber-cores. Some techniques
can be used to process the images to reduce this artifact, which
can be summarized by different forms of low-pass spatial
196 Baris N. Ozbay et al.

filtering [51–53]. However, the fundamental lateral resolution

limit of the imaging system is determined by the core-to-core
spacing and magnification of the imaging system.
l To achieve small mode-field area sizes for single-mode propaga-
tion, it is necessary that the core size is small. Commercial small-
core CIFBs have cores of different shapes, sizes, and propagation
properties [54] and may result in random signal heterogeneity
that shows up in the captured images.
l Core-to-core coupling is a phenomenon that occurs because of
the close inter-core spacing in fiber bundles [55]. This artifactual
spreading of light intensity is most critical with broadband light
in the NIR wavelengths, as used in multiphoton microscopy.
This also limits the density of fiber-cores that can be used, and
consequently the imaging resolution and field-of-view.
l CIFBs are much stiffer than a thin, single-core fiber, with the
mechanical stiffness depending primarily on the diameter of the
CIFB (and thus the number and density of the cores). This poses
a challenge for developing a system for freely behaving rodents,
as it limits their mobility in their behaving environment. Lea-
ched fiber-bundles, in which the cores are only fused at the ends
of the bundle, may be a promising solution to this problem.
While appreciating these challenges, CIFBs greatly simplify the
distal optical setup, which is desirable when implementing the distal
axial focusing mechanism. Additionally, the use of a CIFB may
greatly reduce the cost of adoption for fiber-coupled microscopes
because it may allow researchers to use an existing bench-top laser
scanning microscope for fiber-coupled imaging with few or no
modifications.

2.7 Ultrafast Laser- The time-averaged two-photon fluorescence intensity generated by

Pulse Propagation a pulsed laser source with average power Pavg, pulse width τP, and
Through Fiber pulse repetition rate fP is given by the following equation:
 

 P2avg NA2 2
If ,gen ffi πδ2 η ð8Þ
τP f P hcλexc
where δ2 is the two-photon cross-section, η is the quantum effi-
ciency of the fluorophore, h is Planck’s constant, and c is the speed
of light [56, 57]. This relationship shows that two-photon fluores-
cence signal is inversely proportional to the laser pulse duration.
Chromatic dispersion through an individual core of the CIFB
results in temporal broadening of the pulse, significantly reducing
the fluorescence signal from two-photon excitation [58]. Figure 4
illustrates the principle of chromatic dispersion. Due to the spectral
bandwidth of the pulse, the different frequencies travel with differ-
ent velocities so that, at the exit of the fiber, the pulse is spread out
in time. In normal material dispersion, the lower frequencies (red
 Miniature Multiphoton Microscopes 197

Fig. 4 Illustration of material dispersion. The initial pulse out of the laser is short with all spectral frequencies
in phase. Inside the 1 m glass fiber, the different frequencies travel with different velocities, resulting in a
pulse that is stretched out in time. The resulting pulse is positively chirped, where the lower frequencies (red)
travel faster than the higher frequencies (blue)

wavelengths) travel faster than the higher frequencies (blue wave-

lengths) and the resulting pulse is positively chirped. Dispersive
optics can be used to counter-act the material dispersion. One
method is a Treacy grating-pair compressor, which uses two parallel
gratings to spectrally disperse the light and recombine it in a
geometry where the blue light travels faster than the red [59]. Addi-
tion of the grating-pair compressor before the fiber can effectively
cancel out material dispersion, resulting in a short pulse at the fiber
exit.
In addition, the high peak powers required for efficient
two-photon imaging result in non-linear effects in the fiber. In
the nonlinear regime, there is an intensity-dependent change in
refractive index of the material, called the optical Kerr effect:
nðI Þ ¼ n0 þ n2 ∙I ð9Þ
where n0 is the linear refractive index and n2 is the second-order
nonlinear refractive index of the material.
198 Baris N. Ozbay et al.

As a pulse propagates in a material, the Kerr effect results in a

time varying refractive index, and therefore a modulation in the
phase of the electric field, called self-phase modulation (SPM),
resulting in a frequency shift that varies across the pulse, according
to the following equation:
2πL
Δωðt Þ ¼  dn=dt ð10Þ
λ0
where Δω is the shift in the instantaneous frequency across the pulse
in time, L is the length of the fiber, and n is the refractive index.
Typically, SPM results in spectral broadening when propagating a
high-peak power short pulse in fiber; however, in the special case
where the pulse starts with a negative chirp, the frequency shift
from SPM can cause spectral narrowing [60], resulting in a longer
pulse duration, due to the time-bandwidth product. High-peak
power pulse propagation through optical fibers for multiphoton
imaging has been explored extensively in previous works [61–64].
One solution to obtain short pulses at the fiber exit is to first
spectrally broaden the pulse by propagating in a polarization main-
taining fiber (PMF) before applying negative chirp from the Treacy
grating-pair compressor. As the pulse propagates through the
CIFB, the spectrum narrows but is balanced out to obtain the
initial spectral width from the laser, resulting in the bandwidth to
support a short pulse. In certain cases, it is possible to obtain a pulse
that is shorter than the original pulse duration by generating more
bandwidth.
In addition, the high-peak power pulses incident on the proxi-
mal glass surface of the CIFB also produce unwanted background
from auto fluorescence and inelastic Raman scattering [65]. We
find that the background signal from the fiber surface is greatly
reduced when the pulse is pre-chirped with lower peak intensity,
thus resulting in fewer non-linear interactions at the proximal fiber
surface.
Furthermore, the variability in diameter, shape, NA, and
amount of cladding between adjacent cores warrants an investiga-
tion into the feasibility of propagating ultrashort pulses in a CIFB
for two-photon imaging. Figure 5 shows a comparison of an image
of a uniform fluorescent target with one-photon versus two-photon
excitation through a CIFB. As can be noted, there is more hetero-
geneity in signal for the two-photon case. The issue of core-to-core
variability in transmission of short laser pulses through commercial
CIFBs was further explored by Garofalakis et al. by measuring the
transmitted power, polarization, mode, and pulse duration from
different fiber cores. The authors found that variation in pulse
durations was the predominate cause of this heterogeneity and
concluded that it resulted from a variation in the amount of chro-
matic dispersion in different cores [66]. Further work to under-
stand and correct for these effects could improve multiphoton
imaging through CIFBs.
 Miniature Multiphoton Microscopes 199

Fig. 5 Imaging heterogeneity in CIFB. Images taken using the 2P-FCM showing the raw image of the proximal
end of the CIFB focused on a NIR phosphorescent detector card to demonstrate one-photon excitation and a
uniform green fluorescent test slide to demonstrate two-photon excitation

2.8 Tunable Focus by Liquid lenses are a common variety of electrically tunable lens
Liquid Lens (ETL) that use one or more liquids as a shape-changing refractive
Technology interface to allow for focus adjustment with minimal mechanical
motion [67]. There are several common liquid ETL types that may
be used for performing high-speed optical focusing for laser-
scanning microscopy [68]. Large-aperture liquid ETLs are particu-
larly suited for bench-top systems because of their large focal range,
high speed, and repeatability. An example of a commercially avail-
able 10-mm aperture, 30-mm diameter ETL (EL-10-30-C-MV,
Optotune AG, Switzerland) functions by rapidly changing the
volume of liquid in a container with a flexible polymer surface,
which results in a focal length shift. Large aperture liquid ETLs
have enabled rapid axial focusing, up to 100 Hz, in compact laser
scanning confocal microscopes (LSCM), 2P-LSM, and selective
plane illumination microscopy (SPIM) systems without mechanical
movement of the objective or sample [69–72]. However, shape-
changing polymer ETLs are not suitable for miniaturized head-
attached microscopes because of their large size and susceptibility
to orientation and vibration.
Electrowetting tunable lenses (EWTLs) are another type of
liquid ETL that has applications in microscopy [73]. An example
is the Arctic 316, made by Varioptic, France, with outer diameter
7.8-mm and 2.5-mm aperture. Electrowetting is a method for
changing the wettability of a liquid on a dielectric surface by apply-
ing a voltage across the interface, effectively changing the contact
200 Baris N. Ozbay et al.

angle of the liquid with the surface. To form a lens, two fluids with
dissimilar refractive-indices and hydrophobicity are placed in a
container with dielectric sidewalls. The hydrophilic polar liquid
has dissolved impurities to allow it to react to an external electric
field. The contact angle of the liquid interface to the sidewalls of the
lens can be changed by applying an electric field across the lens.
Eventually an equilibrium is reached between the electrostatic
forces acting on the polar liquid and the surface tension of the
system to create a lens surface with a stable curvature.
Carefully matching the density of the liquids at these scales
makes EWTLs very resistant to the effects of gravity due to orien-
tation and vibrations, which makes it ideally suited for a miniature
head-mounted system. Commercial EWTLs have also demon-
strated very large tuning ranges, on the order of 50 diopters. Fur-
ther, the responses of EWTLs to input voltages are well described
by simple oscillators. Although EWTL focusing speed is not as
rapid as some other ETLs, by engineering the voltage input func-
tion, the lens response time can be brought to less than 20 ms,
which is within the range of what is required for an active scanning
system [74]. EWTLs also have the potential to perform extended
optical functions, such as beam steering and wavefront shaping
[75, 76]. Because of the small aperture size, these lenses are not
frequently used in bench-top microscopy applications. However,
EWTLs are good candidates for robust axial focusing solution for
miniature microscopes.

3 Methods

This section describes in detail the design, construction, and testing

of the two-photon fiber-coupled microscope (2P-FCM), with axial
scanning enabled by an integrated EWTL and lateral scanning
achieved with the use of a CIFB. The use of an EWTL is ideal for
this application as it is lightweight, is compact, has low-power
requirements, and is immune to motion and orientation
[68, 77]. The optics are packaged in a light-weight 3D-printed
enclosure. Pulse propagation through the CIFB is controlled by
careful pre-compensation of the dispersion of glass and verified by
spectrally resolved auto-correlation measurements. Testing and
verification of the 2P-FCM performance is done using imaging
resolution test targets and fluorescent beads in thick agarose pre-
parations. Finally, we describe how to image in vivo neuronal
activity by 3D-imaging of neurons in the motor cortex of a freely
behaving mouse and in the piriform cortex using a GRIN relay lens
combined with the head-attached 2P-FCM. A baseplate is perma-
nently affixed to the mouse skull for attachment and alignment for
repeated imaging over the same brain region.
 Miniature Multiphoton Microscopes 201

3.1 Overall System The experimental setup for imaging with the 2P-FCM is shown in
Design Fig. 6. The distal optics of the 2P-FCM are housed in a two-part
3D-printed enclosure, which can be repeatedly attached to a base-
plate affixed to the animal’s head. A CIFB couples the distal imag-
ing optics to a custom 2P-LSM system to relay the excitation laser
and collect emitted fluorescence from the sample. Laser-scanning
over the CIFB forms an image of the sample while the fluorescence

Fig. 6 2P-FCM imaging system. Pulses from a Ti:Sapphire laser source are spectrally broadened through
polarization maintaining fiber (PMF) and chirped using a grating-pair pulse stretcher. Output pulses are focused
and scanned onto the surface of the CIFB through scanning mirrors, scan/tube lens relay, and 10x Objective.
Fluorescence emission is collected by the CIFB and directed to a photon counting PMT through a dichroic filter.
The collected signal is amplified and transformed to logic levels to be detected by the DAQ counter
202 Baris N. Ozbay et al.

collected back through the fiber cores is detected by photodetec-

tors housed in the 2P-LSM after spectral filtering. The individual
components of the system are discussed in detail below.

3.1.1 Laser-Source The excitation source is a Spectra-Physics MaiTai HP Ti:Sapphire

pulsed laser, with ~80-fs duration pulses tuned to a center wave-
length of 910 nm and operating at 80-MHz repetition rate. The
beam power is controlled by a half-wave plate on a rotation mount
(Newport Conex-AG-PR100P) followed by a Glan-Taylor polar-
izer (Thorlabs GT10-B).

3.1.2 Spectral and In order to obtain the shortest pulses at the sample, the laser is first
Temporal Pulse Pre- sent through a pre-compensation setup optimized for propagating
compensation through a 1.0 m length CIFB (FIGH-15-600 N, Fujikura). The
output from the laser is focused into a 0.5 m length polarization
maintaining (PM) fiber (PM780-HP, Thorlabs) that causes spectral
broadening of the pulse. The output of the PMF is collimated with
a fixed fiber-collimation lens (F220APC-780, Thorlabs). The
1.0 mm beam is then expanded through a 3.75 beam expander
to reduce the irradiance on the gratings. The beam is reflected off of
the two gratings at near Littrow angle separated by 265 mm. It is
double passed through the grating pair using a retroreflector with
the beam exiting at a different height. The gratings are reflective
ruled gold with a density of 300 grooves/mm (49-572, Edmund
Optics). The grating-pair stretcher applies ~66,000 fs2 of negative
GDD to the laser pulse to compensate for the positive dispersion
from the 0.5 m length PMF, 1.0 m length CIFB, and additional
non-linear dispersion at the power levels used in the experiments.
The precise grating separation distance was determined empirically
by maximizing the fluorescence signal while imaging a fluorescent
test slide (Chroma Technology) through the 2P-FCM. After
traveling through the grating pair, the beam size is reduced by
2.5 with a reverse beam expander to optimize the beam diameter
for coupling into the CIFB.

3.1.3 2P-LSM Bench-Top The beam is scanned through a galvanometric mirror scanning
System system (6215H, Cambridge Technologies), relayed through a
50 mm FL scan lens and 180 mm FL tube lens in an Olympus
IX71 microscope. The beam is focused and laterally scanned on the
surface of the CIFB using a 10/0.4 NA Olympus UPLANSApo
objective lens. A XYZ-translation stage (CXYZ05, Thorlabs) is used
to accurately align the fiber to the focus of the objective lens. The
beam propagates through the CIFB and then through the distal
miniature optics and focused onto the sample. The fluorescence
emission is collected by the same optics, back through the CIFB.
Because the miniature optics are not chromatically corrected, green
emission light is projected onto a ~50 μm diameter area on the
CIFB surface. Power transmission through the CIFB was measured
 Miniature Multiphoton Microscopes 203

using a 532 nm light source and was found to have a ~ 67% effi-
ciency through the CIFB when collected through multiple fiber-
cores. The 10 objective collects the fluorescence emission from
the CIFB and is separated from the excitation laser path by a
primary dichroic mirror (T670LPXR, Chroma Technology). A
second dichroic mirror (FF562-Di02, Semrock) splits the red and
green fluorescence that is focused using an achromatic doublet lens
(LB1761-A, Thorlabs) on separate large-area photon counting
photo-multiplier tubes (PMT) (H7422P-40, Hamamatsu). The
output pulses from the PMTs are amplified by a high-bandwidth
amplifier (ACA-4-35db, Becker & Hickl GmbH) and converted to
logic-level pulses by a timing discriminator (6915, Phillips Scien-
tific). The pulses are counted by a data-acquisition card (PCIe-
6259, National Instruments) at a rate of 20 MHz. The counts are
sampled and binned by pixels and converted into an image in
custom software in Labview (National Instruments) that also con-
trols the EWTL driver.

3.1.4 2P-FCM Miniature The imaging system for the 2P-FCM was designed in Zemax
Optical System Design optical design software. Models for the stock lenses and for the
EWTL were obtained from the manufacturers (Edmund Optics,
Thorlabs, and Varioptic). The CIFB (Fujikura Ltd. FIGH-15-
600 N) has an outer diameter of 700 μm, an active image diameter
of 550 μm, and a length of 1.0 m. There are ~15,000 cores with
core-to-core spacing of 4.5 μm and core diameter of 3.2 μm, as
previously reported by Chen et al. measured with scanning electron
microscopy [55].
The miniature optics contained in the head-mounted 2P-FCM
imaging system are shown in Fig. 7. The fiber-coupling lens is an
asphere (FL: 6.2-mm, diameter: 4.7-mm, Edmund Optics 83-710)
to collimate the light diverging from the fiber bundle. The electro-
wetting lens is placed in the collimated beam and the light is
refocused onto the sample by an objective lens consisting of a
plano-convex lens (FL: 7.5-mm, diameter: 3.0-mm, Edmund
Optics 49-177), and an aspheric lens (FL: 2.0 mm, diameter:
3.0 mm, Thorlabs 355151-B). The nominal magnification of this
imaging system is 0.4 and field-of-view (FOV) is ~220-μm,
corresponding to the de-magnified CIFB active imaging diameter.
Similarly, the lateral sampling resolution is the de-magnified core
spacing, which is ~1.8 μm.
A commercially available EWTL (Varioptic Arctic 316) is used
to control the axial focusing of the 2P-FCM imaging system. The
predicted working distance and other imaging properties from
Zemax at these three different voltage settings are summarized in
Table 1. The optical power range of the EWTL is specified as 16
to +36 diopters. The optical system is optimized through the full
focal range of the EWTL to minimize magnification change, maxi-
mize the axial scan range, and maximize the working distance of the
2P-FCM.
204 Baris N. Ozbay et al.

Fig. 7 Optics of the 2P-FCM miniature microscope head that focuses excitation light from CIFB cores onto the
tissue. The CIFB-coupling asphere collects the light from the cores of the CIFB, which are then passed through
the aperture of the EWTL. The plano-convex lens and the objective asphere focus the light onto the tissue
through a #1 coverglass with 0.15-mm thickness

Table 1
2P-FCM optical parameters from Zemax through a range of focal lengths

EWTL control (VRMS) EWTL optical power (m1) Working distance (μm) Magnification NA
60 35 450 0.41 0.43
42 0 570 0.40 0.44
25 -16 690 0.39 0.45

The 2P-FCM has low excitation NA of 0.45; however, the large

effective area CIFB results in a higher collection NA of ~0.6. This is
illustrated in Fig. 8 showing the Zemax model for 910-nm forward
excitation and 532-nm backward emission.

3.1.5 3D-Printed The enclosure for the 2P-FCM optics is designed in Solidworks 3D
Miniature Head-Mount CAD software (Dassault Systemes). The packaging is split into
Design three sections: top, bottom, and baseplate, shown in Fig. 9. The
top-section contains the CIFB ferrule, held in place by two
set-screws, and the fiber-collimating asphere. The bottom-section
contains the objective lenses. The unmounted lenses are held in by
friction in precisely sized openings. The top section has two curved
tabs that interface with slots in the bottom section, which help to
ensure reproducible alignment. The EWTL and the electrode are
sandwiched between the bottom section and the top section with
an O-ring that ensures good electrical contact. The flat-flex
 Miniature Multiphoton Microscopes 205

Fig. 8 Numerical aperture comparison of forward excitation light at 910 nm (top) and backward emission light
at 532 nm (bottom). Note that the blue lines indicate the optical rays entering the system on axis, while the
green and red are off-axis rays. Largest emission and excitation field positions are matched at 220-μm FOV.
Note that the backward emission is defocused on the fiber bundle end so that multiple fibers are used collect
the fluorescent emission

Fig. 9 3D-printed enclosure. (a) A two-part 2P-FCM snaps together to secure the EWTL and electrode. (b)
Photo of 3D-printed parts, top: before any processing with supports still attached and bottom: assembled
2P-FCM
206 Baris N. Ozbay et al.

electrode cable exits the enclosure through a small slot between the
sections. The top-section tabs have a single thread at the end, which
interfaces with the baseplate as shown in Fig. 9a. In this way, the
baseplate is pulled up against the bottom section by the top section.
This greatly improves rigidity when attached to a moving animal.
The baseplate is designed with ridges and holes to improve the
adhesion of the cement for attachment to the animal skull. The
entire enclosure is 3D-printed using a high-resolution projection-
based resin printer (Kudo3D Titan 1), with a resolution of 50 μm.
The material used is a photo-curable resin (3DM-XGreen) dyed
with 0.5% molybdenum disulfide to decrease light scattering and
thus increase feature resolution. A photo of the top and bottom
sections immediately after printing is shown in Fig. 9b. 3D printing
allows optimization of the prototype and easily enables design
changes, such as the inclusion of GRIN lenses for deep-brain
imaging.

3.2 Test Sample Resolution and axial scan range measurements were performed by
Preparation imaging fluorescent beads embedded in agarose (Sigma-Aldrich
A9414) and a USAF 1951 resolution target (Edmund Optics
38-257). 2-μm yellow-green fluorescent beads (Invitrogen
F8853) were used to measure axial scanning extent as well as lateral
and axial resolution.
Low-melting-point agarose was prepared at a concentration of
0.5% in water. The 2-μm diameter fluorescent yellow-green beads
were diluted in the agarose to a concentration of ~2.0  107 beads/
mL. Approximately 2.0 mL of solution was placed on a #1 cover-
glass and allowed to set at room temperature. The beads were
imaged in sequential axial planes by a 20x 0.75 NA Olympus
UPLANSApo objective with a motorized stage and separately by
the 2P-FCM by changing the voltage applied to the EWTL.

3.3 Mouse Imaging All experiments were approved and conducted in accordance with
Setup the Institutional Animal Care and Use Committee of the University
of Colorado Anschutz Medical Campus. Three-month-old male
C57BL/6 mice (neocortex recordings, Jackson Laboratories
stock No 000664) or Nst1-Cre (piriform recordings, MMRRC
stock No 030648-UCD) were anesthetized by intraperitoneal
ketamine-xylazine injection. The skin above the target site was
numbed by lidocaine injection and retracted to expose the skull.
For cranial window recordings in neocortex, the mouse was
injected with an adeno-associated virus driving the expression of
GCaMP6s under the synapsin promoter (AAV5.Syn.GCaMP6s),
similar to procedures in [78]. The coordinates of the injection
targeted the hindlimb somatosensory cortex, 0.2 mm posterior to
bregma and 1.5 mm lateral to the midline, at a depth of 300 μm
 Miniature Multiphoton Microscopes 207

[79]. The injection volume was 0.66 microliters delivered with a

glass micro-pipette through a 0.5-mm hole drilled at the target site.
For GRIN lens recordings in piriform cortex, the mouse was
injected with 1 microliter of AAV5-hsyn-DIO-GCaMP6s into
anterior piriform cortex (AP:0.1 mm, ML:3 mm, DV:4.1 mm).
One month after AAV injection, the mice used for neocortex
recordings were implanted with an optical cranial window near the
injection site, using standard techniques as previously described.
Briefly, mice were anesthetized by isoflurane inhalation and the skin
under the scalp was numbed by subcutaneous lidocaine injection.
The skin above the skull was removed to expose the injection site
and skull surface. A 2-mm square window of skull was removed
immediately anterior to the injection hole to expose the dura mater.
The opening was covered with a 2-mm square #1 coverglass and
secured in place with cyanoacrylate glue. Dental acrylic cement
(C&B-Metabond) was used to cover the skull surface. The presence
of fluorescence signal was confirmed with standard 2P-LSM using a
20 1.0 NA Zeiss Plan-Apochromat water-immersion objective.
For piriform recordings an Inscopix 1-mm diameter 9-mm length
GRIN lens (Inscopix, 1050-004596) was implanted to the region
targeted for AAV virus injection and a custom-made headplate was
placed with dental acrylic cement (C&B-Metabond) 2 weeks after
AAV injection.
The baseplate attachment procedure is similar to what has been
described for other miniature head-attached microscopes. While
the mouse was still anesthetized, the 2P-FCM was held and posi-
tioned above the window with a micromanipulator (Sutter
MP-285) until fluorescence signal could be observed with widefield
epifluorescence through the bundle. The target region was chosen
with two-photon imaging and the 2P-FCM was positioned to the
region with the baseplate attached. The baseplate was then secured
to the existing acrylic with black acrylic cement (Lang Dental Jet
Acrylic). After allowing to set for ~30 min, the 2P-FCM was
removed, leaving the baseplate in place, and the mouse was allowed
to recover.
The imaging setup is illustrated in Fig. 10. The mouse was
lightly anesthetized with isoflurane inhalation. The baseplate was
carefully gripped by thumb and forefinger and the 2P-FCM was
inserted and secured with a quarter-turn. The EWTL electrode was
connected to light-gauge wires that were draped, along with the
CIFB, over a horizontal metal post above the behavior cage. The
mouse was allowed to recover in the behavior cage for imaging. The
cage was illuminated by red light to minimize coupling into the
fluorescence detection path, and a camera (Logitech C615) was
positioned above the cage to monitor behavior during imaging.
208 Baris N. Ozbay et al.

Fig. 10 Mouse attachment. (a) The CIFB is attached to a coupling objective on the proximal end and the
2P-FCM on the distal end. (b) 2P-FCM is attached to the permanent baseplate on the mouse with a quarter-
turn

3.4 Image The images from the 2P-FCM show a honeycomb pixelation pat-
Processing tern due to the packing of the cores of the CIFB. Several methods
have been described to de-pixelate images from CIFBs [53, 80,
81]. The simplest methods involve low-pass filtering with either a
blurring function [82] or masking the image in the frequency
domain [83]. However, two-photon imaging through a CIFB has
the additional complication of the non-uniformity of the fiber
cores. Each core is assumed to have a unique sensitivity, due to
the variability in diameter, shape, NA, and amount of cladding
between adjacent cores. This manifests as discrete variations in
image intensity across the FOV.
This was addressed by programmatically dividing out the sensi-
tivity of each core and interpolating the core values to remove the
pixelation pattern. A flat map of the full field CIFB fiber-cores was
taken by imaging a fluorescent test slide (Chroma Technologies)
with the 2P-FCM, with an example shown in Fig. 11a. The flat-
map stores the centroid coordinates of the cores and their
corresponding sensitivity. The processing was performed with cus-
tom software (Matlab, Mathworks). Each image to be analyzed was
registered to the flat-map, which allowed the identification of the
cores. An example of a raw pixelated image is shown in Fig. 11b.
The relative sensitivity of each core was compensated by dividing by
the flat map values. The honeycomb pattern was eliminated by
using the nearest neighbor interpolation method [84]. A Savitsky-
Golay filter was used to reduce the added single-pixel noise intro-
duced by the core multiplication factor during flat normalization.
 Miniature Multiphoton Microscopes 209

Fig. 11 Image processing of two-photon imaging through CIFB. (a) Flat-field map showing non-uniformity due
to differences in sensitivity in individual cores in the CIFB. (b) Unprocessed image of cells in mouse cortex with
fiber-pixelation. (c) Post-processed image after fiber-cores were corrected with flat-field mask and re-gridded
into a typical square pattern. Grid lines added to emphasize pixels

During the interpolation, the fiber cores were registered to a square

pixel grid for straightforward analysis. An example output frame is
shown in Fig. 11c.
For the processing of temporal scans, each frame was processed
with the same flat-map alignment so that the cores are static in the
field. Once the honeycomb pattern and CIFB-induced intensity
variation were removed, a clustering algorithm was used to identify
regions of interest (ROIs) of high correlation [85]. Significant
changes in cytosolic Ca2+ were identified as changes in fluorescence
larger than three standard deviations above baseline within
each ROI.

3.5 Testing and The lateral and axial resolution and the axial focusing range of the
Calibration of 2P-FCM can be tested by imaging 2-μm diameter yellow-green
Resolution, fluorescent micro-beads embedded in clear agarose. The lateral
Magnification, and resolution is fundamentally limited by the average spacing of the
Axial Scan Range fiber cores in the CIFB. With the core-to-core spacing of ~4.5 μm
and 2P-FCM magnification factor of 0.4, the theoretical lateral
sampling at the object is ~1.8 μm.
The beads are imaged with the 2P-FCM at sequential focal
planes by tuning the EWTL focus in discrete steps to obtain a
Z-stack. The images are processed to remove the fiber-pixelation
pattern as described in the previous section. Figure 12 shows a
comparison of processed images of the beads imaged with a 20
0.75 NA objective and the 2P-FCM. The average lateral and axial
line profiles of 5 beads measured from different focus positions are
fit to a Gaussian function. The axial bead size measured by the 20
objective is 4.5-μm FWHM and with the 2P-FCM it is 9.9-μm
FWHM. The lateral bead size measured by the 20 objective is
1.7-μm FWHM (dotted grey line) and with the 2P-FCM it is 2.6-μ
210 Baris N. Ozbay et al.

Fig. 12 Axial and lateral resolution tested by imaging fluorescent beads with a 20x Olympus objective (dashed
lines) or with the 2P-FCM (solid lines). Reprinted from [92]

Fig. 13 Measurement of axial scan range. (a) Side-projection of ~2-μm diameter fluorescent beads
suspended in clear agarose and imaged with a 20x 0.8 NA Olympus objective (green) using 910 nm excitation
light and a motorized stage or with the 2P-FCM while varying the EWTL power (red). (b) Predicted scan range
as the EWTL optical power is changed modeled in Zemax (grey line) and Z-positions of measured beads (black
circles)

m. The lateral bead size is larger than the diffraction limit as it is

limited by the fiber bundle spacing. With a bead size of ~2 μm,
non-uniform sampling of the bead with multiple fiber cores causes a
larger effective lateral profile. The axial profile measurements of
both the 20x objective at 0.75 NA and the 2P-FCM at 0.45 NA
are similar to what is expected from the diffraction-limited
calculations.
In order to calibrate the electrowetting lens voltage, the sample
is imaged by a 20 0.75 NA objective and 2P-FCM. Figure 13a
shows side projections of beads overlaid in green and red, respec-
tively. The same region of the agarose-bead sample was imaged in
both cases, such that most of the same beads appear in both
 Miniature Multiphoton Microscopes 211

Z-stacks. This made it possible to compare directly their apparent

size and axial location to determine the 2P-FCM scan range. The
predicated axial focus plane through the EWTL focusing range
from Zemax and actual bead positions are shown in Fig. 13b
(gray line and black dots, respectively). The full focusing range
did not span the range predicted (240-μm predicted vs. 180-μm
measured), likely due to under-performance by the EWTL at the
high end of the optical power range.
The optical magnification from the CIFB to the target was
evaluated by imaging the group 6, element 2 square on the USAF
1951 resolution target with the 2P-FCM as shown in Fig. 14. The
imaging diameter of the CIFB is 550 μm. The magnification at
three different optical power settings for the EWTL is measured by
comparing the scaled size of the resolution target through the
CIFB with the actual size. The results are summarized in Table 2.
The magnification is measured to be ~0.4, varying by less than 5%
through the focusing range, agreeing closely with the predictions
from the Zemax model.

Fig. 14 Measurements of magnification of 2P-FCM. Elements of a USAF 1951

resolution target were imaged through the CIFB at three different focus settings.
The known size of the elements is indicated
212 Baris N. Ozbay et al.

Table 2
Measured magnification variation through focus

EWTL control (VRMS) Z-Depth (μm) Predicted magnification Measured magnification

60 35 0.41 0.405
42 200 0.40 0.393
25 275 0.39 0.388

4 Discussion

4.1 2P-FCM Imaging The following experiments on three-dimensional, tilted-field scan-

Capabilities ning, and two-color imaging demonstrate the potential features of
the 2P-FCM as an extremely versatile device.

4.1.1 3D Imaging To demonstrate volumetric imaging, a 1 mm thick sample of fixed

mouse cortical tissue was imaged with the 2P-FCM. The sample
was fixed with 4% paraformaldehyde and has cells that express
enhanced green fluorescent protein (eGFP) driven by proteolipid
protein promoter. The eGFP labels oligodendrocyte cell bodies in
the cortex. Axial scanning was accomplished by sequentially tuning
the EWTL across the full focal range in 36 discrete steps, acquiring
36 image planes to form a Z-stack. The images were processed to
remove the fiber-core pattern as described in the Methods section.
A 3D-volume image was created using ImageJ/Fiji software and is
shown in Fig. 15. Over 200 cells were observed in this volume.

4.1.2 Tilted-Field A simple but effective way of increasing the functionality of a

Imaging microscope that has a rapid axial focusing mechanism is tilted-
field imaging. Further, because there are no moving parts, there is
no concern for the stability of the specimen during the scan. The
tilted-field scan is accomplished by driving the EWTL with a con-
trol voltage that changes linearly with the slow axis voltage during
the scan. Figure 16 shows the results of performing this test on
fixed, thick brain tissue with sparse fluorescence signal from neu-
rons expressing GCaMP6s. Twenty-five total angles were acquired,
12 in each direction, varying the voltage on the EWTL from 40 to
50 VRMS across the slow axis of the raster scan.

4.1.3 Multi-Color Separating fluorescence colors is difficult in many miniaturized

Imaging microscopes because the detection path is exclusive and often also
needs to be miniaturized. Multi-color imaging can be an important
feature for researchers who want to investigate the co-localization
or relative abundance of certain physiological markers. The
2P-FCM offers an advantage in that two-photon excitation at a
single wavelength can excite dyes that have separated emission
 Miniature Multiphoton Microscopes 213

Fig. 15 3D imaging with 2P-FCM of fixed tissue with oligodendrocytes expressing eGFP. (a) 3D volume
acquired by the 2P-FCM (220 μm dia. Lateral  180 μm axial) with over 200 cells in the image. (b) Processed
image of a single slice in the stack after filtering to remove pixelation pattern. (Reprinted from [92])

spectra. A common example is GFP and tdTomato, which are both

efficiently excited at ~900 nm with pulsed light, but have spectrally
separate emission bands. Another feature of a fiber-coupled system
is that the emission signal can be spectrally de-mixed after returning
through the fiber-bundle. The same filters and detectors used in a
benchtop multiphoton microscope for multi-channel imaging can
be readily employed for 2P-FCM imaging.
Figure 17 shows two-color imaging with 900 nm two-photon
excitation of tdTomato and GFP simultaneously with both a 20x
Olympus objective and the 2P-FCM. The cells are GFP-expressing
mature oligodendrocytes and tdTomato-expressing oligodendro-
cyte lineage cells and sparsely labeled astrocytes (Mobp-EGFP;
Olig2-Cre; R26-lsl-tdTomato triple transgenic mice). The two
experiments were performed using the same detectors and filters.
This provides an example of how the 2P-FCM can act as a swap-
pable objective lens for mouse imaging, without needing to change
the filters or detection path to acquire images on different channels.

4.2 2P-FCM Imaging For in vivo 2P-FCM imaging a 3D-printed baseplate is permanently
In Vivo attached to the mouse. During each imaging session, the 2P-FCM
is attached to the baseplate with little force on the skull. A photo of
a baseplate attached to a mouse with a cranial window is shown in
Fig. 18a. Figure 18b shows a photo of a mouse with the 2P-FCM
attached. The CIFB and electrode wire for the EWTL are draped
passively over a horizontal post to reduce the weight on the mouse.
When attached, the mouse is able to move around freely in a small
area (about 1200 square). The movement is somewhat restricted by
the length of the CIFB (1.0 m) and torsional resistance of the
214 Baris N. Ozbay et al.

Fig. 16 Tilted-field imaging enabled by rapid focusing of the EWTL. (a) Maximum intensity projection of fixed
mouse brain tissue expressing GCaMP6s in neurons, acquired by the 2P-FCM. Arrows indicate cell bodies
retained in fields. (b) Side projection of the volume. The same cell bodies are indicated by the arrows. The
planes for the horizontal and angled scan limits are indicated. (c) Tilted field images taken from selected
planes at angles (30,0,30) degrees as indicated in (b). (Reprinted from [92])

CIFB, which prevents the rotation of more than about 180 . It was
found that, after short acclimation (~30 min), mice could traverse
the entire behavioral area and habituated to the restrictions. Future
implementation may include a commutator with rotation encoder
for realignment, which has been shown previously [86].
Figure 19 shows a widefield epifluorescence image of the
region for an implanted mouse through the 2P-FCM. The left
image shows the background fluorescence and vasculature on the
day of implantation. The right image was taken 17 days later
showing that the baseplate is stable and only a slight lateral shift
in alignment is observed.
 Miniature Multiphoton Microscopes 215

Fig. 17 Multi-color imaging. (a) Maximum intensity projection of a region of brain tissue acquired with a 20x 0.75
NA objective. Yellow cells are oligodendrocytes (Green and Red), while red-only cells are astrocytes and
oligodendrocytes. Two astrocytes are marked with arrows and are easily identified by the characteristic bushy
morphology. (b) Same tissue imaged with the 2P-FCM, using the same detectors, filters, and excitation wavelength.
Green and red cells are visible in the field, with likely astrocytes marked by arrows. (Modified from [92])

Fig. 18 Mouse 2P-FCM attachment photos. (a) Baseplate implanted on mouse with cranial window. (b) Mouse
behaving with 2P-FCM attached

The expression level in the neurons virally transfected with

GCaMP6s in the cortex was verified by histology shown in
Fig. 20. Neuronal cell bodies are seen in layers 4/5, as well as layers
2/3 in lower density.
216 Baris N. Ozbay et al.

Fig. 19 Implant stability over 17 days imaged with widefield epi-fluorescence.

Scale bar 50 μm

Fig. 20 Histological coronal section of mouse injected with GCaMP6s virus,

12 weeks after injection, showing good expression in layers 2–5 of motor cortex

4.2.1 2P-FCM In Vivo In vivo two-photon imaging of neuronal activity through the
Mouse Imaging Through 2P-FCM was performed in an awake and mobile mouse expressing
Cranial Window GCaMP6s in cortical neurons. The mouse was allowed to wander
freely in a 700 by 1100 plastic cage. The cage was filled with sawdust as
well as food and various novel objects, such as raisins and tissue
paper, to motivate the mouse to explore the environment while
attached to the 2P-FCM. A 3D projection of a Z-stack taken by
 Miniature Multiphoton Microscopes 217

tuning the EWTL focus, showing the imaging volume was acquired
in Fig. 21b, c. Bright cell bodies and processes were visible down to
160 μm, corresponding to cortical layers 2/3. Time-courses were
acquired sequentially at three focal planes at z ¼ 50, 95, and
140 μm below the cranial window. At the deepest focal plane
(z ¼ 140-μm), the active regions are round objects ~10–20 μm in
diameter, which are likely cell bodies. At the middle depth of
95 μm, there is a mixture of processes and cell bodies, while closer
to the brain surface, activity appears predominately from processes
as shown in Fig. 21d. Figure 21f shows the time courses of the ΔF/
F signal for the five indicated regions of interest at the three
different depths.
The mouse was recorded with a camera during the imaging
session to correlate the imaging results to the motion of the mouse.
Lateral motion artifacts were present during some of the record-
ings. A motion correction algorithm was used to correct for the
laterally shifting field [87]. The results of the motion correction
indicate a time-averaged motion artifact magnitude of <2 μm, but
had peaks up to 10 μm in some cases. The intensity of bright cells
that did not exhibit fluorescence activity did not vary with the
motion artifacts, so we conclude that the extent of the motion in
the axial-dimension is lower than the depth-of-focus for the
2P-FCM (<10 μm). Overall, these motion artifacts were similar
to those reported in head-fixed 2PE imaging studies [88, 89] and
widefield imaging, which benefits from a much larger depth of field
[4]. The 2P-FCM did not become loose or dislodged during the
imaging sessions, which lasted between 1 and 4 h.

4.2.2 2P-FCM In Vivo The 2P-FCM can additionally be used for imaging in deep brain
Mouse Imaging in Deep regions by coupling to a GRIN lens. The baseplate is attached such
Brain Regions Through that the 2P-FCM is aligned at the center of the GRIN lens and with
GRIN Lens the focus of the 2P-FCM positioned at the image working distance
of the GRIN lens. The 1:1 GRIN relay lens used did not change the
magnification of the 2P-FCM image, although aberrations from
imaging through the GRIN lens reduced the field–of-view (FOV)
in comparison to the cranial window imaging. Additionally, actuat-
ing the EWTL changed the axial focal plane imaged by the 2P-FCM
through the GRIN lens as shown in Fig. 22. As an example of
behaviorally relevant imaging of neuronal activity in deep brain
regions, the 2P-FCM was used to image activity in anterior piriform
cortex in a freely moving female Ntsr1-cre mouse exploring a novel
environment with male bedding. GCaMP6s was expressed virally
using AAV_CWSL.hSyn.DIO.Synaptophysin-GCaMP6s.P2A.
mRuby. Imaging data was processed to remove the fiber bundle
artifact using the custom Matlab software and then analyzed by
manually selecting ROIs where ΔF/F transients exceeded 6 stan-
dard deviations threshold. Behavioral video was analyzed with the
218 Baris N. Ozbay et al.

Fig. 21 Two-photon Ca2+-imaging in an awake and freely moving mouse using the 2P-FCM. (a) Widefield
camera image of the epifluorescence taken through the FCM showing vasculature in the FOV. A black
rectangle indicates the acquisition region for the following images. (b) 3D projection of a Z-stack acquired
by sequentially imaging and focusing through the tissue using the EWTL. (c) Side projection of the same
Z-stack indicating the three depths at which time series were acquired: at 50 μm, 95 μm, and 140 μm depth,
recorded at frame rates of 2.5, 1.3, and 2.0 Hz, respectively. (d) Maximum intensity projections of image
frames that coincide with Ca2 + transients, showing structural changes through the focal depths. Scale bar
is 50 μm. (e) Selected ROIs that contain fluorescence transients that exceed the 6 SD threshold. Time traces
for five representative ROIs are selected for each depth. (f) Detailed time-courses of the ΔF/F signal for the
five indicated regions. (g) All identified transients, aligned by the peak ΔF/F signal, shown in gray with
averaged intensity signal in black. (Modified from [92])
Fig. 22 Behaviorally dependent responses from 2P-FCM recording in piriform cortex in a mouse implanted
with a GRIN lens and GCaMP6s expressing neurons. (a) Still frame of video of mouse behavior environment
containing familiar bedding and novel unfamiliar bedding during freely moving behavior with attached
2P-FCM. (b) Example maximum projections at two separate focal planes, shown with relative distance of
focus from GRIN lens. (c) Distance between the female mouse’s snout and the foreign male mouse’s bedding
versus time. Manual correction was made to marker placement when DeepLabCut misplaced or lost markers.
(d) An example frame of the behavioral video labeled by DeepLabCut. The green marker tracks the mouse’s
body, the blue marker tracks the mouse’s snout, and the red marker is placed on the male bedding. (e)
Summary of activity, showing greater activity in some ROIs as the mouse approaches the novel odor. (f) Max
projection showing six manually selected regions used in the analysis
220 Baris N. Ozbay et al.

DeepLabCut [90] software package which provides markerless

tracking of body parts. The mouse was allowed to explore the
environment for 5 min while recording behavioral video and imag-
ing neural activity with the 2P-FCM. Piriform cortex is the largest
recipient of projections from olfactory bulb, and it is expected to
show an increase in neural activity when the mouse smells novel
odors. DeepLabCut was used to track the snout of the mouse
during the image sequence. The measured distance of the mouse
to the bedding was compared with activity for manually selected
ROIs. Distinct increases in activity can be observed when the
mouse is in close proximity to the novel male bedding.

5 Materials

Pulse Pre-compensator
l 1x Polarization Maintaining Fiber – Thorlabs Custom Fiber
Patch Cable: PM780-HP, FC/APC connectors both ends,
keyed to slow axis, FT800SM-Blue Tubing, 0.5 m in length
l 1x Input Coupler – Thorlabs ZC618APC-B – Variable Magnifi-
cation and Focal Length
l 1x Output Coupler – Thorlabs F810APC-850 – 7.8 mm 1/e2
beam diameter
l 2x Gratings – Edmund 37–127 – 300 line per mm blazed at
760 nm
l 1x D-mirror – Thorlabs PFD10-03-M01 – 1” Protected Gold
D-Shaped Mirror
l 1x Retro reflector – Thorlabs HRS1015-AG – Hollow Roof
Mirror, Ultrafast Enhanced Silver Coating
l 1x linear translator – OptoSigma TSD-65171SUU (Imperial)
+/ 25 mm travel
l 1x dovetail optical rail
l Additional optomechanics – kinematic mirror mounts, grating
holders.
Objective Adapter to Hold CIFB
l 1x - SMA Fiber Adapter – Thorlabs SM1SMA
l 1x - Base Cage Plate – Newport OC1-BT
l 1x - Z Stage – Newport OC1-TZ
l 1x - XY Mount – Newport OC1-LH-XY
l 1x - Holder for Objective – Newport OC1-LH1-TZ
l 4x - Cage Rod – Thorlabs ER4
 Miniature Multiphoton Microscopes 221

l 1x - RMS to SM1 Thread Adapter for Microscope – Thorlabs

SM1A4
l 1x - SMA Fiber Adapter – Thorlabs SM05SMA
l 1x - Coupling Objective – Olympus 10x/0.4 NA.
2P-FCM Fiber and Head Mount
l 1x – Coherent Imaging Fiber Bundle – Fujikura FIGH-15-
600 N – 1.0 m SMA connector one end and plastic ferrule on
other (Myriad)
l 1x Electrowetting lens – Varioptic Artic 360 and controller
l 1x - Fiber-coupling asphere (FL: 6.2 mm, diameter: 4.7 mm,
Edmund Optics 83-710)
l 1x - Plano-convex lens (FL: 7.5 mm, diameter: 3.0 mm,
Edmund Optics 49-177)
l 1x - Aspheric lens (FL: 2.0-mm, diameter: 3.0-mm, Thorlabs
355151-B)
l 1x – 3D printed FCM head mount and baseplate
Imaging Test Targets
l 1x – Green fluorescent flat (TedPella 2273)
l 1x – Green fluorescent resolution target II-IV (Max Levy
DA113)

6 Notes

The following includes detailed notes on how to align the

pre-compensation optics before the microscope and align the
2P-FCM for freely moving mouse imaging. Details including
Zemax optical design, solidworks files, and software can be found
at https://github.com/CUNeurophotonics/2PFCM.

6.1 Optimizing One of the challenges is setting up the single-mode fiber and
Alignment into Single grating pair compressor in front of the microscope. In particular,
Mode Fiber aligning the beam through the single-mode fiber can be challeng-
ing. Make sure to terminate the fiber with an APC (angled physical
contact) on both fiber ends. APC termination minimizes back
reflection because the fiber face is cut at an angle as opposed to a
flat surface which can reflect back along the same optical path and
cause the ultrafast laser to stop modelocking. For input coupling
into the single mode fiber, we use a fiber collimator (Thorlabs
ZC618APC-B) that allows for the adjustment of beam diameter
and divergence. Before the input coupler, set up two steering
mirrors in x, y kinematic mounts to optimize for the beam input
position and angle. It is ideal for the last mirror before the input
coupler to be as close to the input coupler as possible so that it
mostly controls the input angle while the first mirror controls the
222 Baris N. Ozbay et al.

Fig. 23 Method for alignment of single mode fiber. Visualization of red back-propagating beam from visual
fault locator with forward propagating beam from femtosecond laser on IR card. Femtosecond laser is incident
on back of IR card and seen as a green spot on the front side

position. To perform the initial alignment of the laser to the fiber

input coupler, set the laser to 780 nm with 10–20 mW of power.
Higher power will damage the fiber if misaligned and 780 nm is
used to be able to see the laser.
One method to get alignment started is to use a visual fault
locator connected to the fiber end face. One can then spatially
overlap the incoming femtosecond laser beam with the back pro-
pagating light from the locator using the input alignment mirrors,
shown in Fig. 23.
After performing the course alignment, remove the fault loca-
tor and replace with a power meter. Proceed to “walk-in” the laser
with the mirrors: Turn the horizontal knob of the first mirror and
use the other mirror’s horizontal knob to maximize the power and
repeat. If this keeps lowering the power from the initial amount you
had, try turning the first knob in the opposite direction and repeat.
After the horizontal alignment is optimized, do this for the vertical
as well. If this does not work, you are likely in a side lobe of the Airy
disk. In that case walk it out of the local peak by moving steadily in
each direction. It should go down and then up in one of the
directions. Then start the walk in again!
At the end of the alignment, the output power from the fiber
should be ~70–80% of the power measured before the input
collimator.
 Miniature Multiphoton Microscopes 223

Fig. 24 Photo of grating pair compressor. Labels indicate (a) fiber output coupler
on kinematic mount, (b) D-mirror, (c) retro-reflector roof mirror on kinematic
mount and linear stage, (d) output mirror, (e) kinematic mount with iris for
alignment, (f) first grating, and (g) second grating

6.2 Setup and Alignment steps for the grating pair compressor (shown in Fig. 24)
Alignment of the are as follows:
Grating Pair 1. Adjust the output beam collimator (A) until the output beam
Compressor from the fiber is traveling parallel to one of the hole lines on the
optical table and at a constant height.
2. Insert a D-shaped mirror mount (B) and adjust until the beam
is traveling approximately 90 degrees to the fiber output, again
parallel to a hole line. Finely adjust the position of the mirror
until clipping is minimized on the D-shaped mirror and beam is
again traveling at a constant height and parallel to the hole line.
3. Turn the linear stage knob holding the retro-reflector
(C) forward until it intercepts the incoming beam. Adjust the
position until the beam is reflected at a lower height so it passes
below the D-shaped mirror. Adjust the tilt of the retro-reflector
to move the reflected beam laterally until it is again parallel to
the hole line, ensuring that the reflection is close to perfect in
the lateral dimension with only a change in height.
4. Insert a mirror (D) to intercept the reflected beam after passing
beneath the D-shaped mirror to send to the microscope input.
Again, adjust the mount until the beam is perfectly
perpendicular.
5. Using a final mirror mount, re-align the output beam with the
beam path into the microscope.
224 Baris N. Ozbay et al.

6. Fit an adjustable iris into a threaded kinematic mount (E) that

usually holds the beam-expander tube. Adjust the height and
position of the threaded kinematic mount and its post holder
until the beam is traveling directly through the center of the
iris. Two irises may be used to ensure that the tip and tilt of the
output beam is correctly aligned.
7. Attach the gratings with posts onto the linear dovetail rail.
8. Move the linear stage back such that the beam fully avoids the
retro-reflector and lands entirely on the first grating surface (F).
The grating should be adjusted primarily in the tilt axis (lateral
movement of beam) until it lands on the surface of the second
grating (G) with no clipping. The second grating may be
moved laterally on the compound rail carrier assembly to mini-
mize the clipping of the input beam if necessary.
9. Adjust the tilt of the second grating until the beam once again
aligns well onto the retro-reflector and back through the
grating system, exit mirror (D), and iris.
10. Move the linear stage to again push the retro-reflector into the
beam path, such that the gratings no longer interact with the
beam. Check that the alignment is still good. Put a card to
monitor the beam position some distance after the iris (E).
Make a mark on the card where the beam intersects. Move the
linear stage out of the beam path again and check that the
grating alignment is maintained, and clipping is minimal to
none. Finely adjust the gratings until the beam is realigned
perfectly with the mark on the card. At this point, the gratings
should be nearly perfectly parallel to each other.
11. Adjust the distance between the two gratings to desired length,
performing adjustments of the rail carriers to avoid clipping
when possible. For 1.0 m fiber bundle and 1.0 m polarization
maintaining fiber the best separation between 300 groove/mm
grating surfaces is ~265 mm. When properly aligned the
pre-compensator should yield 25% of the incoming power
from the laser.

6.3 Optimizing The FCM objective holder, used to align the proximal end of the
Imaging Through CIFB to the microscope focus, is shown in Fig. 25.
Coherent Fiber Bundle 1. Screw in the FCM objective holder into the 2P-LSM using the
appropriate adapter. Adjust the micrometer on the linear stage
(OC1-TZ) to position the surface of the CIFB at the focus of
the objective. Visualizing the fiber surface is easiest when using
widefield epifluorescence. Center the fiber using the xy adjust-
ments on the cage adapter (OC1-LH-XY).
2. If there is dirt on the fiber repeat cleaning with methanol/air
puffs.
 Miniature Multiphoton Microscopes 225

Fig. 25 Diagram of FCM objective holder to mount the FCM to any two-photon
laser scanning microscope. The holder screws into the objective turret on the
microscope with the appropriate adapter. A linear stage (OC1-TZ) adjusts the
focus of objective to the end face of the coherent imaging fiber bundle (CIFB). A
cage mount with xy translation (OC1-LH-XY) adjusts the lateral position of the
fiber to center it on the objective field-of-view

3. Hold the FCM with a Sutter micromanipulator perpendicular

to the plane you will image. Image an IR card with the laser at
its lowest setting (0.1%). Check centering and re-set the focus.
4. If possible, use an iris after the scan lens to control the field-of-
view. Close down the iris to exclude the edge of the fiber which
has strong fluorescence and can saturate the detectors.
5. Use a green fluorescent flat (Ted Pella 2273) to take an image
to normalize the fluorescence of the different cores. Make sure
that the pixels in the image are not saturated.
6. If there are high intensity pixels on the fiber, one way to remove
these is to set the PMT gain to zero, increase the laser power
and scan for several seconds.
7. Image a pollen slide as a first step before moving on to your
sample.

6.4 Installing the Optimize 2P-FCM imaging as detailed above. Then perform the
Pedestal for the 2P- following steps to install the FCM over the cranial window in an
FCM on the Cranium anesthetized mouse.
1. Hold the 2P-FCM attached to the baseplate perpendicular to
the cranial window. Bring the 2P-FCM close to the cranial
window and zero the Sutter micromanipulator. Focus on the
GCaMP-labeled neurons.
226 Baris N. Ozbay et al.

Important During the cranial window surgery place the head

plate as close to the cranium as possible. A thick metabond layer
between the cranium and the metal plate will make it impossible to
place the 2P-FCM in the position necessary to be able to focus at
depth. If necessary, you can thin the bottom of the FCM baseplate.
2. Perform a z stack with the electrowetting lens and do a time
course recording. When you have found the right location, mix
adhesive and place small amounts around the baseplate to bond
it with the cranium. Take care not to bond the baseplate to the
2P-FCM.

7 Conclusions

We describe a head-mounted 2P-FCM that achieves 3D imaging of

neural activity in a freely moving mouse. The imaging volume is
220-μm diameter by 190-μm depth. The device is optimized for
resolving neuronal somata with a lateral resolution of 2.6 μm and
axial resolution of 9 μm. The 2P-FCM is compact, is lightweight,
and includes an electrowetting lens for active axial scanning. We
demonstrate the use of this device for tilted plane imaging, multi-
color imaging, and fast multiplane imaging. The 2P-FCM was
demonstrated for the imaging of neuronal activity in different
planes of cortical layer 2/3 of a freely moving mouse with minimal
motion artifacts. The 2P-FCM differs from previous head-
mounted microscopes [4, 12, 46, 47, 86, 91] because it allows
live-focusing for a range of ~200-μm depth suitable for imaging
neurons in layer 2/3 of cortex.
The goal of this work has been to create miniature fiber-
coupled microscopes (FCMs) that have the capability to compete
with head-fixed imaging setups for awake and behaving mouse
brain imaging. There have been two key technologies that have
driven the progress of this work. First, the coherent imaging fiber-
bundle (CIFB), which is a tool that has large adoption in the field of
clinical endoscopy. CIFBs allow passive imaging that functions
remarkably well for both single- and multi-photon fluorescence
fiber-coupled imaging. Second, the electrowetting tunable lens
(EWTL), because of its light-weight and mechanical simplicity, is
an ideal tool for miniaturized microscope applications.
The design outlined here can be easily assembled and attached
onto a standard benchtop multiphoton microscope, commonly
available for many labs, making the technology inexpensive to
implement. Future work can further this technology for multi-site
brain recording or potentially combined with two-photon hologra-
phy for imaging and photostimulation in the freely moving animal
with 3D access.
 Miniature Multiphoton Microscopes 227

Acknowledgments

We would like to thank Ethan Hughes for assistance with in vivo

animal imaging work, Juliet Gopinath, Victor Bright, and Robert
Cormack for helpful discussions. We acknowledge Nicole Arevalo
for assistance with animal care, and Elizabeth Gould and Wendy
Macklin for providing brain tissue from Plp-EGFP mice. Funding
for this work was provided by the National Science Foundation
DBI-1353757, CBET-1631912, and IIP-1602128 and the
National Institutes of Health BRAIN Initiative NS099577.

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 Chapter 8

Optogenetics and Light-Sheet Microscopy

Laura Maddalena, Paolo Pozzi, Nicolò G. Ceffa, Bas van der Hoeven,
and Elizabeth C. Carroll

Abstract
Light-sheet microscopy is a powerful method for imaging small translucent samples in vivo, owing to its
unique combination of fast imaging speeds, large field of view, and low phototoxicity. This chapter briefly
reviews state-of-the-art technology for variations of light-sheet microscopy. We review recent examples of
optogenetics in combination with light-sheet microscopy and discuss some current bottlenecks and
horizons of light sheet in all-optical physiology. We describe how 3-dimensional optogenetics can be
added to an home-built light-sheet microscope, including technical notes about choices in microscope
configuration to consider depending on the time and length scales of interest.

Key words Computer-generated holography, Optogenetics, Adaptive optics, Light-sheet microscopy,

Zebrafish

1 Imaging Translucent Organisms

Larval fish, flies, and worms are popular model organisms in devel-
opmental biology [1] and, increasingly, in systems neuroscience
[2–4]. Optical translucency make these organisms well-suited to
visualize physiological functions using high-resolution fluorescence
imaging with sub-cellular spatial resolution. Small size and their
ability to thrive when immersed in water make it possible even to
image embryonic development and behaviors in toto over hours or
days [5].
Light-sheet microscopy, also known as Selective Plane Illumi-
nation Microscopy (SPIM), has emerged as the method of choice
for imaging smaller organisms, offering a number of advantages
over point-scanning microscopy in speed, accessible volume, and
phototoxicity. The light-sheet revival over the last two decades is
tightly associated with important milestones in live tissue imaging,
including the iconic example of whole-brain imaging in larval zeb-
rafish (Danio rerio). Launched by early examples of calcium imag-
ing of fictive activity [6], several research groups worldwide now

Eirini Papagiakoumou (ed.), All-Optical Methods to Study Neuronal Function, Neuromethods, vol. 191,
https://doi.org/10.1007/978-1-0716-2764-8_8, © The Author(s) 2023

231
232 Laura Maddalena et al.

routinely record calcium activity from the nearly 105 neurons of the
young awake, behaving zebrafish. The resulting avalanche of data is
beginning to lead to new insights about the communication
between different brain areas (see [3, 7] for recent reviews).
A natural extension to such imaging studies is the integration of
optical methods for perturbation, such as optogenetics [8, 9],
optopharmacology [10], and cell ablation [11, 12]. The expanding
toolkit of molecular probes offers many optogenetic actuators to
remotely activate or inactivate cellular processes. In the context of
controlling neural activity, this progress in engineering molecular
probes, together with the development of suitable optical methods
[13], makes possible to photostimulate action potentials in cells
expressing photosensitive channels and read out the affected neural
activity using fluorescent reporters for calcium [14] or voltage
[15]. Many of these tools have been developed into transgenic
animal strains [16, 17]. Early demonstrations of optogenetic
manipulations in combination with light-sheet microscopy include
optogenetic control over a variety of physiological phenomena,
especially in larval zebrafish, from the beating of the heart [18] to
cellular control of reflexive behaviors [19].
In this chapter, we describe how optogenetics can be added to a
home-built microscope inspired by the open-source project Open-
Spim [20]. As an example, we describe in detail a microscope
configuration suitable for cellular or sub-cellular optogenetics in
larval zebrafish. The method involves adding two-photon photo-
stimulation shaped by computer-generated holography (2P-CGH).
The stimulation module exploits the high numerical aperture
(NA) detection objective of the light-sheet microscope to simulta-
neously excite multifocal points targeted either to sub-cellular
regions or to multiple somata. The light-sheet module provides
flexibility to readout neural activity in tiny organisms from small
volumes to whole brain. Volumetric imaging is achieved with an
electrically tunable lens, allowing independent control of imaging
depth without moving the detection objective and consequently
the axial location of the stimulation foci.
We provide technical notes on optical alignment, alternative
configurations for different applications, and limitations and chal-
lenges of combining optogenetics with light-sheet microscopy.
Finally, we offer some perspectives on extending all-optical physiol-
ogy to higher spatial resolution in vivo.

1.1 Light-Sheet The functioning principle of light-sheet microscopy is to illuminate

Technologies the sample with a thin sheet of light while collecting the fluorescent
signal at an angle (usually orthogonal) relative to the illuminated
plane. The illuminated plane is aligned with the focal plane of the
detection objective enabling an image to be collected by a camera,
as in a widefield microscope. Whereas laser-scanning confocal
 Optogenetics and SPIM 233

microscopy achieves optical sectioning through rejection of

photons generated outside of the excitation focus, light-sheet
microscopy avoids generating out-of-focus fluorescence. This
approach provides optical sectioning while minimizing photo-
bleaching and photoxicity.

1.1.1 Light-Sheet Light-sheet microscopes can be implemented in a variety of con-

Configurations figurations, distinguished by the position and number of micro-
scope objectives, the sheet-forming illumination optics, and the
detection optics. Readers are referred to several excellent review
articles focused on developmental biology and high-resolution
applications [21, 22]. With respect to brain imaging, variations of
light-sheet microscopy have been driven by two main challenges:
(i) Balancing optical sectioning with a large field of view (FOV).
(ii) Maximizing imaging speed to resolve fast dynamics from fluo-
rescent sensors.
Here, we briefly compare light-sheet configurations used for
whole-brain imaging in zebrafish, as shown in Fig. 1.

Selective Plane Illumination

The basic SPIM design (Fig. 1a) uses two orthogonal microscope
objectives for illumination (I) and detection (D) of fluorescence.
The illumination light is typically shaped into a two-dimensional
sheet with a cylindrical lens [23, 24]. To image a volume, the
sample is either translated with respect to the detection objective,
or the light sheet is scanned with a galvanometric mirror while also
keeping the illuminated plane conjugated to the camera with either
a piezo objective or an electrically tunable lens.
Later it was demonstrated that rapid scanning of a pencil beam
(a long, thin illumination profile in one dimension) could generate
a “virtual” sheet, as seen by the camera, with the major advantage of
reducing light exposure to the sample [25]. This approach is alter-
nately called digitally scanned light-sheet microscopy (DSLM). In
this case, volume acquisition requires a second scanning mirror.
For (relatively) small, translucent samples, where light can enter
the sample from any side, the SPIM design is convenient. Because
two (or more) objective lenses are used, this design decouples the
axial (△z) and lateral (△x, y) (△x, △y) resolutions that scale as the
inverse of the numerical aperture for excitation objective,
△z
NAI-1, and the detection objective, △x,△y
NAD-1.
The constraints on axial resolution limit the usable FOV. The
usable length of a Gaussian light sheet is proportional to its thick-
ness. For this reason, longer Gaussian profiles have poor optical
sectioning and increased phototoxicity by illuminating a sample
slice thicker than the detection depth of field. The most immediate
improvement for obtaining longer 1-D uniform excitation profiles
234 Laura Maddalena et al.

Fig. 1 Selection of light-sheet microscope configurations used for whole-brain imaging in larval zebrafish.
(a) Fluorescence is collected along a sheet of light formed by side-ways illumination. (b) In variations of multi-
view light-sheet microscopy, additional objectives illuminate or collect fluorescence simultaneously allowing
for either greater uniformity of illumination or multiplexing image formation from different angles. (c) In
variations of single-objective LS, a tilted sheet is swept laterally across the sample while collecting a tilted
epifluorescence image. (d) Selective volume illumination microscopy is a hybrid of light-sheet illumination and
extended depth of field detection

comes as a trade-off with temporal resolution: tiling the excitation

light sheet [26] allows, in principle, to select only the central
uniform region of the excitation profile, stitching together multiple
images over an arbitrary FOV. However, in all-optical physiology
experiments, the decreased temporal resolution may be
unacceptable.
 Optogenetics and SPIM 235

An alternative way to obtain uniform illumination over a larger

FOV is to illuminate the sample from two sides using an additional
illumination objective opposite of the first (dual-sided light sheet).
Depending on how the sample is mounted in the microscope, it
may also be possible to illuminate from additional angles [27].

Multiview Illumination
In the case of multiple illumination sheets, each sheet needs to
cover only half of the total FOV, so a lower NA sheet can be used
to better preserve optical sectioning. For instance, the IsoView [28]
light-sheet microscope employs two different DSLM geometries to
simultaneously illuminate the sample from two opposite sides,
collecting the view with two cameras (Fig. 1b). In this method,
combining the overlapping images from multiple angles, it is possi-
ble to achieve isotropic spatial resolution [28]. This parallel excita-
tion also represents a robust solution against sample opacity.
Moreover, employing two sets of galvanometric mirrors in each
illumination arm (one for scanning, one for correcting incidence
angle on the sample) allows to employ online optimization algo-
rithms [29] to partially correct for low-order sample-induced aber-
rations. These improvements come at a cost in terms of both
hardware (the number of parts and alignment difficulty) and soft-
ware complexity. Additionally, because every image is collected
twice, the amount of data collected necessitates high-end storage
capabilities and lengthy analysis pipelines in order to properly fuse
the views into a final volume. An added benefit provided by this
geometry is the sample that can be left stationary, while the scan-
ning is performed by the galvanometric mirrors (to move the
excitation profile in 3D) coupled with piezo motors that keep the
detection objective focused on the illumination plane.

Swept Plane (Single Objective)

A single-objective light-sheet configuration, employing epifluores-
cence, can be obtained in several ways [30, 31] by generating a
tilted elongated focus (Fig. 1c). The tilted sheet is swept laterally
across the sample to image a volume. Swept plane approaches are
gaining ground in neuroimaging because they facilitate high vol-
ume speeds, as reviewed recently by Hillman [32]. The single-
objective geometry has the advantage of using the same sample
preparation as confocal or two-photon microscopy and, uniquely,
can also be extended to samples of arbitrary size [33]. While the
swept plane approach partially sacrifices resolution because every
image is formed collecting planes from regions far from the optimal
focus, re-imaging the tilted plane (and some post-processing) can
recover diffraction-limited resolution. Higher NA objectives with
short working distances can also be used when applying light-sheet
microscopy to small samples (e.g., single cells) [34, 35].
236 Laura Maddalena et al.

Hybrid Light-Sheet Microscopes

A clever approach to improve imaging speed is to increase out-of-
focus contributions in a principled way. Manipulating the detection
point-spread function, for instance by adding spherical aberration
[36] or a cubic phase profile [37], extends the effective depth of
field of the detection objective so that information can be harvested
from a thicker illuminated volume. Other approaches for simulta-
neous volume acquisition borrow from light-field microscopy. For
instance, an exciting direction of hybrid imaging called selective
volume illumination (SVIM) [38] merges light-sheet excitation
with light-field microscopy techniques to allow extremely fast
(tens of Hz) volumetric imaging. The trade-off for resolution is
acceptable for somatic imaging [39], and SVIM significantly
improves contrast over light-field microscopy with widefield
illumination.

1.1.2 Engineering Light-sheet microscopy is intrinsically efficient with photons. The

Illumination Improves local intensity required for light-sheet imaging is smaller than that
Resolution and for confocal techniques [40], including spinning disk confocal
Photodamage microscopy. In fact, with scanning light sheet, the total energy
deposited at each point of a 3-dimensional (3D) sample is reduced
by a factor equal to the total number of sections obtained during
the imaging [41]. This minimizes photodamage to the specimen
and also has a positive effect on the imaging speed. Moreover,
detectors used in light sheet, as CCDs or sCMOS cameras provide
a better dynamical range rather than single-pixel detectors used in
point-scanning approaches (e.g., avalanche photodiodes or photo-
multiplier tubes). A poor dynamic range causes problems of detec-
tor saturation that translates to trade-offs in smaller volume or
slower acquisition time. Light-sheet illumination is less prone to
excitation saturation compared to point-scanning techniques, so it
is not necessary to compromise imaging speed with long frame
exposure times.
To further improve on minimizing illumination intensity and
photodamage, engineering the excitation beam to produce quasi-
non-diffracting beams, in particular, Bessel beams [42, 43], has
strongly impacted the field. Bessel-like beams preserve a small-
beam waist over a longer distance compared to beams with a
Gaussian amplitude profile (Table 1), translating to more uniform
illumination over the FOV of the detection objective.
Moreover, when propagating through inhomogeneous sam-
ples, Bessel-like beams have reduced scattering and beam spreading
due to their self-healing property; namely the beam recovers the
initial intensity profile after an obstacle. However, Bessel beams
have a major downside: a large portion of energy resides in side
lobes, which can spread out for a tens of microns beyond the central
peak. In fact, they may generate fluorescence signal from out-of-
focus planes, preventing the theoretical gain in optical sectioning
 Optogenetics and SPIM 237

Table 1
Beam properties. Bessel beam characteristics depend on the geometry of
an annular mask, placed in a plane conjugated with the back aperture of the
excitation objective. Parameters: e = pixel size; NA = objective numerical
aperture; M = magnification; n = refractive index; λ = wavelength; w =
annulus width; J1 = Bessel function of the first kind; α, α′ = constants
proportional to outer and inner annulus radius, respectively

Beam Axial uniformity Central peak

n λ
Gauss △z = NA2
þ M nNA e λ
2 NA



Bessel 2λ
J 1 ðαr Þ J 1 ð α ′ r Þ
2
w
r - r

while increasing phototoxicity. For this reason, researchers have

introduced methods to reduce the contribution of the side lobes
to the image:
– Prevention: Multiphoton excitation can prevent excitation of
fluorescence in the side lobes because of their lower instanta-
neous intensity [44] (as also discussed in Chapter 10 by Ji).
– Displacement: Interference between multiple illumination rays
can diminish illumination intensity in the side lobes. For exam-
ple, lattice light-sheet microscopy [45] employs a spatial light
modulator as active optical element to superimpose an array of
Bessel beams that destructively interfere and consequently
reduce the impact of undesired side lobes. This solution has
been proven effective not only in increasing resolution when
imaging cellular samples, but also when studying larger animals
(e.g., zebrafish embryos), if combined with adaptive optics
techniques to compensate for the aberrations introduced by
the morphology of larger organisms [46].
– Rejection: Electronically, fluorescence generated by side lobes
can be mitigated by only collecting signal confocal to the
scanned beam [47]. This approach only slightly increases system
complexity since modern CCD and CMOS cameras already
allow to calibrate an internal rolling shutter modality, where
only a few lines of pixels are active at a time, following the
central lobe of the Bessel excitation.

1.2 Design Choices Optimal choice of the light-sheet microscope configuration

depends on the research questions of interest, particularly with
1.2.1 Prioritize Scale,
respect to the spatial and temporal resolutions required. Small
Resolution, or Speed
organisms tend to have smaller cells than mammalian tissues, so
resolution is of particular concern in both imaging and photosti-
mulation. The choice of light-sheet approach is often a matter of
238 Laura Maddalena et al.

choosing the best trade-off between speed and resolution, given

the dimensions and transparency of the sample.
In designing the system described below, we considered appli-
cations involving the larval zebrafish. For this sample, the SPIM
design is convenient. The larval zebrafish brain occupies a volume
of approximately 500×800×300 μm, and neuronal somata are typi-
cally 5–10 μm in diameter. The whole brain can be measured in a
single FOV of a 10x detection objective, with which cellular resolu-
tion is easily achieved. However, we also wanted the flexibility to
image sub-cellular resolution in smaller brain regions, so we have
used higher magnification to achieve sampling of better than 0.2
μm per pixel on the camera. This lateral resolution is achieved with a
high-NA water-dipping detection objective (practically limited to
NA< 1.1 by commercial objectives). To achieve sub-cellular axial
resolution over most of the brain, we chose to apply the superior
optical sectioning of a Bessel beam.
For example, considering illumination with a laser source with
λ = 488 nm, and an excitation objective with NA= 0.29, a Bessel
profile can be generated to cover uniformly 160 μm with a central
peak width of ≃ 0.6 μm. To reach the same FOV, a Gaussian beam
would have more than double thickness (around 10 μm, as calcu-
lated following precisely the Rayleigh length formula): of course,
this value is chosen following a trade-off between length and the
acceptable divergence that can be tolerated at the edges of the FOV.

1.2.2 Type of The experimenter has many options to add photostimulation optics
Photostimulation to a light-sheet microscope, including the variety of methods dis-
cussed in Chapters 1, 3–5, and 11 of this book. Both scanning and
parallel approaches to photostimulation can be applied to small,
translucent samples. In the first case, resonant scanners or galvano-
metric mirrors steer a focused beam across multiple regions of
interest (ROIs), whereas, in the latter case, all the ROIs are illumi-
nated simultaneously by using computer-generated holograms
(CGHs) projected through spatial light modulators (SLMs).
The same excitation strategies applied in living animals require
increased optical sectioning and penetration depth, both provided
by two-photon (2P) illumination. For example, Dal Maschio et al.
[48] have integrated a 2P-CGH module with a two-photon-scan-
ning microscope, generating an instrument capable of identifying
behavior-related neural circuits in living zebrafish larvae. The stim-
ulation is targeted to single soma with a diameter of
6 μm and an
axial resolution of
9 μm over a volume of
160×80×32 μm.
Comparable lateral and axial resolutions for circuit optogenetics are
achieved by McRaven and colleagues [49], in their 2P-CGH setup
coupled to a 2P scanning microscope with remote focusing, to
discover cellular-level motifs in awake zebrafish embryos. On the
other hand, De Medeiros et al. [12] have combined a scanning unit
with a multiview light-sheet microscope. This is a flexible
 Optogenetics and SPIM 239

instrument to perform ablation of single cells in zebrafish embryos

and also localized optogenetic manipulations with concurrent in
toto imaging in Drosophila. Here, the effect of the optogenetic
manipulation can be monitored at the embryo scale with cellular
resolution. Another example of SPIM integrated with 2P scanning
stimulation is presented in [27], where whole-brain imaging and
brain-wide manipulations in larval zebrafish reveal causal interac-
tion. All these works demonstrated that in the small brain of the
larval fish (
0.1 μm3), it is possible to both record and stimulate
with millisecond temporal resolution and single-cell precision over
the full volume of the engaged neural circuit.

1.2.3 One Photon or As mentioned in other chapters, multiphoton excitation is the

Two? simultaneous absorption of n lower-energy photons to electroni-
cally excite a higher-energy single-photon transition. For visible-
absorbing optogenetics chromophores, near infrared (NIR) wave-
lengths (700–1100 nm) are typical for two-photon absorption.
NIR has the added advantage of high penetration depth in
biological tissues [50]. Transparent tissue might seem to obviate
the need for multiphoton microscopy. On the contrary, there are
several arguments for the use of two-photon excitation light-sheet
microscopy.
On the imaging side, scanned beam approaches also made
two-photon excitations feasible because the spherical focus can
generate the highest peak intensity for a given power, extending
light-sheet microscopy to imaging in highly scattering samples
[51, 52]. Even for optically translucent samples such as larval
zebrafish, scattering is noticeably reduced. For brain imaging in
larval zebrafish, two-photon microscopy is often preferred because
it is more orthogonal to the visual system [2]. Imaging with visible
light impacts general brain activity, visual sensitivity, and even
innate motor behaviors due to non-visual opsins [53], though by
carefully avoiding direct illumination of the eyes, it is possible to
deliver visual stimuli and even virtual reality [27].
For photostimulation with cellular, or sub-cellular, precision in
3D tissues, it is crucial to exploit multiphoton absorption for
optical sectioning. Since 2P absorption is a non-linear process, its
probability depends quadratically on the intensity of the excitation
light. The main consequence is an improved optical sectioning
because the stimulation is generated only in the vicinity of the
geometrical focus where the light intensity is the highest
[54]. The resolution of the multiphoton excited fluorescence is
described as the full-width half maximum (FWHM) of the three-
dimensional point-spread function (3D-PSF) of the fluorescence
intensity hi of the excitation. The following equations describe the
dependency of the 3D-PSF hi on the 3D-PSF of the illumination
intensity of two-photon excitation.
240 Laura Maddalena et al.

h 2P ðu,vÞ/jI ðu,vÞj2 , ð1Þ

where u and v are, respectively, the axial and lateral coordinates of
the optical system.
It should be noted that all-optical experiments require careful
control to avoid heating effects induced by NIR stimulation
[55]. Compared to experiments in rodents or organotypic tissues,
small translucent organisms such as zebrafish larvae are fragile and
easily burned. Their small size, typically smaller than the beam waist
exiting the microscope objective, means that a significant fraction
of the animal’s skin is exposed to defocused light, even while a
tight, diffraction-limited focus may be achieved beneath the skin.
When exposed to NIR light, even small amounts of dark skin
pigment can lead to unintentional burning. Furthermore,
ectotherms such as zebrafish and Drosophila lack the ability to
maintain a constant internal body temperature and typically regu-
late temperature behaviorally (e.g., by heat-seeking or heat avoid-
ance behavior [3]). Even a 1–2 K rise in temperature may have a
notable effect on the physiology under study. In two-photon imag-
ing, intensity is typically kept around 0.1–0.5 mW/μm2 [56]. In
both 2-photon imaging and photostimulation, a potential control
experiment to evaluate the 1-photon NIR effect is to test photo-
stimulation with a lower peak power density, e.g., in mode-locked-
Ti:Sapphire lasers, switching into continuous-wave mode provides
a means to obtain the same average power with orders of magni-
tude lower than energy density.

2 Materials

All-optical physiological experiments require an imaging system

that combines a fluorescence microscope with a light path for
photostimulation. The microscope serves dual purpose: first to
acquire a baseline fluorescence image or movie to identify the
spatial location of ROIs and second to acquire a continuous read-
out of the effect of photostimulation. Here we describe an example
of a Bessel-beam light-sheet microscope and a 2P-CGH module.

2.1 Light-Sheet The microscope schematic shown in Fig. 2 is a digitally scanned

Module light sheet implemented by scanning a Bessel beam in a 2D plane.
The combination of an axicon with a plano-convex lens generates a
beam shaped as a hollow cylinder. Since this beam is collimated, any
subsequent conjugate plane can be chosen as the entry pupil of the
microscope. The collimated ring is conjugated with two galvano-
metric mirrors (G1, G2, Thorlabs, ax1210-A) and the back aper-
ture of the excitation objective (EO; Nikon, 10X/NA 0.3 CFI Plan
Fluorite).
 Optogenetics and SPIM 241

Fig. 2 Example configuration of light-sheet microscope with optogenetics (a) The system consists of a light-
sheet imaging module (left, blue lines) and an optogenetics module (right, red lines) that share a common light
path through the high-NA detection objective (DO). In the imaging module, a continuous-wave visible
wavelength laser is shaped in a Bessel beam by an axicon (A) and scanned onto the sample through galvo
mirrors G1 and G2 and excitation objective (EO). Fluorescence collected through the detection objective (DO) is
transmitted through a low-pass dichroic mirror (DM) and imaged onto a sCMOS camera by an electrically
tunable lens (ETL).The fluorescent signal is recorded with the confocal slit detection approach schematically
shown in panel b. In the 2P-DH module, a Ti:Sa pulsed laser beam is magnified through a telescope (L1, L2),
impinges on the SLM, placed in a plane where the wavefront is flat. Lenses (L3, L4) relay the wavefront from
242 Laura Maddalena et al.

Fluorescence is collected through the detection objective (DO;

Olympus, 20X/NA 1.0 XLUMPLFLN), transmitted through a
low-pass dichroic mirror, and finally, the image is formed by a
300 mm tube lens (L5). The image is relayed to the sCMOS camera
(Andor, Zyla 4.2) by a 1:1 telescope (L6 and L7, focal 150 mm).
An electrically tunable lens (ETL; Optotune, EL-16-40-TC-VIS-
20D), positioned in the common focus of the telescope, can scan
the signal at different depths.
To effectively eliminate the out-of-focus emission excited by
the side lobes of the Bessel beam, the design implements confocal
slit detection of the fluorescent signal, as shown in the inset of
Fig. 2b. The slit is virtually created using an active window of the
sCMOS camera that is rolled synchronously with scanning of the
Bessel-beam illumination. In this way, we minimize the detection of
fluorescence excited by the side lobes. The cost is a slower imaging
speed.
The ETL in the detection path allows independent control of
photostimulation in three dimensions and volumetric imaging,
without moving either the objective lens or the sample. The acces-
sible volume is determined by the choice of the tube lens L5. In
fact, given the objective the focal of the tube, lens is directly
proportional to the image magnification. For such a reason, longer
focal lengths give access to a smaller volume, while they achieve
better sampling of the fluorescence signal at the camera pixel. The
ETL in the configuration shown in Fig. 2a allows to image a volume
extending over 500 μm. The photostimulation can be performed
on a volume extending over 200 μm in depth, with the SLM being
the limiting factor in the theoretical axial FOV.

2.2 2P-CGH Module We implement computer-generated holography for spatial pattern-

ing of the photostimulation light. As shown in Fig. 2a, the ultra-
short pulses of NIR light, emitted by the Ti:Sa laser (Coherent,
Mira-900F), pass a combination of half-wave plate (HWP) and
Glan–Thompson Polarizer (GTP) to control the average power. A
mechanical shutter (S; Uniblitz, LS2S2Z1) allows to block or
transmit the light. Then the beam, magnified through a telescope
(L1 and L2, focal 25.4 mm and 150 mm), impinges on the SLM
(Meadowlark optics, P1920-600-1300-HDMI) placed in a plane
where the wavefront is flat (where the Gaussian laser beam is
collimated). Subsequent lenses relay the wavefront from the SLM
ä

Fig. 2 (continued) the SLM to the pupil of the DO. In the focal plane of lens L3, the inverse pinhole (IP) blocks
the zero-order diffraction spot. (b) Left: Scheme of confocal slit detection. An active window (yellow line) on
the sCMOS camera is rolled synchronously with the scanning of the Bessel beam illumination (cyan line). In
this way, we minimize the detection of fluorescence excited by the side lobes of the Bessel beam (light cyan
halo). Right: Bessel beam projected into uniform fluorescent solution. The zoom in on the middle region of FOV
shows the central lobe of the Bessel beam as it appears with confocal slit detection
 Optogenetics and SPIM 243

to the pupil of the DO: here, the Fourier lens L3 (focal 250 mm)
and lens L4 (focal 500 mm) magnify the beam to fill the back
aperture of the objective. In the focal plane of lens L3, an inverse
pinhole (IP, diameter 1.4 mm) blocks the zero-order
diffraction spot.
The light paths of the light-sheet microscope and the 2P-CGH
module join at a dichroic mirror (DM; Semrock, FF01-720/SP-
25), with a cutoff wavelength at 720 nm. This mirror reflects the
NIR stimulation light to the sample and transmits the fluorescent
readout to the sCMOS. Two-photon-excited (TPE) epifluores-
cence can be recorded, and this is useful to characterize the photo-
stimulation beam as described in Subheading 3.1.
The choice of focal lengths of lenses L3 and L4 is important in
the design of the experiment. If the SLM image at the objective
back aperture is smaller than the aperture itself, the NA of the
objective is not fully exploited. This might be an intentional choice.
Otherwise, if the SLM image is larger, some stimulation light gets
lost. The best option is to choose a telescope magnification that
matches the dimension of the SLM image at the back aperture to
the aperture itself. In this case, the lateral resolution is only limited
by the objective NA.
The pixelated structure of the SLM chip introduces in the
reconstructed hologram a zero-order diffraction spot that appears
as a bright spot in the center next to the hologram. To separate the
hologram from the zero-order illumination, we can either proceed
algorithmically, or we can block the zero-order illumination physi-
cally. Since the result given by the second method can strongly
degrade the quality of the image, it is preferable to implement a
combination of the two approaches to preserve the image quality.
The hologram can be displaced from the zero order algorithmically
by introducing a constant defocus. Once CGH and zero order lay at
different depths, the zero-order beam is blocked by means of an
inverse pinhole without impairing the quality of the CGH.
In high-resolution applications, the spatial accuracy of photo-
stimulation is paramount. As reported in [57], the precision to
address a CGH to a specific target depends on the number of pixels
in the SLM and gray scale values available. Several companies
produce SLM with over 1000 pixels in the shorter axis, which
provide the spatial accuracy required for sub-cellular manipulations
and also a high number of degrees of freedom if the SLM is used as
an adaptive element to correct for high-order aberrations. The
accessible lateral and axial FOV for a CGH is inversely proportional
to the SLM pixel size. However, fairly large pixel size (in the order
of 10 μm) is preferred because, assuming a constant inter-pixel gap,
the fill factor increases with the pixel size. A larger pixel size also
reduces the cross-talk between pixels. Cross-talk acts as a low-pass
filter on the CGH, and it is due to fringing field effect that causes
gradual voltage changes across the border of neighboring pixels and
244 Laura Maddalena et al.

by elastic forces in the liquid crystal material [58]. In the setup in

Fig. 2a, we use a 1920×1152 pixel liquid crystal on silicon (LCoS)
SLM with a pixel pitch of 9.2 μm. This device guarantees 95.7% fill
factor and a CGH switching rate of 31 Hz.

2.3 Sample In SPIM, samples are usually mounted in tubes made of fluorinated
Preparation ethylene propylene (FEP), a plastic with refractive index similar to
water. The tube is filled with a solution of water and low-melting-
temperature agarose (
1.5–2%) for short-term imaging (1-3
hours). For larval zebrafish, these conditions ensure stability of
the sample while maintaining good physiological conditions given
the time frame of the experiment. However, as reported in [59] for
longer experiments (over 1 day), it is recommended to use lower
agarose percentage (
0.1%) or methylcellulose solutions (
3%) to
ensure stability but also proper growth of the sample especially
between 24–72 h post-fertilization.

3 Methods

3.1 Microscope The microscope is aligned by visualizing the Bessel-beam profile.

Alignment By using the galvanometric mirror G1, responsible for the planar
scan, we can image the full profile of the Bessel beam at specific
3.1.1 Align the Light-
locations:
Sheet Module
1. Prepare a low concentration solution of fluorescent dye such as
fluorescein (Invitrogen, F1300) diluted in demineralized
water.
2. Fill the sample chamber with the fluorescent solution and turn
on the excitation laser at low power (1 mW).
3. Optimize the beam thickness and uniformity in the center of
the FOV by setting the galvanometric mirror G1 at 0 V.
4. Optimize the beam focus at the edges of the FOV by setting
the galvanometric mirror G1 at its extrema, e.g., ±1 V. For
steps 3 and 4, the galvanometric mirror G2 and the ETL
voltages are kept to 0 V. These settings ensure that the axial
scanning range is centered. As schematically shown in Fig. 3a, a
properly aligned Bessel beam appears as a tiny, bright, and
straight line profile across the FOV. The inset shows a zoom-
in on a good example of Bessel beam where a sharp profile with
side lobes can be observed. Contrarily, Fig. 3b shows an exam-
ple of a misaligned Bessel beam. It is prominently tilted at the
edges of the FOV, and its profile is enlarged.
5. Perform an automated calibration to acquire a planar scan. The
planar calibration correlates the vertical displacement of the
Bessel beam across the planar FOV and the voltage given to
the galvanometric mirror G1. During this calibration,
 Optogenetics and SPIM 245

Fig. 3 Main steps of system alignment. (a) Example of good planar alignment. This image shows an overlay of
the Bessel profile at different positions across the planar FOV. Inset shows a shallow Bessel beam with its side
lobes. (b) Example of misaligned beam across the planar FOV. (c) Affine transformation between light-sheet
and 2P-CGH module: (1) 3D plot of the coordinates fed into the algorithm to generate a point cloud CGH of
random points spanning over a 3D volume. Those coordinates are defined in the coordinate system x′y′z′ of
the 2P-CGH module. (2) 3D plot of the coordinates measured on the TPE image of the CGH. Those coordinates
are defined in the coordinate system xyz of the light-sheet module. (3) Result of the affine transformation
between 2P-CGH coordinates and light-sheet coordinates
246 Laura Maddalena et al.

sequentially change the voltages driving G1 and for each volt-

age and measure the position of the Bessel beam onto the
camera. The resulting data are voltages as a function of posi-
tion. The slope of the linear fit on these data yields the relation
between voltage and displacement.
6. Perform the volumetric calibration. This step allows to syn-
chronize the displacement of the Bessel beam in the volumetric
FOV with the ETL focal plane. This is achieved by sequentially
changing the voltages fed to both the galvanometric mirror G2
and the ETL. For each ETL voltage, scan the full range with G2
and collect an image at each step. For each image, measure the
maximum intensity of the central lobe of the Bessel beam.
Select the value of G2 corresponding to this maximum to get
the excitation profile in focus.

3.1.2 Align the 2P-CGH The 2P-CGH module is aligned by projecting the zero-order dif-
Module fraction beam into the same fluorescent solution used for the light-
sheet alignment:
1. Align the NIR beam to impinge on the SLM with a small angle
and to uniformly illuminate the whole SLM chip.
2. Check that the reflection of the zero-order beam from the SLM
is well-separated from the incoming beam and that these beams
have the same height.
3. Center the zero-order diffracted beam onto the irises placed in
front of the downstream optics.
4. Check the position of the beam at the sample using the sCMOS
camera. The beam must appear as a bright spot in the middle of
the camera FOV. If this spot appears far away from the center of
the camera, adjustments in the upstream optics are needed.
5. Install the inverse pinhole in the optical path and visualize a
CGH projected into the sample.
6. Calibrate the 2P-CGH module with the ETL voltages. For
this, load a series of single-point CGHs with different
z positions onto the SLM. For each CGH, adjust the voltage
on the ETL and acquire an image of the TPE fluorescence
produced by the CGH. Record the second moment of the
spot intensity as a function of the voltage on each image series.
The voltage corresponding to the minimum second moment is
considered the right voltage to have the point in focus. This
procedure links the z position of a CGH to the optimal voltage
needed for an image in focus.

3.1.3 Registration To obtain good fidelity between the stimulation target and the
Between Light-Sheet holographic illumination, it is critical that the light-sheet module
Module and 2P-CGH and 2P-CGH module share a common coordinate system. This is
Module accomplished by first calibrating the scan volume of the LS and
 Optogenetics and SPIM 247

then calibrating a 3D hologram. Both volumetric scans are achieved

by incrementing voltage on the ETL. Once the 2P-CGH and light-
sheet volumes are calibrated with the ETL, the two coordinate
systems are then aligned through an affine transformation (see
Note 1). This calibration is carried out before each experiment,
and the resulting affine transformation matrix is applied to the
ROIs location selected on the image to get the input coordinates
for the holographic stimulation:
1. Compensate for possible rotation and flip between the
2P-CGH and the light-sheet coordinate system. In the setup
shown in Fig. 2, the 2P-CGH coordinate system is rotated by
-90 ° and flipped vertically with respect to the imaging system
because of the orientation of the SLM and the odd number of
mirrors between the SLM and the DO back aperture.
2. Calculate a point-cloud CGH of random spots spanning the
volume relevant to the experiment (see Note 1) by using x′y′z′
coordinates defined in the 2P-CGH system. In the specific case
of the setup described here, the volume is covering
140 × 140 × 200 μm (Fig. 2b).
3. Project the CGH into a concentrated solution of
fluorescent dyes.
4. Collect images of the TPE fluorescence induced by the CGH
and measure the xyz spots position in the light-sheet system
(Fig. 2b).
5. Calculate the affine transformation matrix through least square
minimization between the xyz and the x′y′z′ coordinates.
6. Align the xyz coordinates to the x′y′z′ by applying the affine
transformation matrix.

3.2 Workflow of The general workflow for all-optical physiology experiment using
Light-Sheet the system described above would be similar to other methods
Optogenetics described in this volume. After alignment of the system, it is typical
Experiment to first acquire a baseline fluorescence image or a 3D movie to
characterize anatomical structure and possibly baseline activity.
Then, the ROIs to be stimulated are selected. The criteria to choose
the ROIs from the baseline image are set by the experimenter and
fed into an algorithm for targeting light, e.g., CGH calculation.
Subsequently, we program a pulse sequence encoding for the CGH
where the stimulation light is temporally gated by an external
shutter. Afterward, the fluorescence signal is recorded over time.

3.2.1 Imaging Larval After microscope alignment, mount a zebrafish in FEP tubing. Use
Zebrafish brightfield illumination to orient and position the sample appropri-
ately. Obtain a baseline fluorescence image. A high-resolution vol-
ume is recommended to help with brain registration later in image
analysis. Based on anatomical or functional criteria, select regions of
248 Laura Maddalena et al.

interest (ROIs) for photostimulation. Generate the hologram

(s) and set a gating sequence or method to trigger the photosti-
mulation sequence of interest.

3.2.2 Analysis of Large- Figure 4 shows the analysis of a representative data set measuring
Scale Ca2+ Data Set spontaneous neural activity in a 5 days past fertilization (dpf)
zebrafish larvae expressing nuclear localized GCaMP6s in neurons,
as acquired by volumetric calcium imaging on the described Bessel-
beam light-sheet microscope. Fluorescence was captured in 20 vol-
ume sections of the forebrain, spaced 8 μm apart, with an acquisi-
tion rate of 0.67 Hz. Motion correction and the extraction of
activity traces from individual neurons were implemented with the
open-source calcium image analysis package CaImAn [60]. Motion
artifacts were corrected through piecewise-rigid registration. Active
neurons were then detected in the motion-corrected data through
constrained non-negative matrix factorization (CNMF). To initial-
ize CNMF, the images were first filtered with a Gaussian kernel.
Thereafter, the Pearson correlation with neighboring pixels and the
peak-to-noise ratio was calculated for each pixel (Fig. 4a). Local
maxima in the pointwise product of the peak-to-noise ratio image
and correlation image were used as initialization positions. With
CNMF, the contours of found neurons (Fig. 4b) and activity traces
(Fig. 4c) were then extracted. In total, 2077 active neurons were
detected in the imaged brain volume (Fig. 4d,e).

3.3 Choice of CGH The 2P-CGH module shown in Fig. 2a is based on Fourier holog-
Algorithm raphy and requires an appropriate algorithm to calculate the desired
phase hologram. The aim of CGH algorithms is to retrieve the
phase mask, namely the phase of the hologram field Uh, to address
the SLM by knowing the field Uo of the target at the image plane,
where these fields are one and the Fourier transform of the other.
To generate a hologram of ROIs distributed in 3D, we compute a
3D hologram corresponding to a field Uo defined at different
depths z as schematically described in Fig. 5a. Optically, the Fourier
transform of the field Uh is realized through the objective lens when
the SLM is conjugated to the objective back aperture through a
telescope (L3 and L4 in Fig. 2a).
The choice of the algorithm to calculate CGHs depends on
several considerations. Ideally, the algorithm provides high diffrac-
tion efficiency, uniformity over the volume of interest, and accuracy
between the target and the reconstructed hologram. The algorithm
should also be fast given the real-time nature of optogenetics
experiments. The shape and dimensions of the target photostimu-
lation pattern also influence the choice of CGH algorithm. When
the target object has a complex and extended lateral shape (>
1 μm), image-based algorithms are employed. They enable the
generation of extremely complex illumination patterns in very
short times; however, they are limited to illumination light focused
 Optogenetics and SPIM 249

Fig. 4 Analysis of light sheet calcium imaging data from the larval zebrafish forebrain with the open-source
calcium image analysis package CaImAn. (a) Images were obtained from the forebrain of a larval zebrafish
(5 dpf) expressing nuclear localized GCaMP6s. A total of 20 volume sections were imaged with Bessel beam
light-sheet microscopy at an acquisition rate of 0.67 Hz. To detect neurons, CNMF was initialized by filtering
the motion corrected timeseries data with a Gaussian kernel and calculating the peak-to-noise ratio and
Pearson correlation with 4 nearest neighboring pixels. Local maxima in the point-wise product of the peak-to-
noise ratio image and correlation image were then used as initialization positions. (b) Contours of neurons
250 Laura Maddalena et al.

on a limited number of two-dimensional planes [61]. Conversely,

point-cloud algorithms allow one to target diffraction-limited spots
arbitrarily distributed in the three-dimensional field of view of the
optical system.
Many variations of these algorithms have been developed
(Table 2). Here we classify them into two categories (see Note 2):
classic algorithms, and more recent algorithms developed specifi-
cally for optogenetics applications. Random superposition and
Gerchberg–Saxton [62, 63] are the most known algorithms. The
first offers high speed but poor quality of the CGH in terms of
uniformity and efficiency, whereas the second provides improved
quality with an increased computational time. Recently, new algo-
rithms have been developed that are optimized for speed, such as a
compressive-sensing version of the Gerchberg–Saxton algorithm
[64]. Here the compressive-sensing method allows to reduce the
computational time by a factor of 10 without impairing the quality
of the CGH achieved with the standard implementation of
Gerchberg–Saxton. Another advanced algorithm recently available
is computer-generated holography by non-convex optimization
[61]. Arbitrary 3D holograms are generated through non-convex
optimization of custom cost functions. Another advanced algo-
rithm recently developed, called DeepCGH [65], is based on unsu-
pervised convolutional neural networks. Holograms computed via
DeepCGH showed improved computational times and high effi-
ciency suitable for neurostimulation experiments.
As an example, in the high-NA configuration hereby described,
we implement weighted Gerchberg–Saxton algorithm to generate
stimulation ROIs with a lateral dimension between
6 and 10 μm,
on the order of the size of small neurons in the larval zebrafish
brain. The principle of this approach is schematically described in
Fig. 5b, while Fig. 5d shows a 3D view of an extended CGH
projected into a thick Rhodamine slide. On the other hand, when
targeting sub-cellular regions, the compressive-sensing weighted
Gerchberg–Saxton algorithm is faster, but the lateral extension of
the CGH depends on the numerical aperture of the detection
objective. In the case that NA = 1.0, the lateral extension of the
spots is on the order of
1 μm. Similarly to the previous case,
Fig. 5c illustrates the principle of point-cloud CGH, and panel 5e
shows a 3D view of the point-cloud CGH. The compressive-
sensing weighted Gerchberg–Saxton algorithm provides compara-
ble results in terms of spot brightness and uniformity as the point
clouds generated by the GS and WGS approaches while reducing
drastically the computational time [64].

Fig. 4 (continued) extracted with CNMF in a single section. (c) The fluorescence (△F/F) signals of 10 randomly
selected neurons. (d) Maximum intensity projection of the measured volume. (e) Activity map of 2077
fluorescence traces from neurons found in all sections clustered by correlation coefficient
 Optogenetics and SPIM 251

Fig. 5 CGH approach. (a) Optical transformation needed to project a 3D digital hologram. Uh represents the
spatial Fourier transform of the desired pattern Uozi defined at different depths. f is the focal length of the lens
(middle element). (b) Scheme of algorithms for extended CGH. Phase masks producing different features at
focal planes I, II, III are combined by superposition principle at the SLM. Each of the three phase masks is
calculated based on the corresponding image at the focal plane. (c) Scheme of algorithms for point-cloud
CGH. Phase masks producing different features at focal planes I, II, III are combined by superposition principle
at the SLM. Each of the three phase masks is calculated based on the coordinates xyz of the spots at the focal
plane. (d) 3D view of image-based CGH of a grid 160 × 160 × 100 μm. (e) 3D view of point-cloud CGH of the
same grid

3.4 Effect of All algorithms previously described compute CGHs based on the
Aberrations on CGH assumption that the sample has a uniform index of refraction. This
assumption is rarely true in neuroscience since brain tissue is gener-
ally optically turbid and non-uniform. Optical inhomogeneities of
samples cause a distortion of the stimulation pattern known as
252 Laura Maddalena et al.

Table 2
Summary of computer-generated holography algorithms

optical aberrations. In Fig. 6, we show the effect of optical aberra-

tions on a point-cloud CGH. Specifically to the computed CGH
phase, we summed a phase encoding for horizontal coma aberra-
tion with progressively increasing coefficient. The aberrated CGH
has a decreased intensity, and it is displaced from its original posi-
tion. Sample induced aberrations can be compensated through
adaptive optics techniques using the SLM as an adaptive element.

4 Summary

Optogenetic manipulation coupled to light-sheet imaging is a

powerful tool to perturb and monitor living translucent samples
such as zebrafish larvae. Light-sheet imaging can be realized in
different flavors with emphasis on balancing optical sectioning
with the dimensions of the FOV and maximizing the imaging
speed to resolve fast dynamics as reported in Subheading 1.1.
Here, the light sheet is realized by scanning a Bessel beam in a 2D
plane. This illumination offers a more uniform illumination over
the FOV compared to Gaussian beams and a reduced scattering
thanks to the self-healing properties of Bessel beams. However, a
major drawback is the presence of the side lobes of Bessel profiles.
In our microscope design, the side lobes are electronically rejected
by implementing a confocal slit detection of the fluorescent signal.
 Optogenetics and SPIM 253

Fig. 6 Effect of coma on CGH: (a) TPE fluorescence induced onto a rhodamine slide by a one-point hologram
with progressively increasing horizontal coma coefficient. Subscripts on the images indicate the
corresponding peak-to-valley (PV) coefficient in units of the wavelength of horizontal coma. The additional
coma phase introduces a dramatic drop in the fluorescence intensity compared to the case without aberra-
tions (0 μm PV coefficient). (b) Bar plot showing the maximum intensity of each aliased fluorescent spot in
a. Intensities are normalized to the maximum intensity of the aberration-free spot (0 μm PV coefficient). The
maximum intensity is reduced below 50% compared to the intensity of the aberration-free spot. (c) Same
fluorescence images showed in (a) where the aberrated spots intensity is enhanced by a constant factor. (d)
Maximum intensity projection of the fluorescence images in (b) showing the position of each point across the
camera chip. The aberration besides the loss of intensity introduces a loss of the CGH fidelity
254 Laura Maddalena et al.

Unfortunately, this solution sacrifices the imaging speed. To

improve this aspect, an interesting future upgrade of our imaging
system is the implementation of a NIR light source. In fact,
two-photon excitation can successfully mitigate the fluorescence
induced by the side lobes as reported in [44]. Moreover, NIR
wavelengths are often preferred for brain imaging of zebrafish
larvae due to their orthogonality to the visual system of the animal
[2]. The photostimulation module exploits the high NA of the
light-sheet detection objective to focus on the sample two-photon
computer-generated holograms. Those offer flexibility to illumi-
nate either sub-cellular ROIs with dimensions in the order of the
light diffraction limit or whole cell bodies extending over several
microns (≈ 6–10 μm). In the first case, optical aberrations due to
tissue inhomogeneities are a limiting factor to reach diffraction-
limited resolution. Hence, a next step in further developing the
2P-CGH technique is to include adaptive optics methods to com-
pensate for tissue-induced aberrations using the SLM as corrective
element. Another improvement could be to include temporal
focusing in the 2P holography module [66] to increase the axial
confinement of the stimulation light as also reported in Chapters
1, 6, 7.

5 Notes

Note 1 Linear affine transforms will account for all linear transfor-
mations between coordinates, including lateral and axial shifts,
magnification mismatches (independently for all axes), rigid rota-
tions, and coordinate shearing. Non-linear distortions, such as
barrel or pincushion distortion, would require a more complex
non-linear transform. Given that the FOV is limited to hundreds
of microns, non-linear effects rarely appear in a well-designed sys-
tem, and a linear transform provides sufficient accuracy even for
photostimulation of sub-micron structures. The linear affine trans-
form matrix between homogeneous coordinates is represented by a
4x4 matrix [67]; however, it is determined by only 12 values as the
last row in the matrix is always defined as {0, 0, 0, 1}. Hence, the
matrix can be determined through least square minimization
between vectors Xi of the image coordinates and Xi vectors of
12 SLM coordinates. The accuracy of the matrix estimation is
improved using more than 12 coordinates see Subheading 3.1.3.

Note 2 Algorithms for CGH.

Problem Description
The mathematical approach to calculate a CGH differs based on
whether we deal with image-based or point-cloud CGHs. To target
 Optogenetics and SPIM 255

complex and extended shapes spanning over few focal planes, the
electric field of the desired pattern per each focal plane is modeled
as
U o ðξ,η,zÞ = uo ðξ,ηÞe iϕz , ð2Þ
where uo is the square root of the desired intensity at depth z and ϕz
is a random phase. The hologram field is defined as the fast Fourier
transform (FFT) of the field Uo at each focal plane. Then the phase
delay between the fields at different depths is optimized to get an
interference pattern at the SLM plane as close as possible to the
target illumination pattern. On the other hand for diffraction-
limited spots arbitrarily distributed in the 3D FOV, the hologram
field is defined as the wavefront generated by the coordinates x, y,
z of the single spots in the focal planes. Hence, the field at the SLM
is calculated as the superposition of the fields generating each
single spot:
P
N - ið2π 0 0 zn π 02 02
λf ðx n x þy n y Þþ 2 2 ðx þy Þþϕn Þ
Uh = uo,n  e λ f , ð3Þ
n=1

where uo,n is the square root of the desired intensity of each spot at
the focal plane, f is the focal length of the optical system, λ is the
wavelength of the light source, xn, yn, zn are the coordinates of the
n desired spots in 3D space, x′ and y′ are the coordinates of each
SLM pixel, and ϕn is a constant term. This field can be related to the
field at the focal plane via a discrete Fourier transform (DFT) of the
single spots. Also in this case, the aim is to optimize the phase delay
between the fields generating each single spot.

Classic CGH Algorithms

• Random Superposition (RS)
The electric field of the desired pattern per each focal plane is
modeled as in Eq. 2. The hologram is calculated as the inverse
Fourier transform of the field Uo, and it is backpropagated to the
SLM plane according to the Fresnel propagator in Fourier
Optics [68]. Then the interference field of all the holograms is
computed. The phase of this interference field is the hologram
retained as the phase mask to load onto the SLM. The average of
the hologram fields at different depths is based on the assump-
tion of independence between the depths. Thus, this method
does not take into account the interference of the fields at
different planes. This results in a degradation of the recon-
structed holograms, which is evident when we project holo-
grams with more than 2-3 depths [61]. This method is very
fast compared to iterative algorithms.
256 Laura Maddalena et al.

• Gerchberg–Saxton (GS)
The electric field Uo of the desired pattern is modeled as in
the previous algorithm. Then the hologram is calculated as the
inverse Fourier transform of the object field Uo. In the resulting
hologram field, the phase is retained, while the amplitude is
substituted with a Gaussian amplitude. This new field undergoes
another Fourier transform to produce the object field at the
focal plane. The pattern produced at the focal plane is similar
to the desired one, but it has a decreased efficiency; hence, the
phase is kept, while the amplitude is replaced with the one of the
desired patterns. At this stage, the inverse Fourier transform of
the field is calculated to retrieve the phase mask at the SLM
plane. This procedure is repeated iteratively until the algorithm
converges to phase mask that produces the best intensity pat-
tern. This algorithm can be implemented for 3D holograms
including in the calculation the propagation of the fields to
different depth levels according to the diffraction theory [68].

• Weighted Gerchberg–Saxton algorithm (WGS)

This is a modified version of GS where all the features in the
CGH have a better uniformity. For this purpose, an extra degree
of freedom is introduced in the CGH calculation that is the
intensity weight of every feature in the pattern. At each iteration,
the intensity of each feature is optimized comparing the intensity
at the last iteration and the intensity of the target. The compu-
tational cost remains the same of GS.

Recent Algorithms

• Compressive-Sensing Gerchberg–Saxton (CS-GS)

This algorithm is a faster version of the GS for sparse
diffraction-limited spots in 3D. The field at the SLM is defined
as reported in Eq. 3, and it is iteratively calculated for a subset of
cM (where c is the compression factor and M is the number of
SLM pixels) randomly distributed pixels of the SLM with GS
algorithm. The last iteration is a standard GS calculation over all
the M pixels in the SLM. The computational cost scales as
Ni × Np × cM + Np × M, and this cost for cNi << 1 can be
approximated to M × Np, where Ni is the number of iterations,
Np is the number of points, and M is the number of SLM pixels.
It has been demonstrated [64] that a speed up of over one order
of magnitude can be achieved without compromising the quality
of the obtained hologram compared to regular multi-spot GS.
 Optogenetics and SPIM 257

• Compressive-Sensing Weighted Gerchberg–Saxton

(CS-WGS)
This algorithm is a faster version of the WGS for sparse
diffraction-limited spots in 3D. WGS is incompatible with the
compressive-sensing approach leading quickly to divergence,
but it is possible to run CS-GS for k - 1 iterations and then
perform a final iteration with WGS. This method, known as
CS-WGS, has an approximately double computational cost com-
pared to CS-GS when c × Ni << 1. As reported in [69], both
CS-WGS and CS-GS can be implemented with GPU calcula-
tions on a low-cost graphic card. This upgrade permits to calcu-
late a CGH ten times faster compared to the standard version
using the FFT algorithm.

• Non-convex Optimization for VOlumetric CGH

(CGH-NOVO)
This iterative algorithm is to produce phase masks based on
the non-convex minimization of a custom-defined cost function
[61]. Given the intensity distribution of the target pattern V at
different depths z, the corresponding hologram is calculated via
the RS algorithm. A cost function is defined as
LðI ððϕs Þ,V ÞÞ, ð4Þ
where I(ϕs) is the distribution generated by the random phase
mask ϕs at each plane and V is the intensity of the desired pattern
at the focal plane. The minimization of the cost function in Eq. 4
gives the optimal phase ϕ. When I(ϕ) and the cost function have
a well-defined derivative, the problem is solved with a gradient
descendant approach.
CGH-NOVO offers the advantage of choosing a cost func-
tion tailored for the specific application. For instance, one might
be interested in stimulating a subset of neurons, while minimiz-
ing the intensity received by off-target neurons below some
illumination threshold, cost functions optimized for binary pat-
terns are suitable. Another example of the flexibility of the
CGH-NOVO is the possibility to implement a cost function
optimized for 2P illumination. This algorithm can be implemen-
ted using a GPU to speed up the calculations.

• DeepCGH
The DeepCGH algorithm [65] uses convolutional neural
networks (CNNs) with unsupervised learning to compute 3D
holograms with the image-based approach. The distribution of
amplitudes A(x, y, z) of the 3D target pattern at the focal planes
is the input of the CNN. The CNN provides in output an
estimate of the complex field P(x, y, z = 0) at depth z = 0. This
field backpropagated to the SLM plane via 2D inverse Fourier
258 Laura Maddalena et al.

transform gives the phase map Φ of the CGH. During unsuper-

vised training of the CNN, the goal is to minimize a loss func-
~ where A is the intensity distribution of the target
tion LðA,AÞ,
~
and A is the intensity associated with the predicted SLM phase.
As shown in [65], this algorithm offers a 10 times faster compu-
tational cost and up to 41% increased accuracy compared to
iterative methods such as CGH-NOVO and GS. Moreover,
experimentally, DeepCGH holograms can elicit two-photon
absorption with 50% higher efficiency compared to GS or
CGH-NOVO algorithms, given the same experimental condi-
tions [65]. However, this algorithm requires a high-
performance graphic card for GPU calculations.

Acknowledgements

This work was supported by funding from TU Delft and The

Netherlands Organisation for Health Research and Development
(ZonMw).

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 Chapter 9

Widefield Multiphoton Imaging at Depth with Temporal

Focusing
Philip Wijesinghe and Kishan Dholakia

Abstract
Optical imaging has the potential to reveal high-resolution information with minimal photodamage. The
recent renaissance of super-resolution, widefield, ultrafast, and computational imaging methods has broad-
ened its horizons even further. However, a remaining grand challenge is imaging at depth over a widefield
and with a high spatiotemporal resolution. This achievement would enable the observation of fast collective
biological processes, particularly those underpinning neuroscience and developmental biology. Multipho-
ton imaging at depth, combining temporal focusing and single-pixel detection, is an emerging avenue to
address this challenge. The novel physics and computational methods driving this approach offer great
potential for future advances. This chapter articulates the theories of temporal focusing and single-pixel
detection and details the specific approach of TempoRAl Focusing microscopy with single-pIXel detection
(TRAFIX), with a particular focus on its current practical implementation and future prospects.

Key words Imaging at depth, Temporal focusing, Widefield imaging, Multiphoton microscopy,
Single-pixel imaging, Compressive sensing

1 Introduction

Optical imaging is expanding its boundaries with powerful

emerging capacities for super-resolution of subcellular features
[1, 2], widefield imaging across scales [3, 4], and achieving high
temporal resolution of ultrafast biological processes [5, 6]. An
important frontier in this endeavor is imaging at greater depths
through scattering media, which is being addressed by multiphoton
excitation and adaptive optics [7–10]. The prospect of recovering
high-resolution information over large volumes of tissues is partic-
ularly attractive for neuroscience and developmental biology. How-
ever, the high spatiotemporal resolutions needed to observe many
biological processes are challenging to presently achieve using
point-by-point scanning, as in conventional fluorescence micros-
copy, which is often combined with aberration correction schemes
[3]. Recently, a new strategy of refocusing ultrafast laser pulses in

Eirini Papagiakoumou (ed.), All-Optical Methods to Study Neuronal Function, Neuromethods, vol. 191,
https://doi.org/10.1007/978-1-0716-2764-8_9, © The Author(s) 2023

263
264 Philip Wijesinghe and Kishan Dholakia

the time domain rather than solely in the spatial domain [11, 12]
has revitalized the concept of widefield multiphoton imaging at
depth [13–15].
The premise that one can focus light in time rather than in
space has emerged rapidly over the past decades. Spatial focusing,
the concentration of the intensity of a light field in space, is ubiqui-
tous in virtually all-optical imaging systems. It is well-known that
spatial frequencies can be focused in space via the Fourier trans-
forming action of a lens [16]. Similarly, spectral frequencies can be
focused in time, with much of the same equivalence in their Fourier
transform properties, by introducing phase modulation and spatial
dispersion [17]. This has formed the foundation of pulse compres-
sion and has led to chirped pulse amplification [18] that won
Strickland and Mourou a share of the Nobel Prize in Physics in
2018. Defocusing or time stretching, on the other hand, has
enabled the recording of ultrashort phenomena and spectral con-
tent in the time domain [19].
More recently, the simultaneous use of spatial and temporal
focusing was presented in 2005 together by Oron et al. [11] and
Zhu et al. [12], demonstrating an interesting phenomenon wherein
a time-compressed pulse can be made to exist only at the focus of a
lens. Away from the focus, the pulse broadens in space and in time,
with a concomitant rapid reduction in its peak intensity. This
restriction of the pulse to the focal region enables axial confinement
of non-linear optical excitation in a scanless, widefield illumination
scheme. This development, termed temporal focusing (TF), has had
profound impact on multiphoton microscopy, where previously
axial confinement could only be achieved by point scanning a highly
focused beam across the image plane, which has severe limits on
temporal resolution. The initial work was followed by a flurry of
demonstrations of TF in widefield imaging [20–25], excitation
[26, 27], harmonic generation [28, 29], super-resolution [30],
micromachining [31–35], remote focusing [36, 37], tissue ablation
[38], and trapping [39], among others. Unsurprisingly, the capac-
ity for ultrafast widefield excitation has particularly flourished in
optogenetics and neuroimaging [40–49]. This is because the
absence of scanning has readily allowed for the simultaneous exci-
tation and measurement of neuronal firing events on the millisec-
ond timescale and over wide fields of view, previously unattainable
by point-scanning approaches.
While a direct mathematical correspondence can be made
between focusing in space and in time [19], it has become evident
that TF behaves differently to spatial focusing in the presence of
wavefront aberrations and through scattering media [50–55] due
to the added angular diversity of the illumination spectra at the
focus. For instance, the addition of TF has demonstrated a substan-
tial improvement in propagation through scattering media and a
reduction in speckle at the focus [51]. This discovery has enabled
 Widefield Imaging with Temporal Focusing 265

precise patterned multiphoton excitation at depth and has led to

remarkable progress in optogenetics. This area is reviewed in [56]
and in Chapter 10.
However, beyond this improved excitation, the capacity for
imaging at depth has been impeded by tissue or sample scattering
in the detection arm of the optical system. Specifically, widefield
detection with a camera would observe severe spatial cross-talk
from depths beyond one scattering mean-free-path length making
it difficult to recover signals in a conventional manner. A major
development came in 2018 with the addition of spatial demixing via
single-pixel detection [13, 14], enabling widefield images to be
recovered without the need for spatial coherence in the detected
signal. This method, termed TempoRAl Focusing microscopy with
single-pixel detection (TRAFIX) [13], works by decomposing the
imaging process into a different domain, or coordinate space, in
which the spatial information is carried by the scattering-robust
illumination, rather than by the detection itself. A hybrid between
widefield and single-pixel detection can also be realized [15, 57],
trading off speed and the robustness to scattering. These methods
have demonstrated a major reduction in photodamage compared to
point scanning by spreading the excitation power both over a wide-
field and in the time domain [13]. Further, the imaging scheme is
amenable to novel compressive-sensing techniques [14, 58], i.e.,
image reconstruction from a few sparse measurements, fundamen-
tally reducing the needed excitation power (and thus photodam-
age), and imaging time. The capacity for precise all-optical
excitation and multiplexed detection further offers great future
potential for simultaneous volumetric recording of sparse func-
tional signals, for instance, neuronal firing events.
Widefield multiphoton imaging at depth marries a striking
combination of novel physics and a new computationally driven
paradigm for multiphoton microscopy. In this chapter, we describe
the theory and experimental realizations, with a particular focus on
the method of single-pixel imaging with TRAFIX. Importantly, we
also note many of the current challenges and prospective advances
of these techniques.

2 Methods

Achieving widefield multiphoton imaging at depth requires TF in

illumination and spatial demixing, such as single-pixel recording, in
the detection. We present, in turn, the theory of TF and single-pixel
detection and the combined principle of TRAFIX. We further
describe the addition of compressive-sensing and hybrid demixing
strategies.
266 Philip Wijesinghe and Kishan Dholakia

Fig. 1 Illustrations of the pulse shape in space and time in (a) spatial focusing and (b) temporal focusing.
Temporal focusing realized using a (c) scattering plate, SP, and a (d) diffraction grating, DG. L: lens; Obj:
objective; FP: common Fourier plane; IP: image plane. Adapted from [11]

2.1 Temporal Focusing of illumination pulses is the route by which we achieve

Focusing axial sectioning in multiphoton imaging. Axial sectioning is
required to record the signal precisely from the focal plane, with
limited out-of-focus interference: all essential to form highly
resolved three-dimensional images. Spatial and temporal focusing
achieve this by different means. For instance, Fig. 1a shows a
spatially focused Gaussian beam, where the drop-off in the axial
intensity away from the focus is related to the Rayleigh range (zR),
which is proportional to the square of the lateral beam waist. Since
the probability of two- or three-photon excitation is related to the
square and the cube of the field intensity, respectively, there is a
strong confinement of fluorescence at the focus. Typically, a tight
focal spot is needed to achieve sectioning at the micrometer scale.
Figure 1b illustrates the concept of TF. Rather than confining the
pulse intensity in space, the pulse width is broadened out of focus.
Since the chance of multiphoton excitation is also inversely propor-
tional to the pulse duration, axial sectioning is achieved over arbi-
trary spot sizes.
To describe the realization of TF in this regard, we first con-
sider the formation of ultrashort laser pulses [12]. Ultrashort pulses
are characterized by a broadband optical spectrum. The shortest
possible pulse is formed when: (1) each spectral component is in
phase (such that the pulse width is related to the Fourier transform
of the envelope of its spectrum) and (2) the spectral components
spatially overlap. Temporal dispersion, which can be described by a
relative phase delay between different frequencies in the spectral
domain or by a chirp in the time domain (i.e., a change in the
 Widefield Imaging with Temporal Focusing 267

instantaneous frequency with time), leads to a broadening of the

pulse width. On the other hand, spatial dispersion leads to a sepa-
ration of spectral components, limiting the available bandwidth in a
local region, and thus the minimum pulse width. TF operates by
forming an ideal, chirp-free pulse at the focal plane and by deliber-
ately introducing spatial and temporal dispersion away from the
focus, such that imaging can solely take place in the focal region.
The experimental realization of TF is well-described in Oron
et al. [11]. Let us consider a thin scattering plate that is imaged by a
perfect 4f system as illustrated in Fig. 1c. An ultrashort pulse
incident onto the plate is scattered, and the individual rays that
travel through the system are refocused onto the imaging plane.
According to Fermat’s principle, all rays that travel from one point
of the scatterer (x1) and arrive at one point in the image (x2) have
identical path lengths. As such, the pulse will be reconstructed at
the image plane with no relative phase delay. However, at a point, P,
away from the focus, rays arriving at a particular angle, θ, will have a
path length difference related to zðcos -1 ðθÞ - 1Þ=c. The maximum
phase delay due to the varied path length at P increases with θ,
which is limited by the numerical aperture (NA) of the system and
the distance from focus, z. In this scenario, the capacity for broad-
ening the pulse out of focus is dependent on the ratio between the
original pulse width and the path length difference introduced.
Alternatively, an angled pulse wavefront can be incident onto the
scattering plate maximizing the phase delays away from the
focus [11].
Ultimately, a much more facile configuration can be achieved
using a diffraction grating in place of a scatterer (Fig. 1d). The
diffraction grating separates the frequencies of the incoming pulse
at differing angles. These are then refocused by a 4f system. In the
common Fourier plane, the signal may be described as a collection
of laterally shifted monochromatic (single-frequency) beams. For
an ideal Gaussian beam, the amplitude at the common Fourier
plane can be described in the time domain as [12]:
Z 1
ðx - αΔωÞ2 Δω2
A1 ðx,tÞ = e- s2  e- Ω  e iΔωt  dΔω , ð1Þ
-1

where s is the spatial width (1/e2 radius) of each monochromatic

beam and Ω is the spectral width. The first exponent represents a
spatial Gaussian profile, laterally shifted by αΔω, where Δω is an
offset in frequency and α is a proportionality constant set by the
diffraction grating and the lens. The second exponent represents
the amplitude scaling of each monochromatic beam, which follows
a Gaussian spectrum of the original pulse. The last exponent is the
phase shift relative to the group velocity.
268 Philip Wijesinghe and Kishan Dholakia

The amplitude near the focus can be evaluated using Fresnel

diffraction. This is done by performing a Fourier transform of
Eq. 1, evaluated at spatial frequencies established by the focus, f.
We assume that the wavenumber for each beam is approximately
the central wavenumber of the pulse k0. The amplitude is given as
[22]:
2
- x2
A2 ðx,z,tÞ = κ  e s
2
Ω2 2
 e - 4ð1þχÞðtþγxÞ , ð2Þ

where
rffiffiffiffiffiffiffiffi
iπf
½1 þ i zzM  - 2 ,
1
κ =Ω
zR
k0 α=f
γ = ,
1 þ iz=z M
iz=z B
χ = ,
1 þ iz=z M
4f 2 2z
s 22 = 2 2
þi ,
k0 s k0
1 2f 2 1 2f 2 1 2f 2
zM = , zR = , and z B = :
s 2 k0 s 2 þ α2 Ω2 k0 α2 Ω2 k0
Notably, the spatial profile at the focus is equivalent to that defined
by any one of the monochromatic beams, i.e., a Gaussian width of
2f/k0s. However, the modified Rayleigh range zR is dependent on
both s and αΩ. Typically, αΩ ≪ s for widefield illumination, there-
fore, zR is defined by the extent to which the spectral dispersion fills
the back aperture of the objective. Rayleigh-like coefficients zM and
zB, related to the spatial and temporal distributions, respectively,
additionally modify the phase evolution with time and away from
the focus. Here, z is defined as the distance away from the focus
(z = 0). The temporal evolution of intensity in the last exponent
defines the pulse shape, and the width can be given as [22]
pffiffiffiffiffiffiffiffiffiffiffi  12
2 2 ln 2 zM z2 ð3Þ
τðzÞ =  1þ :
Ω zB z2 þ zM zR
The pulse is shortest at the focus, reaching the minimum
transform-limited pulse width of 1/Ω (1/e2 radius). The important
conclusion of these equations is that, compared to a spatially
focused Gaussian beam, TF decouples axial sectioning (zR) from
the lateral beam shape (s2), which in turn allows precise control of
the multiphoton excitation profile in 3D.
An alternative and more intuitive view of TF was offered by
Durfee et al. [59] by examining the evolution of the phase delay
(chirp) of each frequency with respect to the focus and lateral
position. The formulation is detailed in Note 2. Figure 2a visualizes
the evolution of the phase front of three selected frequencies of the
 Widefield Imaging with Temporal Focusing 269

Fig. 2 Profile of a temporally focused pulse in the (a) spectral and (b) time domains. Lines in (a) represent the
phase front delay of different spectral components. The profiles in (b) represent the pulse shapes and their
relative delay with respect to lateral position, x. PFT: pulse front tilt

pulse in positions corresponding to fractions of the Rayleigh range

(zR). At the focus, the wavefronts are flat; however, they are tilted
with respect to each other. This represents a linear phase delay of
each frequency with lateral position (Eq. 9). A purely linear phase
delay in the spectrum results in a time shift in the arrival time of the
pulse (via the Fourier shifting theorem). Figure 2b shows the time-
domain version of this signal. Simply, the pulse will sweep across the
focal plane, in a phenomenon termed as the pulse front tilt (PFT).
Away from the focus, we can see increasing second-order dispersion
(Eq. 10), corresponding to a rapid broadening of the pulse shape
and the reduction in the peak magnitude. Interestingly, by intro-
ducing a group velocity dispersion of the second order to the
original pulse, the focal plane of TF can be shifted within the linear
region set by zR [21, 22, 60]. Using this method, TF can scan a 3D
volume without physically scanning the focus or the sample. Fur-
ther, this principle enables simultaneous TF excitation in 3D via
holographic means [61–63]. These methods are reviewed by
Ronzitti et al. [64] and may also be found in Chapters 1 and 7.
A recent discovery and a very important feature of TF are its
ability to robustly propagate through scattering media [51]. This
underlies its importance for widefield imaging at depth. Spatial
focusing over a widefield constitutes weak focusing, or low
270 Philip Wijesinghe and Kishan Dholakia

Fig. 3 Point-spread function of (a,b) temporally focused and (c,d) spatially focused beams with (a,c) no
scattering and (b,d) though 900-μm of a scattering phantom. Scale bar is 20 μm. Reproduced from [13]

numerical aperture (NA) illumination. In this scenario, the field at

the entrance pupil of the objective lens is tightly confined in space
and is refocused, taking nearly parallel trajectories through the
sample to the focal plane. The wavefront propagating through the
scattering media is aberrated, leading to speckle due to multiple
interference. With TF, an equivalent widefield area at the sample is
illuminated; however, the spectral dispersion leads to a substantially
broader field intensity at the entrance pupil, corresponding to an
effectively high-NA illumination scheme. Each spectral beamlet
takes diverse angular paths through the sample. This leads to a
rearrangement in the speckle patterns of each beamlet and an
effective speckle reduction at the focus [51]. Figure 3 shows exper-
imental evidence of this phenomenon. Widefield illumination in
Figs. 3a and c is robust to scattering with TF (Fig. 3b) and exhibits
severe speckle without TF (Fig. 3d) [13].
The aspect ratio of spatial dispersion with respect to beam size
at the Fourier plane, β = αΩ/s, is a useful parameter in quantifying
the transition between the temporal and spatial focusing regimes
(see Note 2). A key consideration is that at high NA, where the
beam width at the common Fourier plane exceeds the spectral
dispersion, i.e., β → 0, TF is equivalent to purely spatial focusing
[59]. This can be validated by examining Eqs. 9 and 10 (see Note
2). In fact, from a purely theoretical perspective, at the focus, we
can consider TF to be equivalent to a high-NA beam, scanned
point-by-point across the same field of view. However,
pffiffiffiffiffiffiffiffiffiffiffiffiffiffi the PFT in
TF sweeps the focal plane with a duration of τ0 1 þ β2 [22], which
can be several picoseconds for typical multiphoton setups. This is
not practically achievable by point scanning; further, the sweep in
the case of TF is completed by a single pulse. As such, TF offers a
mode of illumination unavailable to conventional spatial focusing
and will likely see to the emergence of novel creative methods for
precise non-linear excitation, which we discuss in Subheading 3.
 Widefield Imaging with Temporal Focusing 271

Fig. 4 Imaging process in scattering media. Imaging performed (a) in parallel with a camera; (b) sequentially
using point-scanning microscopy; and (c) by multiplexing with single-pixel detection

2.2 Single-Pixel Widefield imaging at depth requires some form of demixing of

Detection scattering in the detection that impedes direct recovery of the
signal. To explain the imaging process in a practical sense, let us
first consider the storage of a two-dimensional image on digital
media. An image that is W pixels in width and H pixels in height can
be virtually represented as a 2D matrix of values, vwh, with indices
w ∈ 0, 1, . . ., W - 1 and h ∈ 0, 1, . . ., H - 1. This can be stored in
physical memory as a linear sequence of values, vj, that are indexed
by j = hW + w. Here, by “value,” we mean an 8-bit or 16-bit or any
other datatype that describes the intensity or color of each pixel in
the image. As a matter of fact, any image, volume, or any other
digital “information” can be stored in this linear vectorized fashion.
This representation will be used throughout this chapter.
Figure 4 illustrates various imaging processes through scatter-
ing media using this linear vectorial form. Figure 4a shows conven-
tional imaging with a camera. Here, the sample (x = {xj : j = hW
+ w}) is represented in discretized form, corresponding to its
mapping onto the pixels of the camera. Imaging with a camera, in
an ideal case, is the mapping of the sample onto an image (y = {yj :
j = hW + w}), which can be mathematically represented as y = Ix,
where I is the identity matrix. In turbid media, however, light that
travels from the sample is scattered and is detected by nearby
camera pixels. This cross-talk leads to a loss in spatial information.
At depths exceeding one mean-free path length, the contribution
to yj from xj is lower than the cross-talk from other locations in the
sample, thus obscuring the image.
Point-scanning methods can overcome this effect of scattering
in detection by sequentially illuminating a tight spot in the sample
and collecting the total signal using a single-pixel detector, for
272 Philip Wijesinghe and Kishan Dholakia

instance, a photomultiplier tube. Figure 4b illustrates this process.

Scattering in the detection is no longer relevant since the total
signal is detected. The spatial information is carried by the illumi-
nation. Similarly to camera imaging, we can represent the imaging
process as y = Ix, with y formed sequentially over time, rather than
in one shot.
Figure 4c illustrates the concept of single-pixel imaging, which
operates by multiplexing the sample signal. Rather than sequen-
tially probing one location, the signal from all locations is mixed.
Scattering in detection is not an issue since the total sum of the
signal is measured. The mixing weights are set by a sequence of test
functions or structured patterns illuminated onto the sample. Simi-
lar to point scanning, a sequence of measurements is needed with
different mixing weights. We can describe this arbitrary imaging
process as y = Φx, where Φ is a measurement matrix whose rows
dictate the pattern of illumination onto the sample (each row is a
vector representing a 2D pattern image).
The recovery of the sample, x, from the measurements involves
pre-multiplying y by the inverse of Φ, i.e., x = Φ-1y. For camera
imaging and point scanning, this is trivial as I = I-1. For single-pixel
imaging, the capacity to invert Φ is strongly dependent on the
choice and number of the test functions. Let us first consider an
orthonormal basis as our measurement matrix, for instance, a
Hadamard matrix [65], H, routinely used in single-pixel imaging.
Its inverse is well-defined as H-1 = HT/N, where N = WH is the
total number of pixels in an image. Using this measurement matrix,
it is simple to recover x by multiplying the detected signal by the
transpose of H. All orthonormal measurement matrices share this
property, namely that their inverse is linearly related to its transpose
(a trivial mathematical operation). In fact, one could consider point
scanning to be a form of single-pixel imaging.
For all these imaging methods, N measurements are required:
either with N pixels of a camera or using N sequential recordings
on the single-pixel detector. When comparing single-pixel to point-
scanning detection, there are several advantages. First, point scan-
ning illuminates a tight spot in the sample with a duty cycle of 1/N.
For a maximum allowable irradiance, the signal at the single-pixel
detector is typically weak. Using single-pixel detection, the duty
cycle is typically 1/2, leading to a stronger signal at the detector.
This results in superior signal-to-noise in detection and allows for
lower excitation power for the same image quality, reducing photo-
bleaching [13]. Second, widefield illumination of patterns may be
used, which is amenable to TF illumination schemes. Third, single-
pixel detectors can be used at wavelengths for which camera hard-
ware does not exist or is prohibitively expensive, e.g., electron-
multiplying CCDs in the infrared range. The major advantage of
single-pixel detection is it allows for compressive sensing, which we
describe in the following section. Using compressive sensing, the
 Widefield Imaging with Temporal Focusing 273

number of measurements needed to reconstruct x can be signifi-

cantly reduced by employing a smaller measurement matrix and
more sophisticated inversion methods.

2.2.1 Compressive Compressive sensing (CS) describes the recovery of a signal from
Sensing far fewer measurements than required by the Nyquist sampling
criterion. The idea that one can accurately reconstruct
N independent values of a signal x from M ≫ N measurements is
counter-intuitive. However, let us consider a similar topic of image
compression, where a one megapixel raw image taking 4MB can
routinely be compressed into a 400-kB JPEG with imperceptible
losses in quality. This process relies on the idea of “sparsity.” Any
image x in the Cartesian coordinate space can be represented in a
different domain Ψ by coefficients s, such that x = Ψ s. Images are
considered sparse when there exists a domain where the coefficient
vector s possesses only a few non-zero values. The number of
non-zero values, K ≫ N, indicates that the image is “K-sparse,”
and it follows that the remainder N - K values are zero. In practice,
images are not perfectly sparse; however, s possesses a few large
coefficients with the rest being close to zero. For example, JPEG
compression selects Ψ to be the discrete cosine transform (DCT);
very briefly, a DCT is performed on x and the largest 10% of values
are stored.
CS, developed independently by Candes et al. [66] and
Donoho [67] in 2006, introduced compression directly to the
measurement process by leveraging the assumption that the signal
to be measured is sparse. This is in contrast to making a full
measurement and performing compression at a later time. If we
consider a case where the imaging process y = Φx is compressed by
performing 10% of the measurements needed to achieve Nyquist
sampling (M = N/10), such that Φ has N columns and M rows. It is
evident that this problem is underdetermined and the solution
space of all possible x that can generate y is infinite. For instance,
in point scanning, this would be equivalent to imaging the first 10%
of the field of view and leaving the other 90% up to interpretation.
CS approaches this problem by a careful design of the measurement
process. We realize that x can be decomposed into a different
domain with a sparse coefficient vector, modifying the imaging
process to y = Φx = ΦΨ s = Θs [68] (Fig. 5). A solution to the CS
problem lies in finding the most sparse s that satisfies y = Θs. The
major challenge in CS is twofold: first, is finding an efficient mini-
mization algorithm that can quantify the “sparsity” of s; and, sec-
ond, is the selection of an appropriate measurement matrix Φ. We
consider these aspects in turn.
In early CS work, a minimization algorithm using an l1-norm
proved to be effective in finding a sparse representation of s and
thus in recovering a compressed signal [66, 67]. Using this
approach, the estimated image x^ is recovered as
274 Philip Wijesinghe and Kishan Dholakia

Fig. 5 Visual illustration of the compressive-sensing process in matrix form

PN
x^ = Ψ  argmin jjsjj1 , s:t: Θs = y , and jjsjj1 = i = 1 js i j : ð4Þ

In other words, we assume that s is sparse if its l1-norm is small;

thus, we find the s with the smallest l1-norm that still satisfies the
imaging process Θs = y. This optimization can be solved using basis
pursuit methods [66–68]. A convenient MATLAB toolbox is
provided by Candes et al. 1 alongside their original publications
[66, 69]. Several alternative efficient algorithms have been pro-
posed [70–73]. Typically, these methods sacrifice some aspect of
accuracy to gain a substantial advantage in speed. We examine one
of such algorithms in a later section.
Let us now consider the choice of the measurement matrix.
The CS problem can be solved with high accuracy using Eq. 4
provided that the number of measurements taken exceeds the
sparsity, i.e., M ≥ K [69]. Additionally, the problem is termed
well-conditioned if no two coefficients in s are sampled by a similar
sequence of test weights, i.e., no two columns of Θ are the same.
More formally, the measurement matrix should satisfy the restricted
isometry property (RIP) [66]. A more intuitive method to verify the
suitability of Φ for CS is using the mutual coherence metric, defined
as c = max i ≠ j jϕTi ϕj j , where ϕi is the i-th column of Φ. A low
mutual coherence implies that the columns are nearly orthonormal.
Matrices Φ that satisfy RIP are difficult to generate deterministically
[68]. Fortuitously, randomly generated matrices, for example, with
Gaussian or Bernoulli distributions, are highly likely to satisfy RIP
and mutual incoherence [74]. Thus, random generation can be
found at the heart of many CS methods.

1
https://statweb.stanford.edu/~candes/software/l1magic/.
 Widefield Imaging with Temporal Focusing 275

While CS is broadly applicable to many signal processing tech-

niques, its use in microscopy is met with additional constraints and
challenges. In particular, s is not ideally sparse, and the imaging
process introduces additional noise to the measurements. Thus, CS
is typically an accurate but lossy estimation. Additionally, the pro-
jection of Φ into the imaging plane is limited by the finite spatial-
frequency bandwidth of the optical system and diffraction and
scattering. We discuss this in the context of imaging and propose
modified measurement matrices and recovery methods in the fol-
lowing section.

2.3 TRAFIX TempoRAl Focusing microscopy with single-pixel detection (TRA-

FIX) [13] combines the capacity to project widefield depth-
2.3.1 Setup
sectioned patterns through scattering tissue with multiplexed
detection and compressive sensing, realizing widefield imaging at
depth. This configuration, in particular, has strong prospects for
rapid, low-photodamage imaging and is well-positioned to provide
utility in neuroimaging and developmental studies that cannot be
offered by conventional point-scanning multiphoton technologies.
The practical implementation of TRAFIX involves a diffraction
grating to enable temporal focusing, a single-pixel detector, and a
dynamic light shaping element, for instance, a spatial light modu-
lator [13] or a digital micromirror device [58], for the sequential
projection of test patterns, ϕi. Figure 6 illustrates a typical TRAFIX
configuration. Briefly, an ultrashort laser pulse illuminates a wide-
field area on the dynamic light shaper (DLS) using a beam expander

Fig. 6 TRAFIX setup, illustrating the projection of a sequence of patterns (ϕi) and single-pixel detection of a
series of measurements (yi). BE: beam expander; DLS: dynamic light shaper; RL: relay lens; DG: diffraction
grating; L: lens; EP: entrance pupil; Obj: objective; S: sample; DM: dichroic mirror; SPD: single-pixel detector.
Numbers (1–3) correspond to the image plane on the DG, the common Fourier plane, and the sample image
plane, respectively
276 Philip Wijesinghe and Kishan Dholakia

(BE). The selection of the light source is discussed in Note 1. A

sequence of patterns (ϕi) is displayed on the DLS and relayed by a
relay lens (RL) onto the diffraction grating (DG). The DG sets up
spatial dispersion at the entrance pupil (EP) of the objective (Obj)
using the lens (L). The Obj temporally focuses the patterns through
the sample (S). The total fluorescence signal is filtered by a dichroic
mirror (DM) and collected by the single-pixel detector (SPD) into
a measurement vector yi.
A DLS is used to sequentially shape the light field into a series
of test patterns. A beam expander is used to illuminate a widefield
region of the DLS and to efficiently utilize the available pixels. The
DLS can be embodied by a spatial light modulator (SLM) by
encoding each pattern with a blazed grating, i.e., multiplying each
pattern in the Cartesian space, ϕi → ϕxy, by a wrapped linear phase
γ  xdx + ydy (mod 1), where dx and dy are spatial frequencies of the
grating. Additionally, assuming ϕ ∈ [0, 1], an orthogonal (90∘)
0
dispersion of complementary patterns via ϕxyγ + (1 - ϕxy)γ , where
0
γ  xdx - ydy (mod 1), leads to a clearer separation of the light-field
energy in diffracted orders. Following the SLM, the first diffracted
order should be selected by spatial filtering implemented using a
pinhole. Alternatively, a digital micromirror device (DMD) can be
used to directly deflect a binary pattern. DMDs, typically, have
faster projection rates in the 10-kHz range but are limited to binary
(on–off) light shaping and may exhibit high loss. Nematic liquid
crystal SLMs enable greater control over the light field, including
grayscale patterns, however, at sub-kHz rates. Despite the high
maximum speed of these devices, a practical limit exists in the
speed with which the test patterns can be generated and sent to
the hardware. This throughput limit is set by the efficiency of the
software, which is unlikely to be met by scientific toolsets such as
MATLAB or ImageJ.
The plane of the DLS forms the first image plane of the system.
The image plane is relayed to a diffraction grating that enables
TF. The DG is conjugate to the back aperture of the objective
(common Fourier plane of the 4f system), such that spatial frequen-
cies of each test pattern are linearly dispersed along one axis accord-
ing to the finite bandwidth of the laser pulse. In the previous
sections, we considered TF of a Gaussian beam (Eq. 1). However,
widefield test patterns used for single-pixel imaging possess a
breadth of spatial frequencies. Thus, a consideration has to be
made on the finest spatial frequency in the patterns, relating to
lateral resolution, and the extent of spatial dispersion for
TF. Previous work utilized TF dispersion aspect ratios (β) of
approximately 4–8 [13, 58].
We consider that a given test pattern comprises a superposition
of high-spatial-frequency and low-spatial-frequency light fields.
The high-frequency component occupies a large space in the Four-
ier plane and is spatially dispersed to an effectively low extent, i.e., a
 Widefield Imaging with Temporal Focusing 277

small relative β = αΩ/s, where s is large. The low-frequency com-

ponent occupies a small region of the Fourier space and is dispersed
to a large extent. At the sample plane, the high-frequency light field
is axially sectioned due to the tight spatial focus, in a similar fashion
to diffraction-limited point-scanning microscopy. Its robustness
through scattering is established by the high NA. The
low-frequency light field is sectioned via TF and propagates
through scattering media due to the effective increase in the NA
from the spatial dispersion. Thus, if the combination of the spatial-
frequency bandwidth of the test patterns and the spatial dispersion
from TF fills the entire back aperture of the objective, axial section-
ing over a widefield and robust propagation in scattering media will
be achieved.
The selection of relative dispersion, or β, is achieved by choos-
ing an appropriate DG period and the objective and lens pair. For
instance, a 1200 g/mm reflective blazed grating, a 400-mm focal
length lens, and 20x 0.75NA and 100x 0.7NA objectives (Nikon,
Japan) were used in the original studies of TRAFIX [13, 58]. Practi-
cally, a 4f relay system could be introduced between the DG and
Obj to provide an additional degree of freedom in magnification.
The RL system between the DLS and DG can be used to indepen-
dently control the magnification of the test pattern without affect-
ing the TF dispersion.
The detection system is equivalent to a conventional point-
scanning system, where an appropriate wavelength filter directs
the total fluorescence signal to a widefield single-pixel detector.
Typically, a photomultiplier tube (PMT) may be used and then
low-pass filtered to double the pattern projection rate
[14, 58]. The initial demonstration [13] utilized an electron-
multiplying CCD as an SPD by integrating the total signal, which
provides the added capacity for conventional widefield imaging.
For particularly sensitive applications, photon-counting devices
may be implemented.

2.3.2 Imaging Initial work employed the Hadamard matrix as the set of test
patterns (Φ) [13, 14, 75, 76]. The Hadamard matrix [65] (also
termed the Walsh–Hadamard matrix) is formed recursively. It is
orthonormal and symmetrical, with each column being orthogonal
to any other. It is routinely used for single-pixel imaging because it
is easy to generate and it is its own inverse. The first-order Hada-
mard matrix is unity; the second order is given as
" #
1 1
H2 = ; ð5Þ
1 -1
and the 2n order is formed from the n order as
278 Philip Wijesinghe and Kishan Dholakia

Fig. 7 Images recovered using TRAFIX. (a) Reference image of a fluorescent test sample with no scattering, (b)
imaged through a 400-μm brain slice using widefield detection, and (d) TRAFIX. (c) TRAFIX through a 200-μm
brain slice. (e,g) Reference images of mouse-derived astrocytes compared with (f,h) TRAFIX. Scale bar is
20 μm. (a–d) Adapted from [13]
" #
Hn Hn
H 2n = : ð6Þ
Hn -Hn
The sizes of Hadamard matrices are limited to powers of 2, which
set the allowed image sampling sizes. Each row of the Hadamard
matrix represents an individual test pattern.
Figure 7 shows examples of images generated with TRAFIX
using Hadamard test sequences [13]. Figure 7a shows a reference
image of a test target (x) fabricated from a 200-nm spun-coated
layer of super-yellow polymer. The target was imprinted by photo-
bleaching a negative pattern. When the test target is obscured by a
400-μm section of rat brain tissue (mean-free path length, ls = 55
μm), conventional widefield detection using a camera is impossible
due to scattering in the detection (Fig. 7b). Using TRAFIX, the
image may be retrieved when obscured by 200-μm (Fig. 7c) and
400-μm (Fig. 7d) section of rat brain tissue.
Figures 7e–h demonstrate the proof-of-principle of TRAFIX
on primary mouse astrocytes. No scattering was introduced; how-
ever, the results demonstrate the capacity to reconstruct images
from the faint signal in biological samples. Importantly, a long
recording time per test pattern on the order of 0.1–0.5 s was
required due to the low pulse energy density of the fast-repetition-
rate (80 MHz) laser used. However, even with a non-ideal laser,
three-photon signal recovery was demonstrated with TRAFIX
 Widefield Imaging with Temporal Focusing 279

[75]. Wadduwage et al. [15] built on this concept and demon-

strated a stronger signal and faster widefield detection of mouse
muscle at depth by using a high-pulse-energy laser (see Note 1) and
a hybrid demixing method.
The capacity for CS gives TRAFIX an advantage in the ultimate
imaging speed and in the reduction to photodamage. However, a
special consideration has to be made to the implementation of CS
in microscopy compared to conventional macroscopic compressive
imaging (e.g., photography). Typically, applications of CS in imag-
ing employ Hadamard or randomly generated matrices. The test
patterns from those measurement matrices possess a broad spatial-
frequency bandwidth that is difficult to propagate through high-
NA imaging systems. The exceptionally high, step-wise variations in
intensity that are characteristic to these test patterns, even at low
pixel sampling N, lead to large diffraction effects and an overfilling
of the back aperture of the objective. Figure 8 shows a simulation of
the field intensity at the DG (1), the common Fourier plane (2),
and the sample (3), corresponding to the locations marked in

Fig. 8 Compressive sensing in microscopy. (a–c) The projected pattern (1), the spectrum in the Fourier plane
(2) clipped by the objective pupil (blue circle), and the resulting pattern in the sample plane (3), for the (a)
Hadamard, (b) random, and (c) Morlet patterns. (d) Performance of compression (M/N) using each pattern set,
compared to the reference image (e). Scale bar is 10 μm. Adapted from [58]
280 Philip Wijesinghe and Kishan Dholakia

Fig. 6. The blue circle indicates the entrance pupil size. The Hada-
mard pattern (Fig. 8a) demonstrates a large structured pattern in
the Fourier plane that exceeds the entrance pupil. The random
pattern (Fig. 8b) shows a broad specular bandwidth. In both
cases, the sampling pixel size of each pattern was set as the diffrac-
tion limit.
The limit in the spatial-frequency bandwidth leads to two
important issues in CS. First, the effective low-pass filtering leads
to a different pattern being projected onto the sample plane to the
one assumed in the CS optimization algorithm. This is clear by
comparing locations (1) and (3) in Fig. 8. In this scenario, the CS
problem becomes more poorly conditioned; the noise in the mea-
surement process may be attributed to larger non-existing frequen-
cies. Second, diffraction leads to a large portion of the pulse energy
to be blocked by the entrance pupil. In the widefield regime, pulse
energy is an important parameter to maximize, directly impacting
the signal-to-noise ratio.
An alternative pattern set was proposed to control and selec-
tively probe the spatial-frequency space, generated from Morlet
wavelets mathematically convolved with randomly generated matri-
ces [58, 77]. These Morlet patterns feature two important proper-
ties. First, the Morlet wavelet, based on real-valued Gabor filters,
reaches the limit set by the uncertainty principle, i.e., an optimal
trade-off between spatial and spatial-frequency localization. Sec-
ond, the convolution with a randomly generated, Gaussian-
distributed matrix satisfies the mutual incoherence property
required by CS.
Figure 8c shows the propagation of a Morlet pattern [58]. It is
evident that the Morlet patterns can be designed to fit the entrance
pupil, and the pattern at the sample plane resembles the pattern
projected by the DLS. In this demonstration, the sampling pixel
size N matches the theoretical diffraction limit. Interestingly, the
CS sampling pixels size sets the image resolution, yet, due to the
Nyquist sampling criterion, a bandwidth of twice the pattern fre-
quency is required to propagate the pattern through a microscopy
system. This corresponds to the observations in Fig. 8a–c (2) and
suggests that an image resolution below twice the diffraction limit
is not achievable with Hadamard or Random measurement matri-
ces. Optionally, this can be overcome with digital microscanning
techniques [78], by taking multiple CS measurements with a series
of patterns, spatially shifted by half the sampling size. Morlet pat-
terns, due to the independent selection of bandwidth and sampling
size, are able to reach the diffraction limit.
CS recovery from microscopy data requires additional consid-
eration. The conventional method of CS recovery using l1-norm
minimization using the basis pursuit algorithm is inefficient for
imaging applications for several reasons. Importantly, the CS
 Widefield Imaging with Temporal Focusing 281

problem is linearized into vector form (Eq. 4), which makes it

broadly applicable to signal processing; however, the 2D/3D
nature of imaging can be exploited to improve CS recovery. We
can assume some extent of spatial smoothness to the image. Fur-
ther, the basis pursuit method scales with the total sampling pixels
N, making the recovery of large images (e.g., exceeding 64 × 64
pixels) computationally taxing. Finally, the l1-norm minimization is
constrained to provide a solution that strictly satisfies y = Φx. It is
inevitable that some noise is introduced in imaging, especially when
the multiphoton signal from the sample is faint; thus, concessions
for noise should be made. This is particularly important in the
stability of measurement bases, which we discuss in Note 3. Taking
the above into account, first-order approximations to the CS prob-
lem may be used to achieve satisfactory, and in some instances
improved, performance. In particular, we can utilize Nesterov’s
method [73] (NESTA2), which uses a smooth approximation to
the l1-norm, minimizing ||s||1 s.t. ||y - Φx||2 ≤ ε, for some esti-
mated measurement noise ε. NESTA was found to be substantially
faster, less hardware demanding, and more resilient to measure-
ment noise [58].
Figure 8d demonstrates the CS performance of the measure-
ment matrices in TRAFIX [58]. The sample comprises 4.8-μm
green fluorescent polystyrene beads (G0500, Thermo Scientific)
embedded on a glass microscope slide. A reference image is
provided in the top right of Fig. 8d. Various compression ratios
(M/N) are evaluated. The Morlet exhibits a superior performance
at high compression. The pulse energy density is kept constant
between measurements; thus, the higher intensity in the Morlet-
generated results suggests a higher signal to noise. In fact, equiva-
lent image contrast is achieved at 25%, 67%, and 82% compression,
for the Hadamard, Random, and Morlet patterns, respectively
[58]. Additionally, the Morlet features a stronger performance
through scattering media [58].

2.3.3 Hybrid Demixing An interesting alternative approach, combining patterned illumina-

tion and widefield camera detection, toward the demixing of scat-
tering was presented by Wadduwage et al. [15]. This technique was
termed de-scattering with excitation patterning (DEEP). The prin-
ciple is conceptually similar to another technique presented by
Parot et al. [79], termed compressed Hadamard imaging (CHI).
We recognize that camera detection of TF signal at depth is
obscured by the cross-talk of scattering between adjacent pixels.
However, the contribution due to scattering of the signal from xi to
an imaging pixel yj, where the coordinate j is substantially far away

2
https://statweb.stanford.edu/~candes/software/nesta/.
282 Philip Wijesinghe and Kishan Dholakia

from i, is minimal. Therefore, if cross-talk can be removed between

adjacent pixels, rather than completely from the entire imaging
process, depth imaging can still be achieved.
The DEEP or CHI operates by projecting a sequence of small
Hadamard codes (e.g., 16 × 16 pixels), repeated and tessellated
over a large-pixel-count image. The mapping of the patterns onto
the camera pixels is performed by imaging a calibration phantom
(a thin fluorescent layer). The calibration set is then used to demod-
ulate patterned recording sequences. In effect, this method realizes
a parallel version of single-pixel imaging and is equivalently amena-
ble to CS [15, 79]. Additionally, this can be performed in a line-
scanning configuration [57]. The trade-off exists in choosing the
size of the Hadamard code. The length of the code establishes the
distance in the camera over which the scattering cross-talk can be
eliminated; however, a larger length requires more total
measurements.

3 Future Prospects

TF offers a new paradigm of widefield, axially confined multipho-

ton excitation; yet, as we demonstrate in Subheading 2.1, it shares a
common mathematical foundation with point-scanning methods.
Point-scanning methods sweep the field of view with a series of laser
pulses using mechanical mirrors. TF, however, does so elegantly by
controlling the spatiotemporal evolution of the phase front. In
theory, equivalent field of view, axial confinement, and depth pene-
tration can be obtained using both methods. In practice, however,
TF can do so with a single pulse, including the 3D excitation via
holographic means [64]. As such, TF has become advantageous for
rapid surface imaging and precision optogenetics at depth [56].
The capacity for widefield imaging at depth has been intro-
duced with multiplexed detection schemes. Due to its relatively
young stage of technological maturity, TF imaging at depth, par-
ticularly in two-photon modes, will struggle to achieve the same
speed as point-scanning methods within the next several years (see
Note 4). The maturation of high-speed light shaping technology
and massively parallel detection schemes can improve the prospects
of TF, however. For instance, parallel detection by using each pixel
of an EMCCD as a single-pixel detector combined with local,
repeating pattern projection, could elevate the speed to beyond
30 fps with short measurement matrices.
Recently, three-photon microscopy has re-emerged due to the
availability of near-infrared, high-pulse-energy lasers, three-photon
fluorescent markers, and a desire to perform deep, volumetric
imaging in vivo and through scattering media, such as a mouse
skull [7, 80, 81]. There, due to the low repetition rate of the lasers,
 Widefield Imaging with Temporal Focusing 283

the speed of point scanning is on par with multiplexed detection

methods (see Note 4). Toward this end, the promise of three-
photon microscopy has already been shown with TRAFIX
[75]. In vivo, through-skull imaging is an important goal for
three-photon technology [3]. The lowered goal posts in speed
and the requirement for minimal photodamage present a likely
niche for TF imaging. Beyond this, there are several key capabilities
that can give TF not only a competitive edge, but also deliver
unprecedented performance in select areas, including in neurosci-
ence applications.
A likely key to the success of this technology is the combination
of sparse detection and precision excitation. The concept of sparsity
allows for a fundamentally lower number of measurements to be
made to recover the same information. A clear advantage here
comes in the form of reduced photobleaching and photodamage
[13] to point-scanning methods. TF may find immediate utility in
applications that emphasize long-term, non-invasive imaging over
rapid detection, for instance in developmental studies.
Controlled excitation of arbitrary fields in 3D is possible with
TF holographic methods [24, 61–63, 82]. The combination of
controlled excitation and multiplexed detection lends itself to adap-
tive sampling schemes. For instance, a low-resolution volume may
be formed rapidly; then, based on the features of the low-resolution
volume, sparse volumes of interest may be dynamically probed at
higher resolutions on-the-fly. This would be of great advantage to
sparsely populated samples, for instance, for the tracking of indi-
vidual cellular bodies in 3D biomaterials. In neuroscience, in par-
ticular, the combination of holography and multiplexing may
enable the volumetric tracking of individual sparse neurons and
their connectivity at high spatiotemporal resolutions with no a
priori information to their locations. In fact, a similar compressive
approach was demonstrated using light-field microscopy [83].
Optogenetic excitation with TF has already shown great prom-
ise [56]. Naturally, the addition of multiplexed detection may
enable all-optical functional imaging. For instance, optogenetic
excitation with TF can be combined with TRAFIX recordings of a
reporter (e.g., a voltage sensor), given that the absorption and
emission spectra are well-separated. The detected temporal
response could be demodulated based on the illumination pattern
sequence using the CS method, however, with several caveats.
The key consideration in this endeavor is the repeatability of the
neuronal response. One approach to performing CS in this scenario
would be to project a pattern and record the multiplexed response
over a desired duration. This is then repeated for all other patterns.
The caveat is that the response from all neurons should be repeat-
able each time they are excited; otherwise, CS will not be able to
284 Philip Wijesinghe and Kishan Dholakia

accurately demodulate the signal. It is a big ask for biological

samples to act in such a repeatable manner. The other approach
would be to project all the patterns within the desired temporal
resolution timeframe and repeat this for all time points of the
experiment. The added temporal domain of video imaging is ame-
nable to further improvements to the compression ratio, even
down to a few percent [84]. This approach would remove the
need for repeatability; however, fast pattern projection and fluores-
cent reporter switching rates are needed. This is readily achievable
for calcium imaging, where the desired temporal resolution is on
the sub-second to second scale [5]. For voltage sensing on the
millisecond scale [85–88], pattern projection and reporter switch-
ing rates should be on the microsecond scale. This may be presently
achievable for sparse simultaneous measurements on the order of
tens of neurons; however, widefield imaging is currently intracta-
ble. An ambitious vision could be to map the volumetric structure
of a neuronal sample with an adaptable resolution, identify and
optically stimulate each neuron, and map the sparse connectivity
to its neighbors with functional imaging. In fact, the idea of sparsity
and adaptive resolution is well-suited to neuroscience methods,
where neuronal connectivity is naturally sparse over a widefield
and in vivo behavioral stimuli might only affect a subset of the
interrogated volume.
These considerations form a grand challenge for TF imaging in
neuroscience. Despite the appreciable challenges in the road ahead,
the holy grail of this undertaking is the unprecedented capacity for
low-photodamage, highly parallel, adaptive and rapid interrogation
of neuronal signals, and their connectivity in turbid 3D structures,
and potentially in vivo.

4 Summary

Compressive sensing and temporal focusing have seen remarkable

progress in the past decade, in their own right. The combination of
these two techniques in widefield imaging at depth provides a
broad canvas for powerful future advances, both in the novel phys-
ics of the spatiotemporal manipulation of light and in the new
computationally driven paradigm of imaging. Undoubtedly, from
the rapid uptake and interest of this relatively young field, many
developments are still in store over the coming years. However, a
grand challenge remains to fully exploit the multiplexing capacities
in the illumination and the detection methods, to provide capacity
not available to serial point-scanning approaches, and to demon-
strate it in biological samples.
 Widefield Imaging with Temporal Focusing 285

5 Notes

1. Ultrashort Pulsed Laser Selection

An important practical consideration is the choice of the
laser source. For comparison, a laser source for two-photon
point-scanning microscopy typically features a fast repetition
rate ( 80 MHz) with pulse energy densities at the sample in
the range of 0.1–0.5 nJ/μm2 [89]. Incorporating the same
laser in a widefield configuration leads to a large loss in energy
per unit area, for example, at the maximum power setting of a
point-scanning laser (Chameleon Ultra II, Coherent Inc.,
USA), the pulse energy at the sample is 6-nJ, and the pulse
energy density is 0.6 pJ/μm2 [13] for a modest 100-μm field of
view. This is an order of magnitude lower than point scanning,
which makes imaging biological samples with weak multipho-
ton signals a significant challenge. At the other end of the scale,
low-repetition high-power lasers can be employed. In
Wadduwage et al. [15], a 10-kHz repetition rate regenerative
amplifier laser provided a remarkable 1.5 μJ pulse energy with
0.06 nJ/μm2 over their 160-μm field of view; however, the
laser in that instance was not tunable in wavelength. Point-
scanning methods require a high repetition rate to ensure a fast
raster scanning speed across the sample. For widefield imaging,
this is not required; thus, low-repetition high-pulse-energy
lasers are preferred for a good signal-to-noise ratio.
2. Chirp Evolution in Temporal Focusing
Following Durfee et al. [59], we examine TF from the
perspective of position-dependent spectral chirp. TF is consid-
ered as the superposition of paraxially propagating Gaussian
“beamlets” modified by ray optics to incorporate the tilt of the
spectral components. Consider a Gaussian beamlet that under-
goes a tilt from spectral dispersion, transforming the lateral
position x → x - z sin θx , where θx = αΔω/f. The amplitude is
given as [59]
2
s 2 - ðx - sz2sinðzÞθx Þ
Aðx,z,ωÞ = E 0 ðωÞ e , ð7Þ
s 2 ðzÞ
and the phase as
 
1 ðx - z sin θx Þ2
ϕðx,z,ωÞ = k0 x sin θx þ k0 z 1 - sin θx - ηðzÞ þ k0
2
,
2 2RðzÞ
ð8Þ
286 Philip Wijesinghe and Kishan Dholakia

where s2(z) is the axially dependent beam radius at the focus,

R is the radius of curvature, and η is the Gouy phase, given with
respect to the Rayleigh range, z R = k0 s 22 =2, as
sffiffiffiffiffiffiffiffiffiffiffiffiffiffi  
z2 z2
s 2 ðzÞ = s 2 1 þ 2 , RðzÞ = z 1 þ R2
zR z
 
z
and ηðzÞ = arctan :
zR

The position-dependent chirp can be evaluated from the

phase in Eq. 8 by taking the derivative with respect to
ω = ω0 + Δω, evaluated around the central frequency ω0. The
first-order chirp is [59]
z x 1 x2
ϕ1 ðx,zÞ = þ βτ0 ð Þ þ , ð9Þ
c s2 1 þ z 2 =z 2R 2cRðzÞ
where β = αΩ/s represents the aspect ratio of the spatial
dispersion rate with respect to the beam size (s) at the common
Fourier plane and τ0 is the transform-limited pulse width.
Notably, the first term is the arrival time of the pulse, and the
last term is symmetric with x and represents the curvature of
the beam. The middle term is linear with x and represents a
characteristic trait of TF—a pulse front tilt (PFT) [90]. Note
that a linear phase shift in the spectral domain relates to a
temporal shift in the time domain via the Fourier shifting
property. As such, PFT describes the property of TF whereby
the pulse rapidly sweeps across the lateral dimension of the
focal plane.
Similarly, the second-order chirp is given as [59]

x τ0 β z τ20 β2 1
ϕ2 ðx,zÞ = ð - Þð :Þ ð10Þ
s 2 ω0 z R 4 1 þ z 2 =z 2R
Notably, ϕ2 is dominated by the quadratic term, which is
negligible at the focus and increases with z, with a linear rela-
tionship when z is well within the Rayleigh range z R = k0 s 22 =2.
This second-order chirp leads to pulse broadening from tem-
poral dispersion away from the focus (illustrated in Fig. 2).
3. Stability of Compressive Sensing
Like many other inversion methods, the efficacy of CS
relies on several closely linked parameters: the mutual incoher-
ence and the condition number of the measurement matrix,
and the noise in the measurement. The interplay between these
parameters dictates the qualities seen in the recovered images.
For instance, a compressed Hadamard matrix is not mutually
incoherent (in fact, it is perfectly coherent, i.e., there will be at
least two columns that are identical); however, it is well-
 Widefield Imaging with Temporal Focusing 287

conditioned. As a result, CS with a Hadamard matrix leads to

superior performance when the signal-to-noise ratio is low;
however, the image may exhibit features that repeat spatially
and, overall, will demonstrate poor performance with high
compression. A random matrix is mutually incoherent and has
a satisfactory condition number with compression. Thus, it will
perform well with compression and will fail only when the
signal-to-noise ratio is exceptionally low. As a result, the ran-
dom matrix is often the basis of choice for many CS applica-
tions. A Morlet matrix is mutually incoherent; however, it has a
poor condition number when the spatial bandwidth is highly
constrained. While it is superior in microscopy applications due
to the limited spatial bandwidth, it requires a strong signal-to-
noise ratio, i.e., a strong fluorescent signal from the sample.
4. Imaging Speed
Multiplexed widefield detection and CS promise a reduc-
tion in the total measurements and a capacity to capture rapid
dynamic processes; however, the proof-of-principle demonstra-
tions [13–15] have not yet shown a faster imaging speed com-
pared to state-of-the-art point-scanning methods. This is
largely due to the discrepancy in the maturity of the hardware
and control software that drive these methods. Here, we
explore the theoretical speeds that can be achieved by both
methods.
In point-scanning two-photon microscopy, the imaging
speed is limited by the repetition rate of the laser, which is
80 MHz for typical embodiments. Another limit is set by the
scanning speed. High-speed resonant scanning systems can
achieve a line scan rate of 12 kHz. Let us define the maximum
imaging speed based on a 1 megapixel (MP) image; thus,
12 frames per second (fps) can be achieved practically. The
speed of multiplexed imaging is limited by the speed of the
light shaper. For an SLM, let us consider a conservative high-
speed refresh rate of 300 Hz. For a 1MP image and 10%
compression (M/N), 0.003 fps can be achieved. A DMD can
reach speeds beyond 15 kHz. Similarly, for a 1MP image and
10% compression, 0.15 fps can be achieved. This is substantially
lower than point scanning. Hybrid methods [15, 79] using
electron-multiplying CCDs (EMCCD) with 25 fps (1MP
image) could reach >1 fps with short Hadamard codes.
In three-photon microscopy, the requirement for high
pulse energy restricts the laser repetition rate to typically
sub-MHz regimes. For point scanning, this limits imaging
speed to below 0.1 fps (in our 1MP comparison). The speed
of multiplexed imaging, however, is still limited by the light
shaper. As such, multiplexed imaging may have an advantage in
speed in widefield three-photon imaging.
288 Philip Wijesinghe and Kishan Dholakia

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 Chapter 10

High-Speed Neural Imaging with Synaptic Resolution:

Bessel Focus Scanning Two-Photon Microscopy
and Optical-Sectioning Widefield Microscopy
Guanghan Meng, Qinrong Zhang, and Na Ji

Abstract
Brain is composed of complex networks of neurons that work in concert to underlie the animal’s cognition
and behavior. Neurons communicate via structures called synapses, which typically require submicron
spatial resolution to visualize. To understand the computation of individual neurons as well as neural
networks, methods that can monitor neuronal morphology and function in vivo at synaptic spatial resolu-
tion and sub-second temporal resolution are required. In this chapter, we discuss the principles and
applications of two enabling optical microscopy methods: two-photon fluorescence microscopy equipped
with Bessel focus scanning technology and widefield fluorescence microscopy with optical sectioning ability,
both of which could be combined with optogenetic stimulation for all optical interrogation of neural
circuits. Details on their design and implementation, as well as example applications, are presented.

Key words Optical microscopy, Neural imaging, Synapse, Bessel beam, Two-photon fluorescence
microscopy, Widefield microscopy, Structured illumination microscopy

1 Introduction

With the development of genetically encoded fluorescence reporters

that convert neural activities into fluorescence fluctuations [1–7],
optical microscopy has been widely applied to in vivo studies of
brain function due to its non-invasiveness and subcellular spatial
resolution. Over the past decades, major efforts have been made to
improve the performance of optical microscopes in terms of their
resolution, speed, and imaging depth [8–13]. Depending on the
specific application, a distinct set of criteria should be applied in
order to select the most appropriate imaging modality. In the case
where synaptic responses need to be characterized (e.g., mapping all
the synaptic inputs of a neuron), the ideal method should have high

Authors Guanghan Meng and Qinrong Zhang have equally contributed to this chapter.

Eirini Papagiakoumou (ed.), All-Optical Methods to Study Neuronal Function, Neuromethods, vol. 191,
https://doi.org/10.1007/978-1-0716-2764-8_10, © The Author(s) 2023

293
294 Guanghan Meng et al.

spatial resolution to resolve individual synapses and sufficient tempo-

ral resolution to capture all the ongoing activities in the observation
area/volume. For studies in opaque samples (e.g., adult mammalian
brains), multiphoton fluorescence microscopy is often the method of
choice, because its nonlinear excitation leads to optical sectioning and
its near-infrared illumination light provides optical access to struc-
tures at depth in highly scattering tissue [10–12]. Despite its ability to
image scattering tissues, the main limitation of multiphoton micros-
copy is its speed, which is limited by the point-scanning scheme of its
standard implementation [12], especially when applied to three-
dimensional (3D) volumetric imaging. Bessel focus scanning tech-
nology, where a Bessel-like beam is utilized for fluorescence excitation
[14–17], has been used to improve the volumetric imaging speed of
multiphoton microscopy via an extended depth of field (DOF), and is
discussed in Subheading 2 of this chapter. In contrast, for studies in
transparent samples with minimal tissue scattering, widefield fluores-
cence microscopy can be applied, with the advantages of high imaging
speed and simple hardware implementation (see also Chap. 2). How-
ever, widefield microscopy does not have optical sectioning ability
[18–21]. As a result, significant out-of-focus signal contributes to a
blurry and sometimes overwhelming background, which obscures
the in-focus objects, especially small structures like synapses. One
approach to impart optical sectioning ability to widefield fluorescence
microscopy is via structured illumination (SI) [9, 22–25], where the
sample is illuminated with a structured rather than uniform pattern of
light to distinguish the in-focus information from the background
signal. The details of this technology are discussed in Subheading 3 of
this chapter [9].

2 Bessel Focus Scanning Two-Photon Fluorescence Microscopy

2.1 Background Widefield fluorescence microscopes with optical sectioning ability

enable high spatiotemporal resolution imaging in neurobiological
2.1.1 Multiphoton
model systems that are transparent (e.g., zebrafish larvae and Dro-
Excitation
sophila larvae), but cannot visualize deep structures in opaque
samples (e.g., adult mammalian brains) due to tissue scattering.
In multiphoton excitation fluorescence microscopy, the longer
wavelength of excitation light renders the system more resistant
to scattering and grants optical access to structures at depths up to
1–2 mm in the mouse brain [10–12]. Furthermore, the nonlinear-
ity of multi-photon excitation can significantly suppress out-of-
focus fluorescence generation, thus requiring no additional device
or computation to realize optical sectioning.
 High-Speed Neural Imaging with Synaptic Resolution 295

2.1.2 Challenges of In a conventional multiphoton fluorescence microscope, fluores-

Volumetric Imaging with cence excitation is three-dimensionally confined. Thus 2D or 3D
Multiphoton Microscopy scanning of the focus is required to obtain a planar image or a
volume stack, respectively. Such a point-scanning scheme ensures
optical sectioning ability and high spatial resolution even in opaque
tissue, but limits imaging speed. Therefore, to record neural
dynamics with sufficient frame rates, scanners that can move the
excitation focus at high speed are desired. Lateral beam steering
(translation of the focus in a plane perpendicular to the optical axis)
in multiphoton microscopy is typically achieved by a pair of
galvanometer-based optical scanners (or “galvos”). Conventional
raster-scanning galvos can achieve line scan rates of a few kHz
[12]. By replacing one of the raster-scanning galvos with a faster
scanner (e.g., resonant galvos [26]), line scan rates of tens of kHz
and 2D frame rate of tens of Hz can be realized [12]. However,
information observed from a single optical section can be incom-
plete. Since both neural circuits and individual neurons therein are
3D structures, volumetric imaging with subsecond temporal reso-
lution is required to capture their dynamics completely. To perform
volumetric imaging in multiphoton microscopy, in addition to
scanning the focus in the lateral xy plane, axial movement of the
excitation focus along z direction is required. In contrast to lateral
focus scanning, axial scanning of the focus is more challenging. A
common method to scan the excitation focus axially is to translate
the microscope objective along z direction, the speed of which,
however, is limited by mechanical inertia associated with the objec-
tive. Wavefront shaping devices such as electrically tunable lenses
(ETLs) [27], spatial light modulators (SLMs) [28], or acousto-
optic deflectors (AODs) [29–31] allow the control of wavefront
divergence at the objective back focal plane, which translates the
focus axially. These methods avoid the problems posed by objective
inertia, but introduce additional aberrations to the system and
degrade image quality at large focal shifts, since most microscope
objectives are designed to produce optimal performance for light of
a specific divergence (e.g., multiphoton excitation objectives are
designed for collimated beams) [32]. A dual-objective remote
focusing method, where the excitation light travels through two
microscope objectives with conjugated pupil planes [33–35], has
aberrations caused by the two objectives cancelled out and can
achieve high-speed volumetric imaging at diffraction-limited reso-
lution over a large axial range. The requirement of two objectives
nevertheless increases the system cost and complexity, requiring
custom optical design [36]. Moreover, all the above volumetric
imaging methods are sensitive to axial motions of the sample,
which cause the objects of interest to move out of focus and the
loss of dynamic information. Bessel focus scanning multiphoton
microscopy provides an alternative to the volumetric imaging
methods described above [15, 16, 37, 38] and will be discussed
in detail below.
296 Guanghan Meng et al.

Fig. 1 The concept of Bessel focus scanning multiphoton microscopy. (a) 2D scanning of a conventional
Gaussian focus obtains information from a single plane (the plane in yellow). (b) 2D scanning of a Bessel focus
covers a volume (the volume in orange)

2.1.3 Bessel Focus During most brain imaging experiments in vivo, neurons exhibit
Scanning Technology variations in their fluorescence signal associated with their func-
tional activity, but their positions remain unchanged. Therefore,
when the temporal dynamics of neurons is the subject of interest,
one does not need to constantly track their 3D locations, but can
instead obtain axially projected views of 3D volumes via extended
depth-of-field imaging by scanning an axially elongated focus, e.g.,
a Bessel-like beam [14–16, 39–41] (Fig. 1). Since all structures
along the elongated focus are probed simultaneously within a single
2D scan, the 2D frame rate becomes a projected 3D volume rate.
Although axial resolution is reduced substantially during Bessel
focus scanning, the 3D positions of neurons and neuronal struc-
tures can be obtained from a conventional 3D stack by scanning a
Gaussian focus. Because at the same numerical aperture (NA), a
Bessel focus has a higher lateral resolution than a Gaussian focus
[41], synapse-resolving lateral resolution is maintained even for a
0.3-NA Bessel focus [42]. With Bessel focus scanning, imaging
throughput can improve by tens to a hundred times, with the
image data size reduced by the same amount [15]. Bessel focus
scanning technology is compatible with other fast scanning meth-
ods mentioned in Subheading 2.1.2 and, when combined together,
can further boost the volumetric imaging speed of a multiphoton
microscope [15, 38]. The extended depth of field of a Bessel focus
also makes the imaging process insensitive to axial motion, which
eliminates axial motion artifacts. Together with the much reduced
data size, it substantially simplifies image processing
[15, 42]. Although Bessel focus scanning technology can be
incorporated into both two-photon and three-photon fluorescence
 High-Speed Neural Imaging with Synaptic Resolution 297

Fig. 2 The Diagram of two Bessel focus modules. (a) A two-photon microscope with an SLM-based Bessel
module (gray box). (b) An axicon-based Bessel module. L2 can be translated along optical axis to change the
axial length and numerical aperture of Bessel beam. D is defined as 0 mm when the mask is at the front focal
plane of L2 and positive when L2 moves away from the mask. Ti:Sa Ti:Sapphire laser, EOM electro-optical
modulator, BE beam expander, M mirror, L lens, Obj objective

microscope systems [37], the sections below focuses on Bessel

focus scanning two-photon fluorescence microscopy for volumetric
imaging.

2.2 Materials and A typical two-photon fluorescence microscope includes the follow-
Equipment ing optical components (Fig. 2): a femtosecond laser, an excitation
power control unit such as an electro-optical modulator (EOM)
(i.e., a Pockels cell), a pair of scanners/galvos, a scan lens and a tube
lens, a dichroic mirror, an objective (often mounted on a piezoelec-
tric stage to perform axial scanning), detection filters, one or two
photomultipliers (PMTs).
A Bessel focus module includes the following optical compo-
nents (Fig. 2): two mirrors to switch the light path between Gauss-
ian and Bessel modes, an SLM or an axicon, a lens, an annular mask,
and a pair of conjugation lenses.

2.3 Methods The essential components in the excitation light path of a

two-photon fluorescence microscope are shown in Fig. 2a. The
2.3.1 Two-Photon
output of a Ti:Sapphire laser with a Gaussian intensity profile first
Fluorescence Microscope
passes through a Pockels cell, and then a beam expander (see note 1
in Subheading 2.4). The Gaussian beam is then directed either
straight to the galvos or into a Bessel module, by moving two
mirrors M1 and M2 out of and into the light path, respectively.
The galvo pair could be either placed close together or conjugated
with a pair of scan lenses. The latter can eliminate beam wandering
298 Guanghan Meng et al.

on the second galvo as well as the objective back focal plane during
scanning, but leads to more power loss due to adding additional
optical elements. In some systems, a resonant scanner is
incorporated in addition to the two raster-scanning galvos, with
the three scanners together enabling high-speed imaging in a small
subfield positioned anywhere inside a large field of view
[36, 38]. After the galvos, a scan lens (L4) and a tube lens
(L5) conjugate the scanners to the back focal plane of the objective.
The emission light path (e.g., the dichroic mirror and the detec-
tors) is not shown in the diagram.

2.3.2 Design and Setup An annular illumination pattern at the objective back focal plane
of a Bessel Focus Module generates a Bessel-like focus. This annular illumination can be
created with a phase mask [14], an SLM [15, 42], or an axicon
[16, 39, 40].
In an SLM-based Bessel focus module (Fig. 2a, rectangle box),
a reflective phase-only SLM is placed at the front focal plane of a
lens (L1). A circular binary phase pattern (alternating 0 and π) on
the SLM diffracts the incident Gaussian beam preferentially into the

1 diffraction orders, which form an annular ring at the back focal
plane of L1. An annular aperture mask is placed at the back focal
plane of L1 to selectively transmit the desired annular electric field,
which is conjugated to the galvos by a pair of lenses L2 and L3, and
then to the objective back focal plane.
An axicon-based Bessel focus module (Fig. 2b) has a similar
configuration to an SLM-based module, except that an axicon is
placed at the front focal plane of L1. The conical surface of the
axicon refracts the light according to Snell’s law, which forms a ring
at the back focal plane of L1. The annular aperture mask at the back
focal plane of L1 is not necessary for an ideal axicon (with infinitely
small conical tip), but is necessary in practice to block the unwanted
light refracted through the tip of an imperfect axicon.
When it comes to choosing between an SLM and an axicon-
based Bessel module, several factors need to be considered. An
SLM-based module offers more flexibility in terms of point spread
function (PSF) engineering [15] and allows the NA and axial length
of the Bessel focus to be adjusted independently [15]. Details on
designing Bessel foci with different PSF profiles are discussed in
Subheading 2.3.3, but a brief overview is presented here. In both
methods, the axial length of the Bessel focus can be altered by
varying the size of the Gaussian beam impinging on the SLM or
the axicon, for example, by adding a beam expander or reducer at
the entrance of the Bessel module. With the beam size fixed, a user
can adjust the NA and axial length of the Bessel focus indepen-
dently by changing the phase pattern on the SLM and the dimen-
sions of the annular mask. Therefore, an SLM-based module is ideal
for systems utilizing multiple objectives with different NAs (e.g., a
1.05-NA objective for neocortical imaging or a 0.5-NA
 High-Speed Neural Imaging with Synaptic Resolution 299

microendoscopic lens for deep brain imaging). In contrast, an

axicon-based module does not allow users to adjust NA and axial
focal length independently, without varying the beam size or intro-
ducing a different axicon [39, 40]. Translating one of the conjuga-
tion lenses (L2 or L3) along the optical axis concurrently changes
the NA and axial length of the Bessel focus [16]. However, despite
being less flexible in PSF engineering, the axicon-based Bessel
module is nevertheless an attractive alternative for the following
reasons: first, its transmissive layout occupies less space and makes it
easier to incorporate into an existing system [38]; second, an axicon
module costs much less (~$5000, rather than $30,000 for an
SLM-based module) to set up; third, an axicon works with a larger
wavelength range compared with an SLM (e.g., compatible with
three-photon microscopy [37]).

2.3.3 Design of a Bessel We use MATLAB® to calculate the PSF profiles of Bessel foci and to
Module guide the design of Bessel module. The underlying physics is
described below, and the MATLAB codes can be found in Refs.
[15, 16].

Generation of Annular Concentric binary grating patterns with phase values alternating
Illumination with an SLM between 0 and π are applied to a phase-only SLM to diffract most of
(Adapted from Ref. [15]) the incident electromagnetic field into the
1 orders (see note 2 in
Subheading 2.4), which after lens L1 forms a ring at the mask
plane. The radius of the ring (ρ), determined by the period of the
grating (d), the focal length of L1 ( f1), and the wavelength of the
light (λ), is calculated from the grating equation as:
f 1λ
ρ¼ :
d
For an annular mask to transmit the ring and block the other
diffraction orders, its inner and outer diameters Di and Do should
satisfy the relation:
D o þ D i ¼ 4ρ:
Combining the two equations above, to generate an annular
illumination pattern that centers on an annular mask with inner and
outer diameters Do and Di, the period of the circular binary grating
on the SLM is:
4f 1 λ
d¼ :
Do þ Di
With the size of the SLM pixel defined as p, the period of the
circular binary grating in units of pixels S is:
d 4f 1 λ
S¼ ¼ :
p pðD o þ D i Þ
300 Guanghan Meng et al.

The thickness of the annulus at the mask plane generated by the

above circular binary grating is ~2f1λ/beamD, with beamD being
the diameter of the excitation laser on the SLM [43].

Generation of Annular For an axicon with an apex angle A, the angle of incidence α on the
Illumination with an Axicon conical surface is: α ¼ πA
2 . The refraction angle is then derived
using Snell’s law: αr ¼ nα, given that α is small and n is the refractive
index of the axicon. Therefore, the angle between the refracted
light and the optical axis is:
θ ¼ αr  α ¼ ðn  1Þα:
The radius of the ring at mask plane is:
ρ ¼ θf 1 ¼ ðn  1Þαf 1 :
The annular mask to transmit the ring should again satisfy:
D o þ D i ¼ 4ρ:
Therefore, the apex angle and refractive index of the axicon
should meet:
Do þ Di
ðn  1Þα ¼ :
4f 1

Calculation of Two-Photon Two-photon excitation PSF can be calculated using Richards and
Excitation PSF Wolf integrals [44, 45], from which both the lateral and axial full
width at half maximum (FWHM) of the PSF can be determined.
The information required for PSF calculation includes: the wave-
length of excitation light, the objective information (i.e., NA,
magnification, immersion media), and the electric field distribution
at the objective back focal plane.
The following equations hold for all infinity-corrected
objectives:
FL tube
F L obj ¼ ;
M obj
BPD obj ¼ 2N A obj F L obj
where the FLobj is the focal length of the objective, FLtube is the
focal length of the tube lens, Mobj is the magnification of the
objective, and BPDobj is the back pupil diameter of the objective.
The Bessel annulus at the back focal plane with an outer diameter
Do and inner diameter Di gives rise to an excitation NA as:
Do
N ABessel ¼ N A obj :
BPD obj
Or:
Do
N ABessel ¼ :
2F L obj
 High-Speed Neural Imaging with Synaptic Resolution 301

The PSF profile is determined by Do, Di, and the electrical field
distribution within the annulus, which is discussed below.

Design of the Annular The annular mask is designed to be conjugated to the objective
Aperture Mask in an SLM- back focal plane, with a magnification factor M, which is deter-
Based Bessel Module mined jointly by focal lengths of lenses L2–L5. In most cases, the
(Adapted from Ref. [15]) objective, L4, and L5 (scan lens and tube lens) are already selected
and built prior to the design of the Bessel module as an add-on.
Therefore, one only needs to choose L2 and L3 to fit into the
available space and conjugate the mask plane to the galvos. It has
been demonstrated previously that a 0.4-NA Bessel focus works
well for in vivo two-photon fluorescence imaging in the brain [15],
a 0.3-NA Bessel focus has better performance than 0.4-NA one
when combined with two-photon fluorescence microendoscopy
[42] (due to the substantial off-axis aberrations of gradient refrac-
tive index lenses), and a larger NA Bessel beam is more suitable for
three-photon microscopy [37]. With the objective, L2–L5, and the
desired NA selected, the outer diameter of the annular mask is
determined as:
2N A Bessel F L obj
Do ¼ :
M
With NA (i.e. Do) fixed, the axial length of the Bessel beam is
dictated by the thickness of the ring, which is determined by the
focal length of L1 and the beam diameter on SLM, with smaller f1
and larger beamD generating thinner rings and longer Bessel foci.
The axial length of the Bessel focus can be further adjusted by fine-
tuning Di and S.
The electrical field distribution at the mask, including its ampli-
tude and phase, can be visualized via simulation (Fig. 3). The
annular mask selectively passes the electric field within Di < r < Do,
where r is the radial coordinate at the mask plane, and is usually
centered around the largest electric field amplitude (rmax, blue line,
Fig. 3). Two cases are of particular interest. In Case I, Di and Do are
selected to have the annulus fall on the two amplitude peaks closest
to and on either side of rmax (Fig. 4b, red lines in Fig. 3a, c). In Case
II, the edges of the annulus are located at the two zero-amplitude
crossings that are closest to and on either side of rmax (Fig. 4e, green
lines in Fig. 3a, c). Even though Case I has a thicker annulus than
Case II, the resulting Bessel focus has a longer axial FWHM than
that of Case II (Fig. 4c, f). This is because the negative parts of the
electric field (between the green and red lines, Fig. 3a, c) destruc-
tively interfere with the positive parts (between the green lines) and
broaden the axial FWHM. Reducing the thickness further from
Case II (e.g., between the purple lines) increases the axial FWHM
again. As shown in Fig. 4, to ensure that rmax is located at the center
of the annulus, without changing Do (i.e., the NA of the Bessel
beam), the period of the SLM pattern, S, can be concurrently
altered with Di.
302 Guanghan Meng et al.

Fig. 3 Electrical field distribution at the mask plane in an SLM-based Bessel module. (a) Amplitude and (b)
phase of the (c) electric field at different radial positions. 0 is the location of the optical axis. The red and green
dashed lines in (a) and (c) represent the inner and outer diameters of the annular masks for Case I and Case II
discussed in Subheading 2.3.3.4. (This figure is adapted from Ref. [15])

In summary, the two cases discussed above represent typical

examples of long and short Bessel focus and can be used as a
starting point for the mask design. If space permits, inserting a
beam expander or a beam reducer before the SLM can further vary
the axial length of the Bessel focus (see note 3 in Subheading 2.4).
The MATALB code to facilitate this module design can be found in
Ref. [15].

Design of an Axicon-Based When using an axicon for Bessel beam generation, one should start
Bessel Module with selecting a high-quality axicon (e.g., 1-APX-2-H254-P,
ALTECHNA Inc.; XFL25–010-U-B, ASPHERICON). Once the
axicon is selected, the apex angle α is determined (see Subheading
2.3.3.2); thus, the radius of the ring at the mask plane is only
determined by the focal length of L1 (Fig. 2b). From here, one
 High-Speed Neural Imaging with Synaptic Resolution 303

Fig. 4 Profiles of Bessel foci using masks with different ring thickness but the same outer diameter. SLM
phase pattern, the dimension of an electric field at annular mask, and a measured profile of a 2 μm diameter
fluorescent bead, corresponding to (a–c) Case I and (d–f) Case II in Fig. 3. (g, h) Simulated axial profiles of
Bessel foci generated by annular masks of different widths. (This figure is adapted from Ref. [15])
304 Guanghan Meng et al.

can simulate the electric field at the mask plane and the 3D PSF
(code can be found in Ref. [16]) to guide the selection of L1, L2,
and L3. Example simulation results are presented in Fig. 5. In this
simulation, lens L2 is translated along the optical axis. The location
where the front focal plane of L2 coincides with the back focal plane
of L1 (i.e., the mask plane) is indicated as D ¼ 0 mm (Fig. 5, second
column). When L2 is moving toward the mask plane (negative D),
more power is allocated to the central region of the objective back
focal plane (Fig. 5, first column), which yields a smaller effective NA
and, together with the phase distribution of the pupil function, a
longer Bessel focus. In contrast, moving the lens away from the
mask plane distributes more power to the edge of the objective
pupil function, leading to a bigger effective NA and a shorter focus
(Fig. 5, third column).
The mean radius of the annular mask becomes well defined
once L1 and axicon are both selected (see Subheading 2.3.3.2),
although one can jointly vary the outer and inner radii of the ring
and obtain different ratios of transmittance and axial profiles. The
mask is intended to eliminate the unwanted light passing through
the imperfect, typically round, tip of the axicon, which if not
blocked can interfere with the rest of the refracted electric field
and cause the measured PSF to deviate from simulation. The thin-
ner the annular mask is, the more effectively it can block the
unwanted light, but more power loss will be introduced to the
system. For the case when limited excitation power is available,
one can use a thicker mask, or even no mask, and obtain
non-theoretical yet still usable PSF profiles [16].

2.3.4 Alignment of The Bessel focus module is located between the laser and the
Bessel Module (Adapted two-photon fluorescence microscope. First, obtain locations of
from Ref. [15]) the front and back focal planes of lenses L1–L3 (e.g., from their
Zemax models or vendor specifications). Second, place lenses so
Installation of the Optical that the back focal plane of L3 is at the first scanner (or between the
Components two scanners, if they are not conjugated), the front focal plane of L3
is superimposed with the back focal plane of L2, and the front focal
plane of L2 is superimposed with the back focal plane of L1 (i.e.,
the mask). At last, place the SLM or axicon at the front focal plane
of L1 (see note 4 in Subheading 2.4). It is helpful to have two
mirrors with tip/tilt mounts between L3 and the first scanner to
co-align Gaussian and Bessel paths. Alternatively, if there is not
enough space, set up one mirror with both tip/tilt and translational
controls.

Gross Alignment of the Set up two irises in the optical path shared by the Gaussian and
Bessel and Gaussian Beam Bessel focus modalities between the alignment mirror
Paths (s) mentioned in Subheading 2.3.4.1 and the first scanner. They
should be sufficiently separated and not conjugated with each
other. Ideally, the first alignment iris in the Bessel module should
 High-Speed Neural Imaging with Synaptic Resolution 305

Fig. 5 Engineering Bessel foci by displacing Lens L2 in an axicon-based Bessel module. (a, e, i) 2D and (b, f, j)
1D (along x direction) representations of the amplitude and phase of the electric fields at the objective back
focal plane, (c, g, k) axial, and (d, h, l) lateral two-photon excitation point spread functions for L2
displacements of D ¼ 20 mm, 0 mm, and 20 mm, respectively. (This figure is adapted from Ref. [16])
306 Guanghan Meng et al.

be placed right after the alignment mirror(s) and the second iris as
close to the first scanner as possible to increase the accuracy of the
alignment. Adjust the positions of the irises to center on the (well
aligned) Gaussian beam. For SLM-based Bessel module, apply a
concentric binary grating on the SLM, and the reflected beam
appears as concentric rings. Then adjust the mirror(s) between L3
and the first scanner iteratively so that the Bessel concentric rings
pass through and center on the two irises (see note 5 in
Subheading 2.4).

Fine Alignment For fine alignment, dismount the objective and place a camera (e.g.,
DCC1545M, Thorlabs) at the back focal plane of the objective.
Under the Bessel mode, one should see a ring on the camera. (For a
well conjugated system, where the scanners are all conjugated to
each other, sweeping one or more scanners should not move the
position of the ring on the camera. But if the microscope does not
have its scanners conjugated, place the camera at the plane with
minimal movement.) Adjust the axial positions of L2 and L3 so that
the ring is sharpest on the camera, which indicates that the back
focal plane of the objective is conjugated to the annular mask (see
notes 6–8 in Subheading 2.4).
Placement of annular filter mask: Place the annular filter mask
at the back focal plane of L1. Apply the corresponding concentric
binary grating as calculated in Subheading 2.3.3 and adjust first the
lateral position of the mask until the post-mask ring is symmetric
and then the axial position so that the power passing through the
annular filter is maximized (see note 9 in Subheading 2.4).

2.3.5 Results and Data In this section, representative in vivo demonstrations of Bessel
Analysis focus scanning 2PFM are presented. In an SLM-based Bessel mod-
ule, using the method described in Subheading 2.3.4, the Bessel
beam of different NAs and lengths was employed to image the
cortical neurites of an awake Thy1-YFP line H mouse (Fig. 6).
With the increase of NA or axial length of the Bessel focus, more
energy is distributed to the side rings, which reduces image con-
trast. 0.4-NA Bessel foci provide high signal-to-background ratio
while maintaining the ability to resolve synapses laterally in
two-photon microscopy (Fig. 7). A single 2D scan of a Bessel
focus probes all structures from the entire volume (e.g., a depth
range of 60 μm in Fig. 7), and the calcium activities in individual
dendritic spines can be characterized (Fig. 7c). In contrast, a mini-
mum 36 scans of Gaussian beam (calculated by the ratio of axial
range of structures and Gaussian focus axial FWHM) are required
to cover the same volume (60 2D scans were used to generate
Fig. 7a). By using longer Bessel foci, imaging throughput can be
improved by more than 100 folds, with data size reduced by the
same factor.
 High-Speed Neural Imaging with Synaptic Resolution 307

Fig. 6 In vivo images of cortical neurites taken with Bessel foci of different NA and axial lengths, in a head-
fixed awake Thy1-YFP mouse. An SLM-based Bessel module was used. (a) Images obtained by 2D scans of
Bessel foci and their axial point spread functions. (b) A Gaussian image stack with structures color-coded by
depth obtained with a 1.05-NA Gaussian focus. (From Ref. [15])

With an axicon-based Bessel module, translating L2 allows

Bessel foci of different lengths and NAs to be generated. Functional
recording from neurons labeled with calcium indicator dye
(Cal-520 dextran) in zebrafish larvae is presented in Fig. 8. With
the axial length of the Bessel focus gradually increasing (Fig. 8a–h),
more and more structures are brought into focus using a single 2D
scan.
As mentioned in Subheading 2.1.3, Bessel focus greatly
reduces axial motion artifacts due to its longer axial extent. As a
result, only lateral 2D registration is required for correcting sample
308 Guanghan Meng et al.

Fig. 7 Bessel focus scanning technology improves imaging throughput while maintaining synaptic lateral
resolution in calcium imaging of GCaMP6s+ neurites in an awake mouse brain in vivo. (a) Average intensity
projection of an image stack acquired by 60 2D scans of a Gaussian focus (1.05 NA), with structures color-
coded by depth. (b) A single 2D scan of a Bessel focus (0.4 NA, 53 μm FWHM) probes the same volume. Insets:
zoomed-in views of dendritic spines. (c) Calcium activity traces from axonal varicosities (putative boutons) and
dendritic spines (arrowheads in b). (From Ref. [15])
 High-Speed Neural Imaging with Synaptic Resolution 309

Fig. 8 An axicon-based Bessel module enables 50 Hz volumetric calcium imaging of spinal projection neurons
in zebrafish larvae. (a) Image acquired by Gaussian focus scanning at 127 μm from the dorsal surface of the
head (relative depth z ¼ 17 μm). (b) Averaged calcium transients of neurons evoked by the acoustomecha-
nical tapping stimuli. (c, e, g) Volumetric images obtained by scanning a short (14 μm axial FWHM), medium
(24 μm axial FWHM), and long (39 μm axial FWHM) Bessel foci, respectively. (d, f, h) Averaged calcium
transients of responsive neurons. (i, j) Average intensity projections of a 66-μm-thick image stack acquired by
Gaussian focus scanning. Color in (i) encodes relative depth. Eleven trials were averaged in (b), (d), (f), and (h).
Shadow represents standard deviations. (From Ref. [16])

motion, instead of the much more computationally intensive 3D

registration/interpolation. As a demonstration, a Thy1-YFP mouse
was imaged in vivo and the fluorescence traces of the same dendritic
ROI measured with Gaussian and Bessel focus scanning, respec-
tively, were plotted (Fig. 9). Since the neurons were labeled with
310 Guanghan Meng et al.

Fig. 9 Bessel focus scanning is resistant to axial motion artifacts. (a, b) Images obtained with 2D scans of a
Gaussian focus and a Bessel focus, respectively, from an awake Thy1-YFP line H mouse. (c, d) Brain motion
(upper panel, quantified as the lateral image displacement with time) causes (c) large changes of fluorescence
signal from two YFP+ dendrites (ROI1 and ROI2) in Gaussian focus scanning mode, (d) but not Bessel focus
scanning mode. (From Ref. [15])

yellow fluorescent protein (YFP) rather than activity sensors (e.g.,

GCaMP6), fluctuations in the traces were motion artifacts, which
only showed up in Gaussian but not Bessel traces.

2.4 Notes 1. The beam expander after the EOM (Fig. 2a and Subheading
2.3.1) is optional, but included in all our custom-built 2PFM
systems. The output from typical laser usually has a small (e.g.,
~1 mm) diameter and short Rayleigh length, thus diverging
quickly during propagation. Expanding the beam increases
Rayleigh length and reduces divergence during beam
propagation.
 High-Speed Neural Imaging with Synaptic Resolution 311

2. Whenever a grayscale liquid-crystal SLM is utilized in the sys-

tem, calibration, i.e., the process of generating a lookup table
between the command values (gray level, e.g., 0–255 for an
8-bit SLM) and the phase exerted onto the wavefront of the
light, is necessary to ensure accurate wavefront control. For
Bessel beam generation (Subheading 2.3.3.1), since only 0 and
π phase shifts are required, a simple method can be used: First
carefully align the SLM, L1, and the mask following the steps in
Subheading 2.3.4. Then sequentially apply a series of concen-
tric binary grating images with the correct spatial period, but
different gray-scale values for high phase value rings onto the
SLM. Measure the power right after the mask. The image
giving the maximum post-mask power (i.e., yielding highest
diffraction efficiency) corresponds to the optimal 0-π binary
grating used for generating Bessel beam.
3. Inserting beam reducers or expanders into the beam path may
cause beam shifts, so one needs to double-check the optical
alignment after additional beam reducers/expanders are used
to change the Bessel beam size (Subheading 2.3.3.4). Refer to
Subheading 2.3.4 for detailed alignment instructions. Alterna-
tively, beam reducers/expanders should be mounted with tip,
tilt, and translational control.
4. When installing the SLM for Bessel beam generation (Sub-
heading 2.3.4.1), the incidence angle of the laser beam on
the SLM should be kept small to minimize pixel crosstalk that
would lead to decreased diffraction-efficiency (for our systems,
the incidence angle is 10 ).
5. In the Bessel beam path, and especially close to the planes
conjugate to the objective focal plane, iris(es) used for align-
ment (Subheading 2.3.4.2) should be carefully chosen so that
when fully opened, Bessel beam can pass through without
being clipped. (Closing down an iris near a plane conjugated
to objective focal plane to intentionally clip the Bessel rings can
shorten the Bessel beam, providing a convenient, if not easily
controlled, method of PSF engineering.)
6. Tilted Bessel focus: Even with perfect conjugation along the
optical pathway, one may still encounter an issue that the Bessel
focus is tilted away from the optical axis (i.e., when moving a
sample, say, fluorescent beads, up and down, bead images shift
laterally). It is caused by the annular illumination not being
centered on the objective back focal plane, often due to minor
alignment errors along the Bessel beam optical path. Often, a
small amount of tilting does not impact experimental interpre-
tation and does not need to be corrected. If not, adjust itera-
tively the mirror(s) between L3 and the first scanner
(Subheading 2.3.4) so that the image of the sample does not
312 Guanghan Meng et al.

move laterally as it moves up and down. To position the annular

ring more easily on the objective focal plane, a laser positioner
consisting of two prism mirrors mounted on two translation
stages can be included in the system. The motion axes of the
two translation stages are orthogonal to each other, which
enables both x and y position adjustment.
7. Sometimes tilting the Bessel beam intentionally can help with
the following issue: in Bessel modality, bright structures right
above or below the objects of interest may appear as bright
rings in the acquired image (e.g., when observing the superfi-
cial apical dendritic tree of an L2/3 cortical pyramidal neuron,
the soma in L2/3 can contribute to background). By tilting the
Bessel focus, the unwanted ring-like fluorescence is shifted
away from the structures of interest.
8. Asymmetric side rings in Bessel focus: Make sure that the laser
beam is incident at the center of the circular grating pattern on
the SLM or the conical tip of the axicon. When the beam size
on the SLM or axicon is small (e.g., an additional beam reducer
is used), this effect is particularly severe; thus, careful alignment
is needed (Subheading 2.3.4).
9. In both modules, a single mask with arrays of annular filters can
be mounted on a 3D translation stage with large motion ranges
in x and y, which allows the user to easily swap annular filters for
generating distinct Bessel foci. Even if only one annular mask is
needed, the mask should still be mounted on a 3D translation
stage, although the lateral motion range can be small, to allow
fine adjustment of mask position (Subheading 2.3.4.3).

3 Widefield Fluorescence Microscopy with Optical Sectioning

3.1 Background In standard widefield fluorescence microscopy, excitation light

simultaneously illuminates the sample area, from which the emitted
fluorescence is collected by a microscope objective and forms an
image of fluorescent structures on a camera. The parallel nature of
illumination and fluorescence detection enables widefield micros-
copy to reach high frame rates. However, in standard (i.e., non-
light-sheet illumination) widefield microscopy, emitted fluores-
cence comes from both structures in the focal plane and structures
above and below the focal plane. Its incapability to eliminate the
out-of-focus background fluorescence limits its application to thin
samples such as cultured cells or ultrathin tissue sections. A power-
ful approach that imparts optical sectioning capability to widefield
fluorescence microscopy utilizes structured illumination
(SI) [9, 23–25]. Because a structured illumination has its highest
contrast in the focal plane, the in-focus fluorescence image is
modulated while the out-of-focus background fluorescence is
 High-Speed Neural Imaging with Synaptic Resolution 313

unmodulated. Optically sectioned images can be reconstructed by

taking advantage of the difference in signal modulation [23, 24]. SI
can also be used to down-modulate sample spatial frequencies and
leads to super-resolution imaging [18, 20, 21, 46]. In this section,
we describe how to build, align, and operate a widefield structured
illumination microscope (SIM). Because super-resolution SIM
(SR-SIM) has been reviewed extensively previously [46], we focus
on an optical sectioning SIM (OS-SIM) implemented with a
refined image reconstruction method for accurate structural and
functional imaging of neurons and synapses in vivo [9].

3.2 Material and Figure 10 shows an example schematic diagram of a SIM setup.
Equipment After passing through an acousto-optic tunable filter (AOTF; AA
Opto-Electronic, AOTFnC-400.650-TN) (Fig. 11), an excitation
3.2.1 Optical
laser beam is expanded to match the active area of a spatial light
Components
modulator. To produce sinusoidal illumination patterns at the focal
plane, the laser light reflects off the spatial light modulator (SLM;
Forth Dimension Displays Ltd., QXGA-3DM) displaying binary
gratings. Two achromatic half-wave plates (HWP1 and HWP2;
Bolder Vision Optik, BVO AHWP3) and a polarizing beam splitter
(PBS; Thorlabs, PBS251) direct the laser to the SLM and maximize
its diffraction efficiency off the SLM, by ensuring the right polari-
zation direction. The diffracted light then transmits through the
PBS and has its polarization further controlled by another HWP
(HWP3; Bolder Vision Optik, BVO AHWP3) and a quarter-wave
plate (QWP; Bolder Vision Optik, BVO AQWP3), both of which
are mounted in a fast rotator (FR; Finger Lakes Instrumentation,
A24201). A dichroic mirror (D1; Semrock, Di-R405/488/561/
635-t3–25  36) reflects the illumination laser and transmits the
emitted fluorescence, and an identical compensating dichroic mir-
ror (D2, shown in Fig. 12) is used to minimize polarization scram-
bling (more details below). An objective lens (Nikon, CFI Apo
LWD 25X, 1.1 NA and 2 mm WD) is used for both illumination
and fluorescence collection. The emitted fluorescence is focused
and imaged on a camera (Hamamatsu, Orca Flash 4.0). Focal
lengths of all lenses (L1-L8) in the microscope: 150 mm,
125 mm, 400 mm, 400 mm, 175 mm, 300 mm, 85 mm, and
75 mm.

3.2.2 Fixed Mouse Brain To demonstrate structural imaging with OS-SIM, we prepared
Slices Preparation brain slices from a Thy1-GFP line M transgenic mouse (The Jack-
son Laboratory, stock 007788). After being completely sedated
with isoflurane (Piramal), the mouse was transcardially perfused
first with phosphate buffered saline (PBS, Invitrogen) followed by
4% paraformaldehyde (PFA, Electron Microscopy Sciences). The
mouse brain was dissected and immersed in 2% PFA and 15%
sucrose in PBS solution overnight at 4  C. Then the immersion
solution was replaced with 30% sucrose in PBS. After 24 h, the
Fig. 10 Detailed optical layout of a structured illumination microscope. (a) An amplitude mask in a rotational
mount selectively transmitting first-order diffraction beams. (b) Structured illumination generated by
two-beam interference at the sample plane. See Subheading 3.1 for detailed information of the optical
components

Fig. 11 Laser beam multiplexing with single-edge laser dichroic beam splitters
 High-Speed Neural Imaging with Synaptic Resolution 315

Fig. 12 The polarization scrambling by a dichroic mirror (D1) can be

compensated by another identical dichroic mirror (D2) properly oriented

mouse brain was sectioned on a microtome (Thermo Scientific™,

Microm HM430) to 100-μm-thick slices, and then mounted on
microscope slides to dry. After ~45 min, we placed cover glasses
with mounting medium (Vectashield® Hardset™ Antifade mount-
ing medium, H-1400) on top of the microscope slides with brain
slices.

3.2.3 Drosophila Larvae To demonstrate functional imaging with OS-SIM, we used trans-
Preparation genic Drosophila third instar larvae. A GCaMP6f-based postsynap-
tically targeted genetically encoded calcium indicator was expressed
in Drosophila larval muscle throughout development (genotype:
w1118; OK6-Gal4/UAS-CpxRNAi (BDSC Line #42017);
MHC-CD8-GCaMP6f-Sh/+). Larvae were dissected using a tradi-
tional semi-intact fillet preparation in HL3 solution (concentration
in mM: 70 NaCl, 5 KCl, 0.45 CaCl2·2H2O, 20 MgCl2·6H2O,
10 NaHCO3, 5 trehalose, 115 sucrose, 5 HEPES, and with pH
adjusted to 7.2) before imaging. During imaging, to maintain
viability, the larval fillet was immersed in HL3 containing 1.5 mM
CaCl2·2H2O and 25 mM MgCl2·6H2O.

3.3 Methods In this section, we provide instructions on how to successfully build

a structured illumination microscope. Most instructions apply to
both OS-SIM and SR-SIM. Throughout this section, we provide
useful notes where SR-SIM differs from OS-SIM in
implementation.
316 Guanghan Meng et al.

3.3.1 SIM Setup The illumination and emission light paths for a structured illumi-
nation microscope follow that of a standard widefield microscope,
with added components/modules to generate and optimize the
structured (e.g., sinusoidal fringe) illumination at the sample
plane (Fig. 10). In the following subsections we discuss critical
steps in generating SIM illumination.

Laser Beam Multiplexing, Widefield fluorescence microscopes usually use continuous-wave

Shuttering, and Expansion (CW) lasers as excitation sources. In this section, we demonstrate
the imaging of GFP (green fluorescence protein)-expressing sam-
ples; therefore, a single 488-nm CW laser is shown in Fig. 10. For
multi-color imaging, multiple CW lasers with different wavelengths
can be multiplexed using dichroic beam splitters at 45 angles of
incidence. The output power of multiple CW lasers can be con-
trolled with a single AOTF placed right after the combined laser
beams. When combining multiple laser beams, it is important to
ensure that they are all perfectly directed into the entrance of AOTF
and follow the same light path to the sample plane. We recommend
starting with a single CW laser (with the shortest wavelength, i.e.,
the laser closest to the AOTF) to build the microscope. Once its
alignment is optimized, other CW lasers can be added to the light
path. Each laser beam must have independent tip and tilt adjust-
ments, which can be done by using two successive mirrors between
the laser and the dichroic beam splitter. Beam expansion can be
done by using either pairs of achromatic lenses or integrated beam
expanders. Either way, we need to ensure that each laser beam is
collimated after expansion, which could be qualitatively tested with
a commercially available shearing interferometer (e.g., SI035 from
Thorlabs) that consists of a wedged optical flat (mounted at 45 to
the light path) and a diffuser on top. If the beam is collimated, the
interference fringes produced by the reflections from the front and
back surfaces appear parallel to the reference line on the diffuser.
Some beam expanders have the sliding lens design, facilitating easy
divergence adjustment with a collimation adjustment ring (e.g.,
GBE02-A from Thorlabs). One needs to rotate the adjustment
ring and meanwhile observe the interference fringes on the diffuser
until they are parallel to the reference line.

Beam Modulation Module 1. Structured Illumination Generated by Two Beam Interferences

The structured illumination at the sample plane can be pro-
duced by two-beam interference. In our implementation, a spatial
light modulator (SLM) is placed in conjugation to the sample plane
(Fig. 10). To generate a sinusoidal illumination pattern, the SLM
displays a binary grating pattern. The two first-order diffraction
beams are selected to transmit through an amplitude mask
(Fig. 10a) positioned at the focal plane of L1, and then imaged
onto the objective back focal plane by a pair of lenses (L2–L3). The
 High-Speed Neural Imaging with Synaptic Resolution 317

two beams exiting the objective interfere at the sample plane,

producing sinusoidal patterns (Fig. 10b). It is recommended to
use a flip mount for the amplitude mask, allowing an easy switch
between uniform and structured illumination.
2. Producing the Desired Illumination Patterns
There are a few important parameters to consider when choos-
ing the sequence of gratings displayed on the SLM: the pattern
period, pattern phase, pattern orientation, and the number of SI
images, which are optimized for specific samples and applications.
For a binary grating displayed on the SLM with the period d,
the diffraction angles θm are:
mλ
sin ðθm Þ ¼ ,
d
where m is the diffraction order, and λ is the illumination
wavelength.
At the focal plane of L1 (focal length FL1), the distance
between the
1-order diffraction beam focal spots, h
1 is:
λ
h
1 ¼ 2FL 1 tan θ1  2FL 1  :
d
The focal spots on the mask are then imaged onto the objective
back focal plane by L2 and L3 (focal lengths FL2 and FL3). H
1, the
distance between the two dots at the back focal plane, becomes:
λ FL 3
H
1 ¼ 2F 1   :
d F L2
The SI frequency/period at the sample plane is determined by
the ratio of H
1 to the diameter of objective back pupil. A ratio
equals 1 means the two beams are focused to the edge of the
objective’s back pupil (i.e., occupying the full numerical aperture
of the objective), and then exit the objective at the largest-possible
angles, generating a sinusoidal interference pattern of the highest-
possible spatial frequency at the focal plane (i.e., diffraction-limited
spatial frequency).
To change the phase or the orientation of the illumination
pattern, we laterally shift or rotate the binary grating on the SLM,
respectively. This results in the sinusoidal interference pattern at the
focal plane shifting by a specific phase or rotating to a
corresponding orientation.
For OS-SIM, the SI period should be selected based on the
desired optical section thickness and the sample. In theory, the
patterns that provide the maximal optical-sectioning strength
(i.e., generate the thinnest optical sections) have spatial frequency
that is half of the diffraction limit [23, 47]. In practice, when
318 Guanghan Meng et al.

imaging a thick or densely labeled sample, the strong fluorescence

background sometimes makes it difficult to detect the modulated
signal in the focal plane if the optical section is too thin. In such
cases, the SI spatial frequency should be empirically determined to
optimize OS-SIM image quality. For SR-SIM, SI patterns with
higher spatial frequency lead to higher lateral resolution. SI with
spatial frequency at the diffraction limit, however, loses optical
sectioning capability (because the resulting standing wave illumina-
tion maintains contrast even out of the focal plane). In thick sam-
ples, a compromise between optical sectioning strength and spatial
resolution is required [8] for SR-SIM.
The number of SI images varies with different reconstruction
methods. For example, the OS-SIM implementation proposed by
Neil. et al. [23] requires three SI images with the same orientation
but equally spaced phases. For 2D SR-SIM [20], to achieve the
most resolution improvement, SI images with multiple ( 3) orien-
tations and phases ( 3) are typically used.

Maximizing Diffraction A polarizing beam splitter (PBS) is used to reflect the excitation
Efficiency and Pattern beam toward the SLM and transmit the diffracted beam traveling
Contrast at the Sample away from the SLM (Fig. 10). As the PBS reflects the s-polarized
Plane component and transmits the p-polarized component of the inci-
dent light, an achromatic half-wave plate (HWP) is placed before
the PBS to control excitation beam polarization. The HWP is
rotated to minimize the transmitted power, thus maximizing the
beam power delivered to the SLM. Our SLM has maximal diffrac-
tion efficiency for p-polarized light. Therefore, another HWP is
placed between the SLM and the PBS. With a binary pattern
displayed on the SLM, we rotate this HWP to minimize the
power in the 0th-order diffraction beam.
High modulation contrast is essential for both OS-SIM and
SR-SIM imaging. For OS-SIM, high contrast ensures strong mod-
ulation of in-focus signals, providing maximal optical sectioning.
For SR-SIM, high contrast ensures large magnitude of high fre-
quency components, supporting the extended spatial resolution.
To maximize the illumination contrast, the two interfering beams
should have s-polarization at the sample plane. In principle, one can
use a HWP set at an optimized angle for the grating orientation for
OS-SIM, or mount a HWP in a fast rotator and maintain
s-polarization for all illumination orientations used for SR-SIM.
However, in practice the s-polarization state may not be maintained
when the illumination beams reach the sample plane. This is
because optical components in the illumination light path may
alter beam polarization. For example, a dichroic mirror used to
separate excitation and emission light (Fig. 10, D1) may reflect
and transmit the p- and s-polarization components differently,
and therefore scramble the polarization of the illumination and
make an originally linearly polarized light to become elliptically
polarized.
 High-Speed Neural Imaging with Synaptic Resolution 319

To solve this problem, we can use the combination of a HWP

and a QWP mounted on fast rotators (HWP3 and QWP on FRs,
Fig. 10) to compensate for the ellipticity altering effect and provide
desired polarization at the imaging plane [48]. For each pattern
orientation (i.e., each desired polarization), we need to find the
rotational angles of the two waveplates that maximize the contrast
at the imaging plane. In practice, we take a series of SI images while
rotating the two waveplates, until image contrast is maximized. For
OS-SIM, the angles only need to be optimized for a single orienta-
tion. For SR-SIM, angle optimization is required for all SI orienta-
tions and the waveplates must be rotated during image acquisition
to ensure maximal contrast for all SI orientations. In our system,
this is achieved by mounting the waveplates in two fast rotators
synchronized with SLM display and imaging acquisition.
Alternatively, we can utilize an additional, identical dichroic
mirror oriented at a specific angle relative to the first dichroic
mirror [49] (Fig. 12), with the reflections upon the two dichroic
mirrors interchanging the s- and p-polarization components and
therefore canceling out the polarization scrambling by the two
dichroic mirrors.

Fine Alignment SIM is very sensitive to misalignment, and here we provide two
useful methods to ensure perfect alignment. With both methods,
fine tip/tilt adjustment is made with two successive mirrors before
the SLM.
The first method utilizes the back-reflection pattern from the
objective lens. We first move the mask out of the light path (or flip
down if a flip mount is used). We then let the SLM display a flat
pattern so that it acts as a mirror and allows standard widefield
illumination. To achieve perfect alignment with the illumination
beam entering the objective center along its optical axis, we hold a
piece of white paper with a hole between L3 (Fig. 10) and the
objective, letting the illumination beam transmit through the hole
and reach the objective lens. We then can observe the light reflected
from the lens components inside the objective onto the white
paper. In the case of perfect alignment, the pattern appears as
concentric rings (Fig. 13a); when it is misaligned, the pattern
appears off-centered and scrambled (Fig. 13b).
With the second method, we directly observe the two interfer-
ing beams below the objective lens. For this, we let the SLM display
a grating pattern and put the mask back in path. Here we display on
the SLM a fine grating pattern so that the two diffraction orders
enter the objective lens close to its edge (typically at 80% of the NA;
see Subheading 3.3.1.2 for detailed information). In the case of
perfect alignment, the two beams below the objective appear sym-
metric in shape and with similar brightness (Fig. 14a). Choosing a
fine pattern makes any clipping of the two beams easy to detect. If
the light path is misaligned, the two beams appear asymmetric and
320 Guanghan Meng et al.

Fig. 13 Back-reflected illumination light observed before the objective. (a) Perfect alignment pattern. (b)
Misalignment pattern

with different brightness (Fig. 14b). This procedure should be

repeated for different orientations to ensure perfect alignment
along all directions.

3.3.2 SIM Detection Path In our system, the emitted fluorescence is collected by the same
objective and focused onto a camera for imaging (Fig. 10) with
achromatic lenses (L4–L8). Magnification from the sample plane to
the camera should ensure Nyquist sampling for the desired resolu-
tion. For SR-SIM that can double the diffraction-limited resolu-
tion, the pixel size should be smaller than a quarter of the
diffraction-limited widefield resolution.

3.3.3 Optical-Sectioning In this section, we introduce how structured illumination is utilized

Widefield Imaging and Its for optical sectioning and describe a refined OS-SIM reconstruc-
Application Examples tion method optimized for in vivo imaging.

Refined OS-SIM The basic idea of OS-SIM is that only in-focus information can be
Reconstruction Method effectively modulated. Figure 15 shows how structured illumina-
tion only preserves its contrast for structures in the focal plane (blue
arrows). When a structure is out-of-focus (orange arrows), its
fluorescence signal is not or only weakly modulated. Taking advan-
tage of this difference in signal modulation, we could computation-
ally reject out-of-focus background and retrieve in-focus
information.
One popular OS-SIM implementation was proposed by Neil
et al. [23], which requires three SI images with equally spaced
 High-Speed Neural Imaging with Synaptic Resolution 321

Fig. 14 Transmitted illumination light observed below objective. (a) Perfect

alignment. (b) Misalignment

phases 0 , 120 , and 240 . This “basic OS-SIM” method recon-

structs an optically sectioned image using Eq. (1):
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
Ibasic SIM ¼ ðI0  I1 Þ2 þ ðI1  I2 Þ2 þ ðI2  I0 Þ2 , ð1Þ
where I0, I1, I2 is the intensity of each image at 0 , 120 , and 240
phases, respectively. The pairwise subtractions discard the out-of-
focus (un-modulated) signal, and the summing removes the
non-uniform illumination patterns. As shown in Fig. 16, compared
with the widefield image (Fig. 16a), basic SIM (Fig. 16b) effectively
suppressed the background fluorescence. However, the signal-to-
noise ratio (SNR) is suboptimal because of the positive bias created
by the non-linear operation, making it difficult to resolve fine
structures such as dendritic spines (Fig. 16b inset). We proposed a
method [9] to suppress high-frequency noise while maintaining
322 Guanghan Meng et al.

Fig. 15 Structured illumination only modulates in-focus signals effectively. Images at two sample planes with
different structures in focus. Blue arrows: in-focus structures, orange arrows: out-of-focus structures.
Scale bar: 3 μm

optical sectioning: we first low-pass (LP) filtered the basic SIM

image, high-pass (HP) filtered the noise-averaged (free of positive
bias) widefield image (IWF ¼ [I0 + I1 + I2]/3), and then carried out
a weighted summation of the two in order to reconstruct the final
optical section using Eq. (2):
ISIM ¼ LPðIbasic SIM Þ þ α∙HPðIWF Þ, ð2Þ

With this refined algorithm, we observed a substantial improve-

ment in SNR, which helped to better reveal the morphology of
dendrites and dendritic spines (Fig. 16c).
We imaged the same dendritic structures using a two-photon
fluorescence microscope (Fig. 16d), the most popular OS imaging
method for brain tissue. Comparing the images taken with OS-SIM
and two-photon, we found that they provided comparable optical
sectioning capability. In addition, one-photon excitation of
OS-SIM led to higher resolution, resulting in crisper and sharper
OS-SIM dendritic spine images.
 High-Speed Neural Imaging with Synaptic Resolution 323

Fig. 16 Widefield, basic SIM, refined OS-SIM, and two-photon fluorescence images of Thy1-GFP line M brain
slices. (a–d) Maximum intensity projections (MIPs) of 8-μm-thick widefield (WF), basic SIM, refined OS-SIM,
and 2-photon image stacks (0.1 μm Z step, 21–29 μm depth, 440  396 pixels at 86 nm pixel size),
respectively. Insets are single optical sections. Scale bar: 5 μm; insets: 1 μm

Fast Functional Imaging Applying OS-SIM to functional imaging, we demonstrated the

Using OS-SIM capability of OS-SIM in capturing in vivo calcium events at high
frame rate. We performed functional imaging at Drosophila larval
neuro-muscular junctions (NMJs), where the muscle was labeled
with post-synaptically targeted GCaMP6f-based genetically
encoded calcium indicator [50].
Similar to the brain slice data, OS-SIM provided excellent
optical sectioning, resulting in images with much higher contrast
(Figs. 17a–c). We recorded the calcium activity at the NMJs at
25 Hz OS-SIM frame rate (75 Hz for raw image frames). By
calculating fluorescence change ΔF/F from eight regions of inter-
est, we compared the sensitivity of widefield and OS-SIM imaging
in reporting calcium activity (Fig. 17d). The suppression of the out-
of-focus fluorescence background by OS-SIM gave rise to a ~8
324 Guanghan Meng et al.

Fig. 17 In vivo functional imaging of quantal releases at the neuro-muscular junctions (NMJs) of a Drosophila
larva with OS-SIM. (a–b) Averages of widefield (WF) and OS-SIM image sequences (frames without calcium
activity) of NMJs (at a depth of 20 μm, 492  492 pixels at 86 nm pixel size). Scale bar: 5 μm; insets: 2 μm. (c)
Lateral line profiles across the structure in the insets (b, along red dashed line). (d) Spontaneous calcium
transients from 8 regions of interests (8 s of recording, orange circles in a). Widefield transients were
increased by 8 times for better visualization. (e) Averaged calcium transients over 5 events (black asterisks in
d) measured with widefield and OS-SIM

larger ΔF/F than widefield imaging (Fig. 17e). Furthermore, with-

out the contribution of the often unevenly distributed out-of-focus
fluorescence, OS-SIM allowed accurate measurement of the ampli-
tudes of calcium transients and quantitative comparison of in vivo
activity in different structures.

Parameter Selection in For optimal image reconstruction, parameters should be carefully

Image Reconstruction selected based on the imaged sample as well as the desired section-
ing strength. Theoretically, the maximum sectioning strength is
obtained when using an illumination period twice the diffraction
limited resolution [47]. In practice, there exists a tradeoff between
the optical sectioning strength v.s. modulation depth and signal-to-
noise ratio. As a result, when imaging a sample with strong back-
ground fluorescence, a larger illumination period should be used so
that the modulated signal can be more easily detected. When
extracting low- and high-spatial frequency information from the
basic SIM and widefield images, the crossover frequency, σ, was
chosen to balance artifact suppression and sectioning strength.
Larger σ means that we take more information from the basic
SIM image, while small σ means more information from the wide-
field image, which typically has higher SNR than the basic SIM
image. Thus, when imaging samples with higher SNR such as fixed
brain slices, we used larger σ to better exploit the optical sectioning
capability from the basic SIM image; when imaging noisy samples,
we used smaller σ to sacrifice optical sectioning for better image
 High-Speed Neural Imaging with Synaptic Resolution 325

quality. The scaling factor, α, weights the widefield image to ensure

continuity in the Fourier domain. The value of α was determined by
the modulation depth, which can be either precisely calculated
using the correlation-based algorithm [51] or empirically estimated
by final image quality.

Other Optical Sectioning We described and demonstrated our refined OS-SIM method for
Reconstruction Methods in vivo structural and functional imaging in previous sections.
There are other optical sectioning reconstruction methods as well
as structured illumination strategies. For example, differential illu-
mination focal filtering (DIFF) microscopy [25], a variant of HiLo,
reconstructs one optical section from two images with structured
and complementary illumination patterns. HiLo microscopy
[24, 52], another SIM method, reconstructs one optical section
from one SI image and one uniform illumination image. HiLo has
faster imaging speed and is less sensitive to illumination distortion
and sample motion. For systems that cannot provide precise SI
translation (e.g., by an SLM), HiLo is a better option. The
OS-SIM system described here can also implement these alternative
SIM methods. A method should be chosen after considering the
application, implementation difficulty, and budget.

Other Considerations for In In addition to the often low signal-to-noise ratio, two other issues
Vivo Imaging of applying OS-SIM to in vivo imaging are sample-induced wave-
front distortion and motion-induced reconstruction artifacts. Pre-
viously [9], we demonstrated that aberration correction by adaptive
optics is essential for OS-SIM in both structural and functional
imaging of in vivo structures. For example, the imaging of the
Drosophila larva suffered from spherical aberrations coming from
the muscle layers above the focal plane and the high sucrose con-
centration immersion saline, resulting in severe reconstruction arti-
facts and abnormal calcium dynamics. All our presented results in
this section were aberration corrected. For motion artifacts, we
used a phase-corrected algorithm to remove motion-induced
reconstruction artifacts [9], which is especially important for
in vivo imaging.
Since the demonstrated 25 Hz rate is more than sufficient for
calcium imaging, we did not push for the highest imaging speed
that our camera is capable of. The imaging speed of OS-SIM is
theoretically limited by the frame rate of the camera, with the
maximum full chip (2048  2048) frame rate at 100 Hz. The
frame rate can be increased by simply reducing the line number of
readout. The camera in our system (Hamamatsu, Orca Flash 4.0)
can operate at 400 Hz at 512 lines and 800 Hz at 256 lines. By
implementing an interleaving OS-SIM reconstruction [53], the
OS-SIM frame rate equals that of the raw image frame rate. Thus,
for an imaging area of 256  2048 pixels, the 800 Hz frame rate
makes voltage imaging possible with OS-SIM.
326 Guanghan Meng et al.

4 Discussion

Both Bessel focus scanning two-photon fluorescence microscopy

and optical-sectioning widefield microscopy can perform high-
speed imaging of neural activities at synaptic resolution. OS-SIM
can have faster frame rates; however, its application for deep imag-
ing of optically opaque samples is challenging due to tissue scatter-
ing. Volumetric imaging with OS-SIM requires physical movement
of the objective or sample, thus maybe slower than Bessel focus
scanning two-photon fluorescence microscopy, which allows video-
rate volumetric recording of neurons at hundreds of microns deep
into the highly scattering mouse brain. One should choose from
the two methods based on sample and application, for example,
whether tissue scattering is a concern and whether activity informa-
tion over large volume is required. For thin or transparent samples,
OS-SIM enables functional imaging with synaptic resolution at
hundreds of hertz; for opaque samples, Bessel focus scanning
two-photon fluorescence microscopy would be the method of
choice for volumetric activity imaging. Furthermore, both methods
can be combined with other cutting-edge techniques and labeling
strategies, e.g., adaptive optics [13, 54–56] and near-infrared sen-
sors [57–59], to further enhance the imaging resolution and depth,
respectively.
Recent advances in optogenetic actuators and microscopy tech-
niques to activate them have allowed all-optical manipulation of
neuronal activity at single-cell resolution (see also Chap. 3 and 11 of
this book). They can be combined with both microscopy methods
described here to realize all optical writing and reading of neuronal
activity. The high volumetric imaging throughput of Bessel focus-
ing scanning two-photon fluorescence microscopy makes it partic-
ularly suited to study the effect of selectively activating a
subpopulation of neurons on the activity dynamics of extended
3D networks [38]. If large imaging depths or high volumetric
rates are not required, OS-SIM designed to have a large FOV can
be combined with optogenetic stimulation to monitor activity over
a mesoscopic area [60].

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 Chapter 11

Optical and Analytical Methods to Visualize and Manipulate

Cortical Ensembles and Behavior
Luis Carrillo-Reid, Weijian Yang, and Rafael Yuste

Abstract
The development of all-optical techniques and analytical tools to visualize and manipulate the activity of
identified neuronal ensembles enables the characterization of causal relations between neuronal activity and
behavioral states. In this chapter, we review the implementation of simultaneous two-photon imaging and
holographic optogenetics in conjunction with population analytical tools to identify and reactivate neuronal
ensembles to control a visual-guided behavior.

Key words Two-photon imaging, Two-photon optogenetics, Holographic microscope, Neuronal

ensembles, Pattern completion, Population vectors, Control of behavior

1 Introduction

One of the main questions in modern neuroscience is how the

activity of identified neuronal populations generates behavioral
and mental states [1]. Recent advances in optical techniques that
allow the simultaneous recording and manipulation of neurons
with single-cell resolution [2, 3] combined with population analyt-
ical tools [4–8] suggest that neuronal ensembles are units of brain
computation [9, 10]. Neuronal ensembles are groups of neurons
with coordinated activity that may underlie sensations, perceptions,
emotions, and memories. In order to prove causality between the
activity of specific neuronal ensembles and learned behaviors, it is
becoming clear that the ability to manipulate and record hundreds
of neurons simultaneously needs to be guided by analytical tools
that allow the identification of neurons that could have a determin-
istic impact in brain states. Holographic two-photon imaging [11],
two-photon optogenetics [12], and analytical tools [13] offer the
possibility to target neuronal ensembles to control behavioral
states. The demonstration of the causal relation between neuronal
ensemble activity and behavior has been achieved recently in

Eirini Papagiakoumou (ed.), All-Optical Methods to Study Neuronal Function, Neuromethods, vol. 191,
https://doi.org/10.1007/978-1-0716-2764-8_11, © The Author(s) 2023

331
332 Luis Carrillo-Reid et al.

different brain areas [4, 14–18], paving the pathway to design more
sophisticated experiments to understand complex mental states in
health and disease [19].
The optimal design of all-optical experiments to control
learned behaviors with single-cell resolution requires the simulta-
neous reading and writing of neuronal activity. This could be
achieved in many ways [20], but in this chapter we will focus mainly
on scanning and parallel optical techniques guided by analytical
tools. We describe the implementation of scanning two-photon
imaging and parallel two-photon optogenetics using a spatial light
modulator (SLM). We also describe the main concepts necessary to
identify and target neurons with pattern completion capability
[6, 21] that can recall neuronal ensembles related to a visually
guided behavior [4].

2 Implementation of Simultaneous Two-Photon Imaging and Two-Photon

Optogenetics

2.1 Background Optical methods are powerful to record and manipulate neuronal
activity at single-cell resolution across a large population—a key
2.1.1 Light-Sensitive
requirement to investigate neuronal ensembles. Comparing with
Sensors and Actuators of
electrophysiology, optical methods can simultaneously sample and
Neuronal Activity
target a large group of neurons with high spatial specificity in a
noninvasive manner and can be conveniently implemented for
in vivo studies. The cornerstone of optical methods is the optical
microscope, which uses light to record and manipulate the neurons
that are illuminated. As the neurons in the brain do not typically
respond to light, they can be loaded or transfected with light-
sensitive sensors and actuators that can respectively report and
manipulate their neuronal activity upon light illumination. Using
these neuronal activity sensors and actuators, an optical microscope
can simultaneously read and write neuronal activity across a large
field of view with high spatial specificity.
Calcium [22–26] and voltage indicators [27, 28] are two com-
monly used sensors for neuronal activity. These indicators are
embedded with fluorophores, which can absorb the illumination
or excitation light, and emit fluorescence. The efficiency of this
light-absorption, fluorescence-emission process is modulated by
the intracellular calcium concentration and membrane potential
respectively in calcium indicators and voltage indicators (See also
Chaps. 1 and 2). Thus, the neuronal activity can be deduced from
the time-lapse recording of the fluorescence in individual cells. The
commonly used calcium indicators include GCaMP6 [26],
jGCaMP7 [29], jRGECO [30], etc. Compared with calcium indi-
cators, voltage indicators are less mature with smaller signal-to-
noise ratio and prone to photo-bleach though much progress has
been made in the past years [31] (Chap. 2). The counterparts of
 All-Optical Probing of Neuronal Ensembles 333

these activity sensors are actuators, which could be chemical-based

neurotransmitter cage-compounds [32, 33] or opsins [34, 35]
serving as light-sensitive ion channels. When absorbing the light,
the cage-compounds could release the neurotransmitters (unca-
ging) and the opsins could open or close the ion channels (opto-
genetics) and thus control the membrane potential. This
mechanism thus allows light control of neuronal activity in individ-
ual cells.

2.1.2 One-Photon and Depending on how light is interacting with the light-sensitive
Multiphoton Excitation sensors and actuators, optical methods can be classified into two
categories: one-photon and multiphoton (i.e., two-photon
[36, 37] or three-photon [38, 39]). In one-photon case, the inter-
action is linear. The fluorescence emission rate of the sensors and
the actuation strength of the actuators are proportional to the
intensity of the excitation light before saturation. While
one-photon excitation is straightforward, it lacks spatial specificity
in 3D. As the excitation would form a double cone-shape pattern
along the axial direction (Fig. 1), all neurons with the sensors or
actuators within the double cone may absorb the light. It is thus
very challenging to just excite a single neuron in a 3D volume.
Furthermore, in terms of imaging, the out-of-focus fluorescence
emission typically becomes background on the image captured at
the focal plane and thus reduces the image signal-to-noise ratio. As
the light-absorption and fluorescence-emission cycles create pho-
totoxicity, one-photon excitation pays a high price to image the
focal plane by creating phototoxicity within the entire double cone
volume. Multiphoton excitation can greatly overcome these limita-
tions, as the interaction between the light and the sensors or
actuators is nonlinear. In the two-photon case, the excitation effect
is proportional to the intensity square of the excitation light before
saturation, leading to a strong gradient of the excitation effect
along the optical axis in the double cone. Thus, it is feasible to
control the incident light so only the light intensity at the focal
point (i.e., the tip of the double cone) is strong enough to excite
the neuronal sensors or actuators (Fig. 1). This greatly improves the
spatial specificity or resolution and reduces the phototoxicity in
out-of-focus region. Another advantage of two-photon excitation
is that the excitation light has a longer wavelength, which reduces
the light scattering effect in scattering brain tissues. Thus
two-photon excitation can penetrate much deeper in the brain
compared with one-photon excitation. The drawback of
two-photon excitation is that much more laser power is required
due to a lower efficiency of multiphoton absorption. As the excita-
tion light eventually turns into heat, more heat will be generated
inside the brain. This is typically not a concern as in a typical
imaging experiment, the laser power used is less than the brain
damage threshold [40], even at very deep layers. In the past two
334 Luis Carrillo-Reid et al.

Fig. 1 Comparison between one-photon and two-photon excitation. In one-photon excitation (a), a double
cone is formed by focusing the visible excitation light (indicated by blue color); fluorophores (green) in the
entire double cone could be excited. In two-photon excitation (b), while a double cone is still formed by the
focus of infrared excitation light (indicated by red color), the fluorescence generation (green) is localized at the
vicinity of the focal region. Reprinted with permission from [101], Springer Nature. Experimental illustration of
the one-photon and two-photon excitation (0.16 NA for both cases) is shown in the bottom panel. (Reprinted
with permission from Ref. [102], Springer Nature)

decades, two-photon microscopes have become one of the work-

horses in neuroscience. Recently, three-photon excitation has been
successfully demonstrated for both calcium imaging [39] and opto-
genetics [41]. Comparing with two-photon excitation, three-
 All-Optical Probing of Neuronal Ensembles 335

photon excitation has an even higher nonlinearity and could further

increase spatial specificity and penetration depth, though the laser
power should be managed so it will not exceed the brain damage
threshold.

2.1.3 Basic Setup of Since the multiphoton absorption rate is generally low, and it has a
Multiphoton Microscope nonlinear relationship with the light intensity, a femtosecond laser
is required for multiphoton excitation. The femtosecond laser deli-
vers a periodic pulse train, typically with a repetition rate of
1~80 MHz. The temporal width of a pulse is below 300 fs, yielding
a very high instantaneous peak power and thus a high multiphoton
absorption rate. Unlike one-photon excitation where a large area or
volume of sample could be simultaneously illuminated and their
fluorescence could be detected through a camera in the case of
imaging, the illumination of multiphoton light is typically through
a rapid scanning of the laser beam on the sample. In multiphoton
imaging, a single pixel detector (versus pixel array in a camera) is
used to record the emitted fluorescence (see also Chap. 2). By
correlating the temporal signal from the detector and the scanning
trajectory, the image can be built. The microscope setup (Fig. 2a)
typically includes a raster lateral (xy) scanning system composed of a
galvanometer scanner and a resonant scanner (30~60 Hz frame
rate), or two galvanometer scanners (4~10 Hz frame rate). For
volumetric imaging, an axial (z) scanning system is implemented
by adding a piezo-electric controller on the objective lens or insert-
ing axial focusing devices, such as electrically tunable lens [42],
spatial light modulator [43], or remote focusing unit [44–46] (~
ms focus switching time). Different axial planes are sequentially
scanned (Fig. 2b). While this configuration of lateral and axial
scanning is straightforward, it may not be the most efficient as it
blindly samples the brain tissue. There could be a large volume of
“empty” extracellular space without neurons, and among all the
neurons, typically only a subset of them are labeled with the calcium
indicators. Random access scanning techniques [47–51] can over-
come this issue. The laser focus spot can jump rapidly between
different regions in the sample in 3D (<20 μs transition time
between different regions) (Fig. 2c). This is enabled by acousto-
optic deflectors (AODs). The challenges of this technique are to
overcome the spatial and temporal distortion of the ultrashort laser
pulses, caused by the angular dispersion from the AODs’ phase
grating and the group delay dispersion of the AOD crystals, respec-
tively. These limit the imaging field of view. While these distortions
can be compensated [51], the optical setup is complex. AODs also
have a limitation of high insertion loss. Recently, many advanced
scanning mechanisms including beam multiplexing [43, 52–55] are
proposed and demonstrated to increase the imaging throughput.
For extensive reviews, see [56–59].
336 Luis Carrillo-Reid et al.

Fig. 2 Basic setup of two-photon laser scanning microscope. (a) Schematics of a

typical two-photon laser scanning microscope. The Pockels cell is used to
modulate the laser light intensity. The xy-scan system and z-scan system are
used to scan the laser focal spot laterally and axially in the sample, respectively.
The dichroic mirror transmits the infrared excitation light and reflects the visible
fluorescent light to the photomultiplier tube (PMT). (b) Schematics of the typical
scanning trajectory in volumetric imaging. (c) Schematics of an exemplary
scanning trajectory using random access scanners. The green regions indicate
neurons of interest. (Reprinted with permission from Ref. [56], Springer Nature)

The setup of multiphoton photostimulation is similar as multi-

photon imaging, but without the detection module. The laser spot
on the sample is typically controlled by a pair of galvanometer
mirrors, and raster or spirally scanned across each target neuron
(1~100 ms) before jumping to another neuron. Alternatively, the
combination of beam shaping and temporal focusing techniques
[60–64] can be implemented so that a disk pattern with tight axial
confinement can be projected to the entire neuron. The scanners
no longer perform raster or spiral scanning, but direct the disk
excitation pattern to different neurons sequentially [3]. Using the
holographic approach, illumination patterns could be simulta-
neously projected to multiple targeted neurons, so the scanners
are no longer required [62–66]. We will discuss this topic in
depth, as well as holographic illumination technique that can simul-
taneously photostimulate multiple neurons, in the next section.
In the following, we focus on our implementation of a micro-
scope that can simultaneously perform calcium imaging to record
neuronal activity and optogenetics to manipulate neuronal activity.
 All-Optical Probing of Neuronal Ensembles 337

2.2 Simultaneous An all-optical method [20] refers to simultaneously recording and

Two-Photon Imaging manipulating the neuronal activity through light. Here, we describe
and Two-Photon the combination of two-photon calcium imaging and two-photon
Optogenetics optogenetics in a microscope. To flexibly image and manipulate
neuronal ensembles, it is desired to have the following features in
2.2.1 Overall the microscope:
Consideration
(i) Calcium imaging and optogenetics should be independently
controlled and performed.
(ii) Two-photon imaging could image neuronal activity over a large
neuronal population with cellular resolution and high temporal
resolution (4~30 Hz for calcium imaging).
(iii) Two-photon optogenetics could simultaneously photostimu-
late multiple neurons with cellular resolution and high temporal
precision (sub-millisecond to milliseconds).
To simultaneously, while independently, perform calcium
imaging and optogenetics, the microscope should have two inde-
pendent beam paths, respectively, for calcium imaging and optoge-
netics (Fig. 3a) [2, 3, 66–69]. These two beam paths then merge
together before the light is directed into the brain tissue. Each
beam path should be implemented with an individual femtosecond
laser with a different wavelength, which corresponds to the central
excitation spectrum of the calcium indicators and opsins. It is
important that they have a different excitation spectrum, so as to
prevent crosstalk between imaging and photostimulation (see also
Chaps. 1, 2, and 5). In other words, when performing calcium
imaging, the imaging laser should not excite the opsin; when
performing optogenetics, the photostimulation laser should not
excite the fluorescence of the calcium indicator. There are two
common options to choose the calcium indicator and opsin pair:
green calcium indicators such as GCaMP [26, 29] with red-shifted
opsins such as C1V1 [70], or red-shifted calcium indicators such as
jRGECO [30] with blue-activated opsins such as ChR2 [34]. While
both of these pairs have minimal overlap in the excitation spectrum,
it is critical to keep the imaging laser power low and perform
control experiments to ensure that the imaging laser does not
increase the spiking rate of the neurons (see Note 1). In case the
photostimulation laser creates fluorescence artifacts, the imaging
data should be processed to eliminate the artifacts. For a successful
experiment, it is also important to have a high co-expression rate of
the calcium indicators and opsins on a large neuronal population.
The cooperation of both constructs into a single virus could be a
promising approach [14, 71]. An example of co-expression of
GCaMP6s and C1V1 using two different viruses is shown in
Fig. 3c.
338 Luis Carrillo-Reid et al.

Fig. 3 Simultaneous two-photon calcium imaging and two-photon holographic optogenetics. (a) Two-photon
microscope setup with two independent beam paths for calcium imaging and photostimulation. An electrically
tunable lens is equipped in the imaging path so the focal plane can be rapidly switched for high-speed
volumetric imaging. The dichroic mirror 3 is used to spectrally separate the fluorescence into green and red
channel. In the photostimulation path, a half-wave plate is placed before the spatial light modulator to align
the polarization of the laser to the active axis of the spatial light modulator. The spatial light modulator then
creates a hologram in the sample to photostimulate the regions of interest (e.g., a group of neurons). A pair of
relay lens (4f system) is used to transfer the light field to a set of galvanometer mirrors which can spirally scan
the holographic pattern. At the intermediate plane of this pair of relay lens, a zeroth-order beam block is used
to block the residue light that is not modulated by the spatial light modulator. The imaging laser and
photostimulation laser have different wavelengths, and they are combined through the dichroic mirror 1 before
being delivered to the sample through the scan lens, tube lens, and objective lens. HWP half-wave plate, PMT
photomultiplier tube. (b) Schematics for simultaneous volumetric calcium imaging and 3D holographic
patterned photostimulation in mouse cortex. (c) An exemplary field of view showing neurons co-expressing
GCaMP6s (green) and C1V1-mCherry (magenta). (Reprinted and adapted from Ref. [69])

To characterize neuronal ensemble activity, it is necessary to

image a large population of neurons, typically on the order of
hundreds. For visual cortex, a minimal field of view of
200
200 μm2 is necessary, though
400
400 ~ 600
600 μm2 would be more desirable. Using
low magnification objective lens, the field of view can go above
1 mm2. Furthermore, volumetric imaging can be performed to
image neuronal ensembles in 3D and across different functional
 All-Optical Probing of Neuronal Ensembles 339

layers (Fig. 3a, b). Due to the sequential scanning nature of

two-photon microscopy, there is a tradeoff between the field of
view and spatiotemporal resolution. Thus, while it is desirable to
increase the field of view so as to image more neurons, one should
maintain cellular resolution with a good signal-to-noise ratio (i.e.,
able to distinguish the calcium transients), as well as a frame rate of
at least 4–10 Hz. In the setup shown in Fig. 3a, we use a galva-
nometer and a resonant scanner pair for lateral scanning, and an
electrically tunable lens for fast focal plane switching. Volumetric
imaging of 3 planes could be achieved at a volume rate >6 Hz, with
a field of view ~500
500 μm2 per plane [69].
A typical experiment starts with two-photon calcium imaging
on the brain tissue. The animal is head-fixed and stabilized on the
microscope stage and could perform behavior tasks. The spatial
footprint as well as the neuronal activity of individual neurons
within the image field of view can then be extracted from the
recording. The neuronal ensemble can be identified from the activ-
ity pattern (see Subheading 3). Depending on the applications, the
users may choose a specific population of neurons to perform
optogenetics experiments. It is thus desirable to perform photo-
stimulation on multiple neurons simultaneously. Two-photon
holographic illumination enables photostimulating a group of
user-selected neurons [2, 12, 14, 66, 68, 69, 72, 73]. We discuss
the topic in the following section.

2.2.2 Holographic Holographic illumination refers to projecting to the brain tissue a

Illumination computer-generated holographic pattern. Neurons falling within
this pattern can then be photostimulated. Computer-generated
hologram is a light field of an arbitrary shape in 3D in an imaging
volume, and this shape can be dynamically controlled and changed
through a spatial light modulator (Fig. 4a). In the simplest imaging
system, which contains a single lens, to generate a hologram in the
imaging space (i.e., around the front focal plane of the lens), one
can spatially modulate the light field at the back focal plane of the
lens. This light field then propagates through the lens, and coher-
ently interferes at the front focal plane and forms the holographic
pattern (Fig. 4a). As the light field at the front focal plane and back
focal plane of a lens forms a Fourier transform relationship, one can
calculate how the light field should be spatially modulated at the
back focal plane based on the desired pattern at the front focal plane
(Fig. 4b, c). The typical spatial light modulator is based on liquid
crystals and is configured to only modulate the phase of the light
field. A Gerchberg-Saxton algorithm (Fig. 5) [74], which is an
iterative approach, can be used to calculate the phase pattern on
the spatial light modulator, given the amplitude of the desired
pattern in the imaging space and the amplitude of the light field
incident on the spatial light modulator. If the holographic pattern
only contains a group of points with different weights, a
340 Luis Carrillo-Reid et al.

Fig. 4 Computer-generated hologram. (a) Principle of computer-generated hologram. The collimated laser
beam is incident onto the spatial light modulator (SLM), which spatially modulates the wavefront of the light.
The light field then propagates through a lens and forms the desired 3D image in the imaging domain through
interference. f, focal length of the lens. (b) By modulating the spatial phase profile at the back focal plane of
the object lens, different focal spot patterns can be formed in the imaging domain. (Reprinted with permission
from Ref. [103], Elsevier). (c) Example of a two-photon SLM hologram. The four panels illustrate the binary
target image, the spatial light modulator phase hologram generated by Gerchberg-Saxton algorithm, the
squared image (to mimic two-photon excitation) of the projected pattern back calculated from the phase
hologram, and the experimentally measured two-photon fluorescence image generated by the SLM. A stylized
picture of Cajal is used as a target image. (Reprinted from Ref. [11], Frontiers)

superposition algorithm [55, 75], which is essentially a single itera-

tion of the Gerchberg-Saxton algorithm, can be used to calculate
the phase pattern (Table 1).
In two-photon microscopes, we could not directly modulate
the phase at the back focal plane of the objective lens, as the back
focal plane is typically inside the objective lens housing. Relay lens
pairs (4f system) can create the conjugate planes of the back focal
plane of the objective lens, and the spatial light modulator can be
 All-Optical Probing of Neuronal Ensembles 341

Fig. 5 Gerchberg-Saxton algorithm. This algorithm is used to calculate the phase hologram φs for the spatial
light modulator (SLM) based on the amplitude of the target pattern At in the imaging space and the amplitude
of the incident light field As onto the SLM

Table 1
Analytical expression of the phase hologram and the Zernike polynomials and Zernike coefficients in
superposition algorithm
M 
P
A i e 2πj fx i uþy i vþ½Z 2 ðu,vÞC 2 ðzi ÞþZ 4 ðu,vÞC 4 ðzi ÞþZ 6 ðu,vÞC 6 ðzi Þg
0 0 0 0 0 0
Phase hologram on the SLM: ϕðu, v Þ ¼ phase
i¼1
[xi, yi, zi] (i¼1,2. . .M ), the coordinate of the cell body centroid (M targeted cells in total); Ai, the
electrical field weighting coefficient for the ith target (which controls the laser power it receives);
Z 0m ðu, v Þ and C 0m ðz i Þ, the Zernike polynomials and Zernike coefficients, respectively, which sets the
defocusing and compensates the first-order and second-order spherical aberration due to defocusing.
pffiffiffi   
Zernike polynomials (defocus) Z 02 ðu, vÞ ¼ 3 2 u2 þ v 2  1
2  
Zernike coefficients (defocus) pffiffi α 1 þ 1 sin 2 α þ 9 sin 4 α þ 1 sin 6 α þ . . .
C 02 ðz Þ ¼ nkz8πsin3 4 80 16

Zernike polynomials (first-order pffiffiffih  2   i

Z 04 ðu, vÞ ¼ 5 6 u2 þ v 2  6 u2 þ v2 þ 1
spherical aberration)
4  
Zernike coefficients (first-order C 04 ðz Þ ¼ nkz pffiffiα 1 þ 3 sin 2 α þ 15 sin 4 α þ . . .
sin
96π 5 4 18
spherical aberration)
h  i
Zernike polynomials (second-order Z 0 ðu, vÞ ¼ pffiffiffi 3  2
7 20 u2 þ v2  30 u2 þ v2 þ 12 u2 þ v2  1
 
6
spherical aberration)
 
Zernike coefficients (second-order C 0 ðz Þ ¼ nkz sinp6ffiffiα 1 þ 5 sin 2 α þ . . .
6 640π 7 4
spherical aberration)
n refractive index of media between the objective and sample, k the wavenumber, z the axial shift of the focus plane in the
sample, u, v coordinates on the spatial light modulator phase mask, nsinα the numerical aperture (NA) of the objective

placed in one of those conjugate planes (Fig. 3a). Furthermore, as

there is residue light that is not modulated by the spatial light
modulator (termed zeroth order beam), it will create a strong
focus at the imaging space (see Note 2). The relay lens pairs help
to resolve this issue as a small beam block can be placed at the
intermediate plane (which is conjugate to the focal plane at the
imaging space) to block the zeroth order beam (Fig. 3a). Using the
holographic illumination, various patterns can be generated in the
imaging space (Fig. 4b, c).
342 Luis Carrillo-Reid et al.

Holographic illumination essentially spatially multiplexes the

excitation beams, and allows multiple neurons being photostimu-
lated simultaneously. However, this comes with an increase of laser
power on the brain tissue. To alleviate this issue, a laser with a lower
repetition rate can be used. When the pulse repetition rate is
reduced, the energy in each laser pulse is increased, while keeping
the overall average power the same. As the two-photon excitation
efficiency is proportional to the square of the laser peak power, the
increase of excitation efficiency due to the increase in pulse energy
outweighs its reduction due to the reduced number of pulses per
unit time. Thus, using a laser with lower repetition rate, a higher
overall excitation efficiency can be achieved while keeping the same
average power. In other words, to achieve the same excitation
efficiency, the average laser power can be reduced. In the holo-
graphic photostimulation system described, we used a laser with a
repetition rate of 1 MHz [69]. This reduces the overall laser power
by 80 times compared with a commonly used 80 MHz femtosec-
ond laser.
Due to the chromatic dispersion and spatial discretization of
the SLM pixels, the SLM has a spatially varying diffraction effi-
ciency [75]. The diffraction efficiency drops as the deflection angle
increases, limiting the addressable 3D field of view of SLM. To
alleviate this effect, the diffraction efficiency can be first measured,
and then compensated in the Gerchberg-Saxton algorithm or
through the weight factor Ai (Table 1) in the superposition algo-
rithm [55, 75]. The laser intensity across the field of view can then
be made uniform. By using an XY galvanometer set to provide a
lateral offset to the centroid of the SLM’s addressable field of view,
the effective lateral field of view can be further extended. While
neurons across this enlarged field of view cannot be targeted simul-
taneously, groups of neurons located at different sub-fields can be
targeted by switching the offset of the XY galvanometer and the
phase pattern on the SLM [76].

2.2.3 Spiral Scan Versus As mentioned in Subheading 2.1, photostimulation can be per-
Scanless Approach in formed by raster or spirally scanning the laser focal spot across the
Holographic neuron, or by projecting a disk pattern which matches the mor-
Photostimulation phology of the neuron so the entire neuron can be stimulated at
once. The same applies in holographic photostimulation. In the
first approach, a group of focal spots are created in the hologram,
and each focal spot lies on the centroid of the individual targeted
neuron [2, 12, 69]. A set of galvanometer scanners then spirally
scan the entire group of focal points (with certain repetitions), so
each focal spot spirally scans across the corresponding targeted
neuron. In the second approach, a group of disk patterns are
created in the hologram and projected to the brain tissue [72, 77,
78]. Each disk spatially overlaps with a targeted neuron. Since the
hologram already generates multiple disks for all the target
 All-Optical Probing of Neuronal Ensembles 343

neurons, this method can work without scanners, and thus this is a
scanless approach. In two-photon excitation, these two approaches
have their own advantages and limitations. The spiral scan approach
spatially concentrates the laser intensity into focal spots; as
two-photon excitation effect is proportional to light intensity
square, this approach has a high excitation efficiency. As the entire
neuron is not photostimulated at the same time, to reach the
threshold of evoking action potentials, it relies on the accumulation
of the excitation effect along the spiral trajectory. The decay con-
stant of the opsin kinetics (tau-off) should thus be longer than the
duration of each spiral (i.e., the opsin channels can stay open during
the one single spiral scan, which could be less than 1 ms); otherwise
the excitation effect cannot be accumulated. The scanless approach,
on the other hand, disperses the light intensity across the entire
neuron, and thus it requires a higher average power to reach the
same excitation strength as the spiral scan approach. However, as
the entire neuron is being stimulated at once, it does not pose a
limitation on the kinetics of the opsin. It is also expected that the
jitter of the delay between the onset of photostimulation and onset
of action potential is smaller. As the opsin decay constant (tau-off)
is typically larger than the duration of a single spiral scan (which can
be <1 ms), the scanning approach is in favor as it takes lower laser
power to photoactivate the neurons. In our experiments, we used
C1V1 as the opsin. Using spiral scan, it takes about 1.8
less power
to photoactivate the neurons than the scanless approach, with the
same photostimulation duration (Fig. 6) [69]. For opsins with
faster kinetics, a faster spiral scan or the scanless approach may be
preferred.

2.2.4 Detailed In this section, we explain the details of how the microscope can be
Implementation of the constructed with two beam paths for two-photon imaging and
Microscope for two-photon holographic photostimulation (Fig. 3a). We aim to
Simultaneous Two-Photon help the readers understand the inside of a commercial microscope
Imaging and Two-Photon system and meanwhile provide basic guidelines for those who want
Holographic to home-build the microscope. Here, we use GCaMP6 as calcium
Photostimulation indicators, and C1V1-mCherry as the red-shifted opsin. The
mCherry can be used to indicate if the opsin is expressed in each
neuron. We combine the holographic illumination with spiral scan
approach to minimize the laser power onto the brain.

Setup of the Two-Photon Imaging Path

(1.1) The imaging laser is typically a wavelength tunable Ti:Sap-
phire laser with a repetition rate of 80 MHz, and a pulse width
of 70~140 fs. For GCaMP imaging, the wavelength can be set
to 920~940 nm. A Pockels cell is set up after the laser to
modulate the laser power. An optional pulse compressor
could be set up after the Pockels cell to optimize the pulse
width at the sample.
344 Luis Carrillo-Reid et al.

A Stimulation 20 ms 10 ms 5 ms 1 ms
Duration
4 mW 5.6 mW 8 mW 18 mW
Spiral Scan
1X Relative Power

Scanless Disk 4 mW 5.6 mW 8 mW 18 mW

1X Relative Power

7.2 mW 10 mW 14.4 mW 32.4 mW 'F/F

Scanless Disk 0.1
1.8X Relative Power
2s

B **** **** ** n.s.

**** n.s.
**** **
1

0.8
Normalized ∆ F/F

0.6

0.4

0.2

Spiral Scanless Scanless Spiral Scanless Scanless Spiral Scanless Scanless Spiral Scanless Scanless
Modality
Scan Disk Disk Scan Disk Disk Scan Disk Disk Scan Disk Disk
Relative
Power 1x 1x 1.8x 1x 1x 1.8x 1x 1x 1.8x 1x 1x 1.8x
Duration 20 ms 10 ms 5 ms 1 ms

C 1

0.8
Normalized ∆ F/F

0.6

0.4

0.2

Modality Spiral Scanless Scanless Spiral Scanless Scanless Spiral Scanless Scanless Spiral Scanless Scanless
Scan Disk Disk Scan Disk Disk Scan Disk Disk Scan Disk Disk
Relative
Power 1x 1x 1.8x 1x 1x 1.8x 1x 1x 1.8x 1x 1x 1.8x
Duration 20 ms 10 ms 5 ms 1 ms

Fig. 6 Comparison between spiral scan and scanless holographic approaches for photostimulation. In the
scanning approach, the laser spot is spirally scanned over the cell body; in the scanless approach, a disk
pattern is generated by the SLM, covering the entire cell body at once. (a) Photostimulation triggered calcium
response of a targeted neuron in vivo at mouse layer 2/3 of V1, for different stimulation modalities. For each
 All-Optical Probing of Neuronal Ensembles 345

(1.2) An optional electrically tunable lens could be set up as an axial

focal control device. A set of XY galvanometer mirrors, or a
resonant scanner paired with a galvanometer mirror, can be set
up to raster scan the laser focal spot on the sample. The electri-
cally tunable lens and the scanning mirrors are located at the
conjugate plane of the back focal plane of the objective lens. 4f
systems are used to relay these planes and to magnify the beam
size so that the beam can illuminate a large portion of the
aperture of the electrically tunable lens and scanning mirrors.
(1.3) A dichroic mirror is set up to combine the imaging path and
photostimulation path and direct the beam to scan lens and
tube lens, which forms a 4f system to magnify the beam at the
back focal plane of the objective lens. This beam size should be
optimized for specific objective lens so as to achieve a good
excitation numerical aperture (NA) for cellular resolution. For
population imaging, the objective lens is typically chosen to be
16~40
, and the excitation NA is typically 0.45~0.6. The
collection NA should be as large as possible so as much as the
emitted fluorescence could be collected.
(1.4) A dichroic mirror is set up before the objective lens to
transmit the infrared excitation light and reflect the fluores-
cence emission to photomultiplier tubes (PMTs). A dichroic
mirror can further separate the fluorescence spectrum into
different channels (typically green and red).

Setup of the Two-Photon Photostimulation Path

(2.1) The photostimulation laser can be a high repetition rate fiber

laser or a low repetition rate pulse-amplified laser (including
optical parametric amplified laser). The latter laser is preferred
for holographic photostimulation as its higher pulse energy
allows lowering the average photostimulation power per cell,

Fig. 6 (continued) modality, the multiplication of stimulation duration and the square of the laser power was
kept constant over four different stimulation durations. The average response traces are plotted over those
from the individual trials. (b) ΔF/F response of individual neurons on different photostimulation conditions
(layer 2/3 of V1, over a depth of 100 ~ 270 μm from pial surface; one-way ANOVA test). For each neuron and
each stimulation duration, the laser power used in the scanless disk modality is 1 and 1.8 times relative to that
in the spiral scan. For each neuron and each modality, the multiplication of the stimulation duration and the
square of the laser power was kept constant over four different stimulation durations. (c) Boxplot summarizing
the statistics in (b). The central mark indicates the median, and the bottom and top edges of the box indicate
the 25th and 75th percentiles, respectively. The whiskers extend to the most extreme data points (99.3%
coverage if the data are normal distributed) not considered outliers, and the outliers are plotted individually
using the “+” symbol. In this experiment, the mice were transfected with GCaMP6f and C1V1-mCherry.
Repetition rate of the photostimulation laser is 1 MHz. The spiral scan consists of 50 rotations to cover the
neuronal cell body, and the scanning speed is adjusted to make different stimulation durations. (Reprinted
with permission from Ref. [69])
346 Luis Carrillo-Reid et al.

thus enabling photostimulating a large number of neurons with

the overall available power budget. For red-shifted opsin such
as C1V1, the preferred laser wavelength is 1040~1080 nm.
Similar as the imaging path, a Pockels cell and an optional
pulse compressor are set up after the laser.
(2.2) A spatial light modulator is set up at a conjugate plane to the
back focal plane of the objective lens. Following the Pockels
cell, a half-wave plate is set up to rotate the polarization of the
laser so the light polarization aligns with the active axis of the
spatial light modulator. A 4f system is used to magnify the beam
size so it fills up the active region of the spatial light modulator.
A 4f system is placed after the spatial light modulator with a
zeroth-order beam block set up at the intermediate imaging
plane. After this 4f system, a set of XY galvanometer mirrors are
set up, which is also at the conjugate plane of the back focal
plane of the objective lens. Here, the XY galvanometer can not
only perform spiral scanning but also provide a lateral offset to
the centroid of the SLM’s addressable field of view, thus effec-
tively enlarging the overall field of view, as discussed in earlier
sessions. Thus, the XY galvanometer is preferred over galvo/
resonant scanner pair, as the latter cannot provide a static beam
shift in the resonant scanner’s scanning direction.
(2.3) The photostimulation path then combines with the imaging
path at the dichroic mirror and is then directed to the sample
through the scan lens, tube lens, and objective lens.

Control Electronics and Software

(3.1) The electronic control system typically includes analog

inputs/outputs to control the Pockels cells, electrically tunable
lens, spatial light modulator, and scanners in both beam paths,
as well as high-speed digitizers to record the imaging data from
the PMT. For home-built microscopes, ScanImage [79] is one
commonly used control software.
(3.2) The phase pattern on the spatial light modulator can be
calculated through a superposition algorithm or a Gerchberg-
Saxton algorithm as detailed in Subheading 2.2.2 so a user-
defined excitation pattern can be projected to the sample. An
open source control software can be found in [80].

Coordinate Calibration

(4.1) It is critical to register the photostimulation beam’s target

coordinate with the imaging laser’s image coordinate. An auto-
fluorescent plastic slide can serve as the sample, and a 2D
holographic pattern can be projected to burn spots on the
surface of the autofluorescent plastic slide. The imaging laser
can then visualize the burned spots. This can also calibrate the
 All-Optical Probing of Neuronal Ensembles 347

axial focus offset between the two beam paths. An affine trans-
formation can be extracted to map the coordinates between the
hologram generation algorithm and the actual imaging system.
This can be repeated for a few defocusing depths set in the
spatial light modulator, and a linear interpolation of the
mapping can be applied for the depths in between.
(4.2) Due to the chromatic dispersion and finite pixel size of SLM,
the SLM’s beam steering efficiency, also called diffraction effi-
ciency, drops with larger angle, leading to a lower beam power
for targets further away from the center field of view (in xy),
and nominal focus (in z). This efficiency drop can be analyti-
cally calculated [55, 75] or experimentally measured by raster
scanning the photostimulation beam on the autofluorescence
slide and detecting the fluorescence strength. A linear compen-
sation can be applied in the weighting coefficient among differ-
ent points in the target pattern to counteract this
non-uniformity (see Note 3).

Optogenetics Experiment

(5.1) Before each set of experiments on animals, it is a good practice

to verify the system (laser power, targeting accuracy, power
uniformity among different beamlets from the hologram) by
generating groups of random spots through holograms, burn-
ing the spots on an autofluorescent plastic slide, and comparing
the resultant image with the desired target.
(5.2) The calcium imaging is first performed followed by the
extraction of the spatial footprint and temporal dynamics of
each recorded neuron. To confirm the co-expression of calcium
indicators and opsins in an individual neuron, one can image
the fluorophore linked to the opsin. A desired group of neurons
can then be selected for optogenetics experiments. Laser power
and stimulation time should be adjusted to produce responses
observed physiologically (see Note 4).
(5.3) While the excitation wavelengths between the calcium indi-
cators and opsins are different, the calcium indicator may still
generate fluorescence background during photostimulation,
particularly when a large number of neurons are photostimu-
lated simultaneously. Imaging processing should be implemen-
ted to remove or suppress this background.

3 Identification and Targeting of Neuronal Ensembles Related to Behavior

3.1 Background The implementation of simultaneous two-photon imaging [11]

and two-photon optogenetics [12] allowed the recording and
manipulation of tens of cells simultaneously [14, 66, 69]. However,
the identification of the neurons that could have a robust impact on
348 Luis Carrillo-Reid et al.

the overall population activity also represents a critical step in the

design of experiments aiming to control learned behaviors [4, 21,
81].

3.1.1 Multidimensional It has been demonstrated that the activity of neuronal ensembles
Reduction Techniques could be defined as an array of multidimensional population vec-
Applied to Population tors, where the dimensionality of the array is given by all recorded
Recordings cells in the field of view [5, 8, 82]. Similar population vectors could
be visualized by diverse computational techniques that define clus-
ters in a reduced dimensional space, where each cluster depicts a
neuronal ensemble [5, 83]. A neuronal ensemble represents a
group of neurons with coordinated activity that repeats at different
points in time [10]. The characterization of brain states using
multidimensional population vectors is independent of the record-
ing length and could be implemented in chronic experiments where
the activity of identified neuronal ensembles could be compared at
different days.

3.1.2 Targeting Two experimental designs are possible to control behavior target-
Visualized Neuronal ing visualized neuronal populations (see Note 5). One solution is to
Populations with Two- target all available neurons that could respond to a given behavioral
Photon Optogenetics cue with single-cell precision [14, 16, 18]. The other solution is to
target neurons with pattern completion capabilities that could
recall physiologically relevant neuronal ensembles related to behav-
ior [4, 6, 10, 19, 21].
In cortical circuits, the synchronous activation of several neu-
rons could simultaneously result in two unwanted scenarios: (i) the
generation of epileptiform activity or (ii) the forced engagement of
GABAergic circuits. In both scenarios, the physiological recalling of
a specialized neuronal ensemble is compromised (see Note 5).
On the other hand, the identification and targeting of neurons
with pattern completion capabilities could keep the balance
between excitation and inhibition inherent to the microcircuit
under study, allowing the recalling of physiological neuronal
ensembles that work as attractors [84] evoking a given behavior
[84, 85].

3.2 Implementation In this part of the chapter, we describe the procedures and concepts
of Analytical Methods to analyze population activity extracted from calcium imaging
to Recall Neuronal recordings. We will focus on the steps after individual neurons
Ensembles Relevant to have been identified from imaging recordings and their activity
a Learned Behavior has been inferred from calcium transients.

3.2.1 Motion Correction, Simultaneous population recordings allow the characterization of

Identification of Neurons, population activity with single-cell resolution but generate large
and Spike Extraction datasets representing an analytical challenge. A common practice
used for analysis of simultaneous population recordings is to create
a binary representation of neuronal activity where 1’s indicate firing
 All-Optical Probing of Neuronal Ensembles 349

Fig. 7 Spike inference from holographic calcium imaging recordings. (a) Representative experiment where two
focal planes were recorded simultaneously using holographic two-photon microscopy. (b) Regions of interest
(ROIs) detected from the recordings in a. Each ROI corresponds to an identified neuron. (c) Representative
changes in fluorescence obtained from ROIs shown in b. (d) Binary arrays representing the activity of one
neuron obtained from inferred spikes. (Modified from Ref. [5, 10])

and 0’s indicate silent periods [82, 86]. Recently, several indepen-
dent research groups have released open-source code to preprocess
calcium imaging recordings to extract activity information from a
series of images [87–90].
There are four main steps that need to be considered before
performing population analysis on binary arrays (Fig. 7):
(i) Motion correction: Lateral displacements are commonly cor-
rected using TurboReg [91], fast Fourier transform [92] or
field approaches [93]. Translations in depth should be avoided
or the data should be discarded.
(ii) Identification of neurons: Automatic identification of regions of
interest (ROI’s) can be performed using different approaches
[87, 88].
(iii) Determination of changes in fluorescence: After the identifica-
tion of ROI’s, fluorescence traces for each neuron should be
extracted.
(iv) Activity inference from calcium transients: The first time deriv-
ative, deconvolution, or fitting could be used to infer spiking
activity from calcium transients [82, 87, 88]. Note that infer-
ring co-activity between pairs of neurons from raw calcium
transients introduces an artifact from the decaying phase of
the changes in fluorescence. Such common mistake generates
spurious correlations between neurons as demonstrated
before [7].

3.2.2 Binary Arrays from To construct binary arrays, inferred activity should be threshold
Inferred Activity usually 3 S.D. above noise level (Fig. 7d). This procedure has been
shown to match bursting activity of neurons above 2 action poten-
tials [82]. The onsets from inferred activity can be used to represent
the overall population activity. A binary matrix [N
T] is con-
structed, where N denotes the total number of neurons in the field
of view and T represents the total number of time points recorded.
350 Luis Carrillo-Reid et al.

a b population vectors
1 0 1 0 0 1 0 0 0 1
a
similarity index
t1
1
0
1
0
1
0
1
0
1 1
0
1 1
0
1 1
0
b
1 1 1

vectors dimensionality
0 1 0 1 0 0 0 1 0 0
1 0 1 0 0 1 0 0 0 1
0 0 0 0 0 0 0 0 0 0
frames in time

0 0 0 0 1 0 1 0 1 0
0 0 0 0 0 0 0 0 0 0

neuron
t2 1 1 1 1 1 1 1 1 1 1
0 1 0 1 0 0 0 1 0 0
1 0 1 0 0 1 0 0 0 1
0 0 0 0 0 0 0 0 0 0
population vector
θ
0 0 0 0 1 0 1 0 1 0
...

0 0 0 0 0 0 0 0 0 0
0 1 0 1 0 0 0 1 0 0
1 0 1 0 0 1 0 0 0 1
0 0 0 0 1 0 1 0 1 0
tn 0 1 0 1 0 0 0 1 0 0 neuronal ensemble
t1 t2 population vector tn cluster of vectors
n dimensions

Fig. 8 Neuronal ensembles represented as multidimensional population vectors. (a) Schematic representation
of different frames where active neurons are shown in black-filled circles (left). Binary matrix illustrating the
overall population activity, where each row is a neuron and each column denotes a population vector. The total
number of recorded neurons gives the dimensionality of the array. (b) Neuronal ensembles representing
recurrent groups of neurons that are active at different times can be understood as clusters of similar
population vectors in a multidimensional space. The cosine similarity could be used as a metric to calculate
the angle between population vectors. If the angle is close to 0 , the population vectors are similar, therefore
almost the same neurons fired at different times. If the angle is close to 90 , the population vectors are
different. (Reprinted from Ref. [10])

Each row in the binary matrix depicts the activity of one neuron and
each column in the binary matrix represents the activity profile of a
neuronal population (Fig. 8a). To visualize the overall network
activity, the binary matrix can be plotted as a raster plot where 1’s
are dots. The population synchronicity can be extracted from the
time histogram of the raster plot [5, 8].

3.2.3 Multidimensional To identify groups of neurons with coordinated activity, population

Population Vectors Defining vectors from a physiologically meaningful time window are con-
Neuronal Ensembles structed; time windows from 100–500 ms have been shown to
reflect physiological functions and generate similar results. Popula-
tion vectors capture the coordinated activation of specific groups of
neurons. Once the population vectors are defined, comparing the
distribution of coordinated activity that could appear randomly
against the observed coordinated activity can show population
vectors above chance levels [5, 7]. Only population vectors with
more active cells than the ones expected by chance should be
considered for further analysis. Significant population vectors cap-
ture network activity defining a multidimensional space in which
the number of dimensions is dictated by the total number of
identified neurons with coordinated activity above chance levels
[4–6, 8, 10]. It is important to highlight that to characterize
population activity, each dot in a multidimensional space should
be a population vector instead of an individual neuron (Fig. 8b).
 All-Optical Probing of Neuronal Ensembles 351

3.2.4 Similarity It has been shown that the use of significant population vectors can
Measurements on be used to discriminate similar patterns of activity repeated at
Population Vectors different times [4–6, 8]. The representation of network activity as
population vectors allows an exhaustive comparison of all popula-
tion vectors to visualize different experimental conditions. Similar-
ity maps of population vectors represent a valuable tool to visualize
recurrent activity of neuronal ensembles. To construct similarity
maps, all possible combinations of vector pairs need to be com-
puted. Since population vectors representing the activity of a given
group of neurons point to a similar place in a multidimensional
space, the angle between population vectors could be used to create
a similarity map [8, 82]. The cosine of the angle between a pair of
population vectors is defined by their normalized dot product: cos
(θ)¼V1 l V2 / llV1ll llV2ll. Thus, if the angle is close to zero, the
vectors are similar whereas if the vectors are different, they tend to
be orthogonal (Fig. 8b).

3.2.5 Identification of Similarity maps constructed from all possible vector pairs could be
Neuronal Ensembles from understood as a low-dimensional representation of the original
Population Vectors network activity, where the angles between all population vectors
as a function of time are highlighted. Thus, high similarity values in
the same row denote recurrent groups of neurons firing together. A
cluster of population vectors pointing in a similar direction gives
the definition of a neuronal ensemble using the population vector
approach. Similarity maps can be transformed to a binary matrix S
of size [T
T] (Fig. 9a, b). The factorization of S using singular
value decomposition (SVD) factorizes S as the multiplication of
three factors: V, ∑, and VT, where V and VT are orthonormal basis
and ∑ contains the singular values [5, 8]. To detect neuronal
ensembles from recurrent patterns of activity observed in the
matrix S, the rate of singular values decay is determined (Fig. 9c).
Taking the singular values above chance levels can reproduce the
original matrix S with high accuracy (Fig. 9d). Then each factor of
the SVD decomposition represents a linearly orthogonal compo-
nent and each factor defines a neuronal ensemble (Fig. 9e). In the
case of primary visual cortex, each neuronal ensemble represents a
different orientation of drifting-gratings, a different natural scene
or the Go signal from a visually guided Go/No-Go task [4, 5,
7]. Another approach to identify neuronal ensembles is the projec-
tion of the multidimensional population vectors into a low dimen-
sional space, in such reduced dimensional space clusters of
population vectors depicted with cluster analysis define neuronal
ensembles or network states (Fig. 8b) [82]. The advantage of using
SVD factorization is that the total number of neuronal ensembles
could be systematically detected from the magnitude of the singular
values. Thus, recurrent population vectors can be assigned to a
given neuronal ensemble and population vectors with low repeti-
tion rate are excluded.
352 Luis Carrillo-Reid et al.

a b c d SVD σ1 1 1 +...+ σ6 6 6
120
1 data
shuffled

similarity index
2X shuffled

magnitude
vectors (t)

vectors (t)

vectors (t)
60

1 cutoff (6th)

1
0 ∗
1 200 400 1 200 400 1 5 10 30 1 200 400
vectors (t) vectors (t) log(singular value) vectors (t)

e σ1 σ2 σ3 σ4 σ5 σ6
1 1 2 2 3 3 4 4 5 5 6 6

Fig. 9 Neuronal ensembles defined by similarity maps of population vectors using singular value decomposi-
tion (SVD). (a) Similarity map illustrating the angles between all possible pair combinations of population
vectors. Note that the recurrent activation of a given group of neurons is visualized as increased similarity
index in the same row. An angle between two vectors close to 0 represents a similarity value close to
1, whereas an angle between two vectors close to 90 represents a similarity value close to 0. (b) Binary
matrix computed from the similarity map in a, representing significant patterns of activity factorized by SVD.
Black patterns in the same row indicate recurrent coactive neurons at different times. (c) Magnitude of
singular values used to define the number of neuronal ensembles repeated above chance levels. Red line
indicates the double of singular values from shuffled data. The cutoff indicates the number of neuronal
ensembles that account for >90% of the data. In general microcircuits of ~100 neurons stimulated with four
drifting-gratings can be defined by ~6 neuronal ensembles. (d) Binary matrix of the first 6 factors obtained
from SVD of b. (e) Factors from SVD that reproduce the overall response of the imaged focal plane to visual
stimuli. Bars on top indicate orientations of drifting-gratings. Empty squares represent spontaneously active
neuronal ensembles that appeared in the absence of visual stimuli. (Modified from Ref. [5])

3.2.6 Pattern Completion In the neuronal ensemble framework, pattern completion refers to
Properties of Neuronal the ability to recall a whole neuronal ensemble by the activation of
Ensembles few neurons with strong functional connectivity [6]. It has been
recently shown that the stimulation of the same neuronal popula-
tion for several times (~100) was able to imprint an artificial neuro-
nal ensemble in layer 2/3 of primary visual cortex of awake head
fixed mice [6]. Imprinted ensembles were composed by neurons
that had low probability to fire together, but after the imprinting
protocol, the stimulated neurons fired spontaneously in the absence
of stimuli (Fig. 10a). The mechanism governing the creation of
artificial neuronal ensembles could be explained by Hebbian synap-
tic plasticity [94], where the connectivity between neurons firing
together was strengthened (Fig. 10b). Indeed, neurons responding
to a given drifting-grating or belonging to a neuronal ensemble
have increased probability to be connected [8, 95]. Imprinted
 All-Optical Probing of Neuronal Ensembles 353

a ensemble training imprinted ensemble pattern completion

b natural ensembles recalled ensemble
(population photostim) (ongoing activity) (single cell activation) (functional connectivity) (strengthened connectivity)

single cell photostim single cell photostim

Fig. 10 Imprinting and recalling of artificial neuronal ensembles in layer 2/3 of primary visual cortex. (a) Spatial
map of neurons activated with two-photon optogenetics several times (~100). Red neurons represent
repeatedly responding neurons (left). Scale bar: 50 μm. Imprinted ensemble (red neurons) shows spontaneous
activity in the absence of any stimuli (middle). The activation of one neuron with pattern completion capability
(blue arrow) was able to recall the imprinted ensemble (red neurons). (b) Cartoon illustrating how imprinted
and recalled ensembles could be explained by the strengthening of connections of pre-existing ensembles and
photostimulated neurons. (Modified from Ref. [10])

ensembles could also be recalled by the stimulation of individual

neurons with pattern completion capabilities (Fig. 10), suggesting
that pattern completion could be a general property of different
brain areas [21].

3.2.7 Recalling Neuronal To prove the causal relation between neuronal activity and a learned
Ensembles Related to behavior, it is necessary to identify representative members of neu-
Behavior ronal ensembles that could trigger the behavior [81]. During the
behavioral training phase photostimulation should be avoided (see
Note 6). It has been recently shown, in a visually guided Go/No-
Go task, that neuronal ensembles responding to drifting-gratings in
layer 2/3 of primary visual cortex increase their reliability for the
Go signal whereas reduce their reliability for the No-Go signal
[4]. Increased reliability is reflected as strengthened functional
connectivity of the Go ensemble allowing neurons with pattern
completion capabilities [21] to recall the whole Go ensemble and
trigger the perception of the Go signal evoking the learned behav-
ior (Fig. 11).

4 Considerations for the Implementation of a Visually Guided Go/No-Go Task

– Training and experiments should be performed at the same hour

of the day.
– That same person should handle the animals.
– Training should be performed every day, avoiding skipping
training sessions on weekends or holidays.
354 Luis Carrillo-Reid et al.

a
Go signal Go ensemble
visual stimuli layer 2/3 V1 licking

b
p.c neurons recall ensemble
opto stimuli layer 2/3 V1 licking

Fig. 11 Controlling behavior using pattern completion properties of Go ensem-

bles. (a) In mice trained in a visually guided Go/No-Go task, the Go signal
activates a neuronal ensemble related to the correct execution of the task
(green neurons). The activation of the Go ensemble produced licking. (b)
Activation of as few as two pattern completion neurons (red neurons) belonging
to the Go ensemble is able to recall the whole Go ensemble and produce licking
even in the absence of visual stimuli or behavioral cues. (Reprinted from Ref. [4])

– The weight of the animals should be systematically documented

and maintained at the same level overnight. In general, 1 mL of
water should increase weight by 1 g.
– Changes in weight 2% of the total mass could significantly alter
behavioral performance.
– To avoid continuous licking, plain water should be preferred
over sweetened water.
– The lick port should be placed 1.5 mm from the mouth. A lick
port too close to the mouth could cause increased non-task
related licking.
 All-Optical Probing of Neuronal Ensembles 355

5 Notes

1. A proper calcium indicator and opsin pair should be chosen to

minimize crosstalk. It is also critical to control imaging laser
power so that neurons are not depolarized by the imaging laser
during imaging [Subheading 2.2.1].
2. The zeroth-order beam block in the photostimulation path
should be positioned at the intermediate plane of the 4f system,
right after the spatial light modulator. While it can block the
zeroth-order beam, it also makes the neurons in the center field
of view inaccessible to the photostimulation beam. Thus, the
zeroth-order beam block should have an intermediate size: if
too small, the zeroth-order beam cannot be totally blocked,
but if too large, there could be a large number of neurons in the
center field of view not accessible by the photostimulation laser
[Subheading 2.2.2, Fig. 3a].
3. As the opsin expression level may differ between different
neurons, the weight factors for different target neurons should
be adjusted in the hologram calculation [55, 75] so that their
calcium transients evoked by two-photon optogenetics have a
similar response in amplitude [see Subheading 2.2.4. (4.2)].
4. The amplitude of calcium transients in individual neurons
evoked by two-photon optogenetic stimulation should be sim-
ilar to the amplitude of calcium transients evoked by physio-
logical stimuli, otherwise the effects described could be due to
over stimulation of neurons [see Subheading 2.2.4. (5.2)].
5. The responses induced by the activation of large numbers of
neurons in a given optical field could be attenuated by the
engagement of GABAergic circuits. The total number of simul-
taneously photostimulated neurons in a given optical field
should be maintained in the same range as the number of active
neurons evoked by physiological conditions (sensory stimuli)
[see Subheading 3.1.2].
6. During the training phase of the behavioral task, pairing of
sensory stimuli and optogenetic stimuli should be avoided.
Otherwise, the effects attributed to optogenetic stimuli could
be due to mice learning to identify the optogenetic activation
of neurons [see Subheading 3.2.7].

6 Outlook

A general development direction of all-optical methods is to

increase the throughput (i.e., field of view and spatiotemporal
resolution) of both imaging and photostimulation, while reducing
the laser power incident onto the brain. Beam multiplexing
356 Luis Carrillo-Reid et al.

techniques are promising approaches to increase the imaging

throughput [56–59]. Larger spatial light modulators with higher
pixel count could increase the beam diffraction efficiency and thus
the field of view [14]. Faster spatial light modulators could allow
rapid switching of the hologram and thus could enable faster
photostimulation of neuronal ensembles in specific spatiotemporal
patterns. As the simultaneously photostimulated neurons across a
3D volume increase [14, 66, 69, 72], there could be off-target
effects through photostimulating the dendritic arbors crossing the
cell bodies of target neurons. Somatic restricted viruses where the
opsin only expresses in the cell body could alleviate this issue
[14, 66, 72, 96].
To access deeper brain regions noninvasively, adaptive optics
[97, 98] and three-photon excitation [38, 39, 41, 99, 100] could
be used. A spatial light modulator in the photostimulation path
could implement adaptive optics, and a spatial light modulator in
the imaging path could allow for similar corrections. Three-photon
calcium imaging has also been successfully demonstrated [39, 41,
99, 100]. One current challenge is that it needs high power for
three-photon optogenetics in deep brain regions. The development
of high-efficiency opsins optimized for three-photon excitation
could overcome this challenge.
The development of all-optical techniques to simultaneously
read and write patterned activity in neuronal populations could
help the understanding of perception, memory formation, behav-
ioral states, and pathological conditions with single-cell resolution
in the next decades. The systematic documentation of the causality
between neuronal ensemble activity and brain states requires fur-
ther development of analytical tools to visualize and target specific
neurons that can control the overall network activity [81]. The
adaptation of artificial intelligence algorithms used in big data to
understand population activity could be used to design and guide
in vivo experiments [13].
Scaling of these techniques to several brain areas to measure
and control thousands of neurons in different parts of the brain will
require the identification and targeting of neurons with pattern
completion capability [21, 81]. This approach could control
thousands of neurons by the activation of a small percentage of
them, reducing sample heating and deterioration of spatial resolu-
tion that comes with increased number of parallel stimulations.
Finally, the development of transgenic mice with genetically
encoded calcium or voltage indicators and opsins that use different
wavelengths in molecularly identified subpopulations of neurons
[20] will allow chronic recordings and stimulation of several brain
areas with single cell and molecular identity precision, which repre-
sent the next challenge for neuronal ensemble research.
 All-Optical Probing of Neuronal Ensembles 357

Acknowledgments

CONACYT (INFR-2018-294756 [LCR], CF6653 [LCR]), Bur-

roughs Wellcome Fund (Career Award at the Scientific Interface
#1015761) [WY], National Eye Institute (R01EY011787 [RY]),
National Institute of Mental Health (R01MH115900) [RY],
National Institute of Neurological Disease and Stroke
(R01NS110422 [RY], R34NS116740 [RY], R01NS118289
[WY]), the US Army Research Office (W911NF-12-1-0594
MURI) [RY], and the National Science Foundation (CAREER
1847141 [WY], CRCNS 1822550 [RY]). Rafael Yuste is listed as
an inventor of the patent: “Devices, apparatus and method for
providing photostimulation and imaging of structures” (United
States Patent 9846313). Weijian Yang and Rafael Yuste are listed
as inventors of the patent: “System, method and computer-
accessible medium for multi-plane imaging of neural circuits”
(United States Patent 10520712).

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 Chapter 12

Illuminating Neural Computation Using Precision

Optogenetics-Controlled Synthetic Perception
Jonathan V. Gill, Gilad M. Lerman, Edmund Chong, Dmitry Rinberg,
and Shy Shoham

Abstract
Connecting neuronal activity to perception requires tools that can probe neural codes at cellular and circuit
levels, paired with sensitive behavioral measures. In this chapter, we present an overview of current methods
for connecting neural codes to perception using precision optogenetics and psychophysical measurements
of synthetically induced percepts. We also highlight new methodologies for validating precise control of
optical and behavioral manipulations. Finally, we provide a perspective on upcoming developments that are
poised to advance the field.

Key words Optogenetics, Holography, Olfactory, Psychophysics, Two-photon excitation

1 Introduction

A fundamental question in systems neuroscience is how sensory

stimuli are encoded by the activity of neurons. This question has
been the subject of intense scrutiny across sensory systems, leading
to fundamental discoveries describing the nature of neural repre-
sentations across many levels of information processing and
abstraction [1, 2]. The rapid pace of this progress has been matched
with an improvement in tools used for recording neuronal activity
at a large scale (e.g., two-photon (2P) calcium imaging), leading to
the now-routine generation of datasets comprised of hundreds to
thousands of simultaneously recorded neurons in awake behaving
animals [3, 4]. A consistent observation made from recordings
taken across sensory systems is that neural responses to sensory
stimuli include changes in both the rate and timing of action
potentials within populations of neurons, as well as modulation
with respect to ongoing, internally generated rhythms (sniffing,
whisking, locomotion, etc.) [5–10]. Despite the ability to measure

Eirini Papagiakoumou (ed.), All-Optical Methods to Study Neuronal Function, Neuromethods, vol. 191,
https://doi.org/10.1007/978-1-0716-2764-8_12, © The Author(s) 2023

363
364 Jonathan V. Gill et al.

complex sensory responses, it remains unclear what role individual

features of neural activity (e.g. rate, timing, etc.) play in perceptu-
ally guided behavior.
One way to pose this question is to ask what features of
sensory-evoked activity are read by downstream brain areas to
guide behavior. To answer this question, one must establish a causal
link between features of neural activity (e.g., the firing rate and
timing of specific neurons) and behavioral report (e.g., detecting a
sensory stimulus). Perturbations of neural activity are therefore a
key tool for connecting specific circuits and features of spiking
activity to perception. Pioneering this conceptual framework, an
influential study by Newsome and colleagues demonstrated that
the perceptual effects of stimulation could be quantitatively esti-
mated, employing experiments in which a monkey’s motion-
perception-related response was biased by electrical stimulation of
the middle temporal visual area (MT) [11]. In these experiments,
the monkey could be biased toward reporting a certain direction of
motion in an ambiguous stimulus if stimulation was applied to a
columnar area of cells which were maximally responsive, or ‘tuned’,
to the corresponding motion direction. One reason why the effect
of this stimulation was both predictable and reliable was that the
spatial scale of the manipulation (hundreds of micrometers current
spread) was on the order of the neural representation (a cortical
column).
The advent of optogenetics has permitted increasingly precise
interventions across a broad range of neural circuits and behaviors,
culminating in the development of two-photon (2P) optogenetics
[12–16]. When combined with 2P imaging and holographic tar-
geting, this technique permits the activation of arbitrary sets of
pre-selected neurons with up to sub-millisecond precision in vivo
[17–22]. Neural representations encoded at fine spatiotemporal
scales can now be interrogated using “precision optogenetics,”
increasing the potential for an even more nuanced understanding
of the connection between neural codes and behavior [23–29]. A
detailed description of optical methods for combining 2P photo-
stimulation and imaging appears in other chapters of this book. In
what follows, we specifically focus on methods developed for com-
bining precise photostimulation with psychophysical measurements
of evoked synthetic percepts and on complementary methods used
to confirm a precise relationship between optical manipulations and
the observed behavior. These methods underlie recent scientific
studies on the coding logic of olfactory perception, whose results
are described in detail in our recent publications [25, 30,
31]. While the particular features of interest may be determined
by the sensory system under study, this chapter highlights general
frameworks for connecting synthetically induced activity with per-
ceptual and behavioral readout.
 Illuminating Neural Computation Using Precision Optogenetics 365

2 Paradigms for Psychophysical Measurement of Synthetic Perception

The rapid development of optogenetics within the past decade has

introduced an extensive toolbox of genetically encoded, light-
activated ion channels capable of temporally precise, bidirectional
modulation of activity within predefined circuits and cell types
[16, 18, 24, 32]. In order to link specific circuits and cell types
with their role in generating a sensory guided behavior, a common
approach has been to manipulate neural activity while an animal is
presented with a stimulus and test the consequence on behavior
compared to the normal condition. In this way an experimenter can
enhance, eliminate, or “tweak” neural activity to discover how
circuits and cell types may be involved in making a particular
perceptual judgement.
This approach has undoubtedly enhanced our understanding
of which brain circuits and cell types are essential for certain per-
ceptual tasks. For example, inhibiting the visual cortex impairs
orientation discrimination [33], and broad, unstructured optoge-
netic activation of olfactory inputs interferes with concurrent odor
recognition [34]. But these manipulations are relatively uninforma-
tive about precisely how sensory percepts are formed by those
circuits and cell types, that is, how they are encoded by and decoded
from their activity.
To address this question, we must manipulate sensory repre-
sentations at the spatiotemporal scale with which they are encoded.
Previous technologies making use of electrical or one-photon
manipulations lacked the ability to perturb sets of individual,
pre-selected cells, limiting their potential to directly connecting
specific neurons and spiking patterns with sensory-guided behavior.
However, spatiotemporal excitation technologies from the
emerging field of precision optogenetics are capable of addressing
representations that are encoded at either the microcircuit or
distributed scales. In what follows, we will describe the application
of two key techniques.
The first, 2P holographic optogenetics, is currently the only
method capable of selectively stimulating individual neurons which
can be targeted based on their response properties, cell type, loca-
tion, and projection targets, along with many other selection cri-
teria [35]. The second, one-photon (1P) patterned optogenetics, is
capable of stimulating groups of superficial neurons spread out over
many millimeters in arbitrary patterns, which can precisely manipu-
late discrete representations organized over larger spatial scales,
such as olfactory bulb glomeruli [30, 36]. Critically, precision
optogenetics has the ability to bypass the sensory organ or periph-
eral circuits involved in sensory-guided behaviors and directly
address the perceptual impact of specifically targeted neurons.
In this way, an experimenter can create “synthetic” stimuli, whose
366 Jonathan V. Gill et al.

cellular composition and timing are exactly known. This allows the
experimenter to take a step beyond validating the participation of
individual circuits for behavior and to determine precisely what
features of the activity of neural circuits guide perception. This is
accomplished by holding individual features constant (e.g., what
neurons are stimulated), while parametrically varying other features
(e.g., timing), and performing sensitive psychophysical measure-
ments of the perceptual impact.

2.1 Using Detection The first experimental strategy is to assess the influence of specific
to Test the Relevance features within neural activity patterns on the detectability of an
of Neural Codes artificial stimulus. Animal survival relies on the detection of brief,
faint cues to signal the presence of food, mates, and predators.
A multitude of studies across species have revealed the exquisite
sensitivity of sensory systems to their preferred stimuli
[37, 38]. Exploring how critical information is conveyed by sensory
circuits at the perceptual limit provides a lens to examine the
essential coding features connecting neural activity to behavior.
However, even in this minimal regime, sensory-evoked responses
can be complex, simultaneously encoding stimuli in the identity,
rate, and timing of specific neurons. By replacing external stimuli
with targeted activation of neurons in sensory areas, we can directly
test which features of the observed activity affect the detectability of
the artificial stimulus. We can then infer the features of activity
essential for perception.
Early studies using either electrical or 1P optogenetic stimula-
tion revealed that rodents can detect changes in the spike rate of
single neurons [39, 40], or across populations of hundreds of
neurons [41] in the somatosensory and visual cortices, using sti-
muli that lasted for hundreds of milliseconds. Additional studies
have explored the role of relative spike timing [41, 42], and sensi-
tivity to latency [43, 44] in populations of hundreds to thousands
of optogenetically activated cortical neurons. These studies were
successful in demonstrating that rodents can perceive minimal
perturbations in sensory cortical neurons, and perceptibility may,
or may not, vary along certain feature dimensions, like timing.
However, the techniques used in these experiments lacked the
ability to target specific sets of neurons according to their func-
tional properties or tuning. By optogenetically labeling olfactory
sensory neurons expressing a specific receptor (M72-ChR2), a key
related study in the olfactory system revealed that mice could detect
brief activation (10 ms) of a single glomerulus [45]. This well-
defined input channel to the olfactory bulb is a site of convergence
where ~5,000 olfactory sensory neurons provide input to ~25–30
mitral and tufted cells (projection neurons), demonstrating that the
elementary features of olfactory perception operate at, or below,
this spatiotemporal scale.
 Illuminating Neural Computation Using Precision Optogenetics 367

The recent maturation of 2P holographic optogenetics repre-

sents an important opportunity for probing the relevance of neural
coding features for detection. This approach has recently been
applied across a range of sensory systems (olfactory, visual, somato-
sensory), where 2P photostimulation of a predefined set of neurons
was used as the stimulus to be detected. Because specific neurons
can be targeted using this technique, a larger feature space can be
explored. For example, a recent study observed that the functional
connectivity and orientation tuning of a subset of photostimulated
neurons was related to their detectability [23] (see also Chap. 11).
Extended photostimulation durations (1s) of a subset of visual
cortical neurons “recalled” a larger ensemble response tuned to a
particular visual stimulus, and as few as two of these “pattern
completion” neurons were detectable. That is, strongly photosti-
mulating a specific small group of neurons (as few as two neurons)
evoked the activation of a larger, behaviorally detectable pattern.
Another recent study found that mice could detect 250–500 ms
photostimulation of ~14 somatosensory (barrel) cortical neurons
and that this ability improved with experience but did not depend
on the precise neurons targeted [26].
Both of these studies found evidence that mice can detect the
photostimulation of a few neurons delivered over a relatively long
timescale (250–1000 ms), potentially evoking hundreds of spikes.
However, for a range of behaviors animals have been shown to
make decisions about salient perceptual cues within very short
temporal windows [46–48]. For example, rodents are capable of
detecting odorants in less than a single sniff (<100 ms), at
extremely low concentrations (as low as 10
12 M) [34, 37, 49,
50]. In this case the very first volley of action potentials generated
by inhaling an odor are sufficient to inform an animal’s response.
To understand what features of olfactory bulb activity guide
rapid detection of faint stimuli, Gill, Lerman et al., 2020 extended
the use of 2P holographic optogenetics to probe the detectability of
single spikes distributed across small ensembles of olfactory bulb
neurons (Fig. 1) [25, 51]. In this study, head-fixed, water-restricted
mice were trained to detect the synchronous activation of a group
of olfactory bulb neurons composed of mitral cells (excitatory), or a
mixture of mitral cells and granule cells (inhibitory). The animals’
respiration was monitored so that photostimulation delivery could
be timed to a fixed delay after inhalation (20~40 ms), mimicking
the sampling of an odor (Fig. 1a, b). A specific, predefined set of
neurons were used as the stimulus across several sessions, allowing
the experimenters to assess the effects of learning or plasticity on
detectability (Fig. 1c). Mice were able to detect the synchronous
activation of ~30 neurons at high performance within several
sessions.
After mice reached a consistent level of performance, the stim-
ulus was parametrically varied along three feature dimensions:
number of neurons, relative timing (synchrony), and latency from
368 Jonathan V. Gill et al.

A B C
2P Imaging 2P Stimulation 5 Go
920 nm + 1028 nm Tone 1 4
6
Lick Hit
2 3 7
sound cues Sniff
8
12
9
10
No-Lick Miss
11 13
16x Photostim. 14
30 cells
No-Go
16 23
sniff signal 15
Response 18
22
19 20
Lick
False
17
Interval 25
21
Alarm
27
24 26 28
Lick 29 No-Lick
Correct
water rewards 30
WT
Reject
1s no stim.

Fig. 1 Testing detection of precision photostimulation. (a) Schematic of photostimulation detection experi-
ment. A head-fixed mouse with a chronically implanted window above the olfactory bulb is positioned in front
of a lickspout and pressure sensor to monitor respiration (sniff). (b) Trial structure for detection experiment. A
tone signaled the start of trials and photostimulation was timed to a fixed delay relative to sniff (c) Left,
neurons in the mitral cell layer (MCs and GCs) co-expressing ChrimsonR-tdTomato (red) and GCaMP6s
(green). Thirty neurons were targeted for simultaneous photostimulation (white circles). Scale bar – 40 μm.
Right, outcomes for responses to the “go” and “no-go” trials. Red circles indicate stimulation of a particular
cell, while empty circles indicate no stimulation. (Adapted from Ref. [25])

A B C inhalation
inhalation inhalation
N=9 ∆t = 10 ms

Neuron #
Neuron #
Neuron #

Decreasing Synchrony
Decreasing Number

Increasing Latency 25
N=5 ∆t = 30 ms

45
N=2 ∆t = 50 ms

0 45 100 0 100 0 65 100

time (ms) time (ms) time (ms)

Fig. 2 Independent control of multiple activity features. (a) Schematic of stimuli which vary in the number of
neurons targeted, but not the timing of activation. (b) Schematic of stimuli which vary in the synchrony across
neurons, but not the number of neurons. Stimuli were presented with a mean latency of 45 ms across
conditions. (c) Schematic of stimuli which vary in the latency of photostimulation relative to the onset of
inhalation, but not synchrony, or number of neurons. (Adapted from Ref. [25])

inhalation (Fig. 2). In this way, the contribution of each of these

features to detectability could be independently assessed. We found
that mice were capable of detecting single spikes distributed across
<20 neurons, with an average psychophysical threshold of 10–15
neurons. Synchrony was tested by staggering the timing of spikes
when the full pattern of neurons was targeted, revealing a strong
 Illuminating Neural Computation Using Precision Optogenetics 369

dependence on the relative timing of photostimulation across neu-

rons. Finally, we specifically manipulated the latency from inhala-
tion and found that detection did not depend on sniff phase.
These results demonstrated that the exquisite sensitivity of the
olfactory system goes far beyond a single sniff or glomerulus, with
mice capable of detecting just a few spikes, so long as they occurred
within less than 30 ms of each other. The independent control of
coding features using precision optogenetics was critical for discov-
ering a previously unknown contribution of relative spike timing to
olfactory perception. Future studies will surely extend this
approach to test an even broader range of features across sensory
systems using even more sensitive behavioral tasks to ultimately
reveal the building blocks of perception.

2.2 Technical Behavioral Task and Training The previously described experi-
Implementation of ments all made use of a similar strategy for assessing the detectabil-
Detection Experiments ity of 2P photostimulation. They used a go/no-go behavioral
paradigm, in which an animal makes a binary decision about the
presence or absence of a stimulus. In this paradigm, an animal is
trained to respond in some manner (e.g., licking, lever press, nose
poke) for a reward, or withhold a response (e.g., do not lick, press,
or poke), to signal whether a “go” stimulus cue is present or absent
(“no-go”). Typically, the “go” stimulus is randomly, or pseudo-
randomly presented on a fraction of trials (typically 0.5), where on
the remaining fraction of trials an animal experiences either a “dis-
tractor” stimulus or nothing. Animal behavior is progressively
shaped to associate the availability of reward with the presence or
absence of the stimulus. Once the task is acquired, the experimenter
can then change the parameters of the stimulus in either a trial or
blockwise manner to determine the sensitivity of animals to each
parameter based on the frequency of correct choices or error type.

In the previously described experiments, 2P photostimulation of a

predefined set of neurons was used as the “go” stimulus. In all cases
the behavioral experiments were conducted in head-fixed, water-
restricted mice outfitted with a cranial window. Detection experi-
ments begin by limiting the amount of water available to an animal
to ~1 mL/day, depending on their body weight. After 3–5 days of
water restriction, mice will consume their daily allotment, at which
point they can begin training to receive water while head fixed.
Habituation to head fixation and “lick training” can occur simulta-
neously, by gently head-fixing naı̈ve mice, and making available a
metal spout (lick tube) that will deliver a small droplet of water
when contacted by the tongue. Mice can freely lick the tube to
receive 2 μL droplets, until they have learned to lick enough times
to receive the full 1 mL of water for the day. This has the advantage
of forming an association between head fixation and the availability
of reward which can decrease overt distress during behavioral
370 Jonathan V. Gill et al.

training and imaging sessions. A useful tool for detecting licking is a

capacitive touch sensor coupled to hypodermic tubing which can be
used to trigger the release of water through a pinch valve or
solenoid controlled by a microcontroller or computer.
After animals reliably lick for water, it is necessary to shape their
behavior to acclimate them to the timing and conventions of the
go/no-go task. This can be done by initially training mice to
recognize very salient stimuli and thus learn the associations of
the task before moving on to more difficult conditions. As
pre-training for a 2P photostimulation detection task, this typically
takes the form of training animals to detect a high intensity target
stimulus for the sensory modality under study (e.g., a high concen-
tration odorant, a high contrast oriented grating, a large amplitude
whisker deflection), with another clearly separable stimulus used as
the distractor, or “no-go” stimulus, or nothing at all. Alternatively,
1-photon light of the appropriate wavelength for the opsin
expressed in the neurons of interest can be used to train animals
to recognize artificial photostimulation during this training stage.
This has the advantage of more closely mimicking the 2P photo-
stimulation detection task, since both involve artificial activation of
neurons in a given area, but has the disadvantage of not clearly
mapping onto a specific perceptual stimulus, which makes it diffi-
cult to study generalization from real to artificial stimuli. Either
way, performing a shaping procedure is essential so that learning
trajectories during 2P photostimulation can reflect each animal
learning to detect activity in a small ensemble of neurons, and not
merely learning the basic contingencies of a go/no-go task.

Perceptual Testing Once the animal’s behavior has been shaped,

detection of the 2P photostimulation can be tested. If neurons are
targeted across days, care must be taken to align each session’s field
of view with a common template (described in Subheading 3.2).
Responses of targeted neurons should be measured outside of the
behavioral task for each session in order to determine whether
changes in detectability are related to learning, or a change in the
ability to photostimulate the targeted neurons.

After detection ability for a set of neurons has been established,

the experimenter can vary features of the evoked activity to test
their contribution to detection. For manipulations of neuron num-
ber, the stimulus is replaced with a hologram targeting all, or a
subset of the neurons from the set. It is important to maintain the
same power per neuron across conditions. For manipulations of
timing, it is important to first measure the average latency and jitter
of spikes evoked by 2P photostimulation (described in more detail
in Subheading 3.1). To control the timing and power of 2P photo-
stimulation, a Pockels cell can be used for rapid and precise control
of light delivery. Alternatively, a shutter can be used; however, it is
 Illuminating Neural Computation Using Precision Optogenetics 371

essential to confirm that the animal cannot hear the shutter if

detection is being tested. For experiments testing relative timing
of neuronal activation, or synchrony, it is important to use a spatial
light modulator (SLM) capable of rapidly switching between holo-
grams targeting subsets of the neurons (ideally <10 ms
switching time).
If using a go/no-go paradigm, manipulations can be per-
formed in a trial, or blockwise manner. While changing the condi-
tion randomly (e.g., number of targeted neurons) on each “go”
trial may seem like an unbiased way to test the feature space, it is
often best to test a single condition per block of trials. The reason
for this is that performance errors can take two flavors, false alarms
and misses. Mice tend to make the majority of their incorrect
choices as false alarms, ensuring that they do not miss a potential
“go” trial or opportunity to obtain a reward. By testing one condi-
tion per block of trials, usually composed of 50% go and 50% no-go
(no stimulus), false alarms are readily interpretable, as the false
alarm rate may increase as the stimulus condition becomes more
difficult to detect (e.g., less neurons targeted), even if the number
of “hits” does not decrease. If different conditions are randomly
interleaved in a trial-by-trial manner, one relies only on miss rate to
determine the differences in detection performance, since all con-
ditions share the same false alarm rate, significantly reducing the
sensitivity of this measure.

2.3 Measuring The second experimental strategy developed recently to connect

Perceptual Distance of neural activity features to perception is to measure the perceptual
Synthetic Percepts distance of synthetic percepts evoked by optogenetic stimulation.
Experiencing a familiar object, for example, a rose, evokes a com-
plex spatiotemporal pattern of activity in sensory areas. What fea-
tures of this activity determine the identity of the object as “rose”
and not another object like “tulip” or “orange”? Are some features
generally better at explaining the differences in how stimuli are
perceived, regardless of the specific stimuli being compared? By
determining these features, we can expose the computational stra-
tegies underlying perceptual identity.
Traditionally, it has been difficult to determine the perceptual
relevance of different coding features using natural
(non-optogenetic) stimuli. One reason for this difficulty is that
different features of neural activity often covary with one another
as stimulus identity is changed. For example, presentation of a rose,
tulip, and orange may each evoke changes in both the firing rate
and timing of overlapping sets of neurons. Which of these features
(cell identity, rate, timing, etc.) is essential for the animal to dis-
criminate “flower” from “fruit,” or to identify “rose” specifically?
While expanding the stimulus set to include more diverse examples
may help tease out responses unique to each class or exemplar,
biophysical constraints often impose correlations between features,
372 Jonathan V. Gill et al.

and it can be difficult or impossible to design stimulus sets that fully

disentangle their contribution.
The use of natural stimuli to determine perceptually relevant
coding features is further hampered by generating inferences purely
through correlation. While some features of activity may appear to
predict perceptual choices made by the animal, they may not have
any actual influence on the behavior. For example, the spike rate of
neurons in a particular brain region may be highly predictive of
whether an animal will classify a stimulus as “flower” or “fruit,” but
it need not be the case that any of these neurons is actually relevant
for the discrimination. If the experimenter was to manipulate the
spike rate of the neurons (activating or silencing them), they may
find no effect on the choices of the animal, despite the strong
correlation of the spike rate to the behavior in the normal condi-
tion. In this way, merely observing activity and relating features to
behavior has limited power for testing causal models of perception.
Precision optogenetics therefore provides an opportunity to
independently manipulate activity features and determine their
differential impact on perception. Further, this technique provides
the opportunity to test whether behavior is guided by combina-
tions, or conjunctions of features (e.g., rate and tuning, or sequen-
tial order and phase). By creating fully synthetic stimuli using
precision optogenetic stimulation, one can manipulate activity fea-
tures while an animal performs a recognition, or discrimination
task, in which they signal the perceived identity of a stimulus
through their behavioral choice. By measuring the frequency with
which animals categorize induced activity patterns to be the same,
or different percepts, one can determine the relative perceptual
distance between synthetic stimuli. Finally, this approach allows
the experimenter to determine what individual or combinations
of features define the axes of perceptual identity for a particular
neural circuit.
This strategy has recently been used in our work to probe the
perceptual relevance of different coding features in the mouse
olfactory system. Chong, Moroni, et al. (2020) used a combination
of genetic labeling (OMP-ChR2, expressing ChR2 only in olfac-
tory sensory neurons [52]) with precise optical targeting using a
high-resolution digital micromirror device to project light patterns
onto the surface of the olfactory bulb, evoking activity patterns
with single glomerulus resolution (Fig. 3a) [30]. In this way, we
created synthetic odor stimuli by activating sets of glomeruli with
high spatial and temporal precision. We trained water-deprived
mice to recognize a single spatiotemporal pattern as a target
“odor,” and to report activation of this pattern by licking a water-
spout. The mice were trained to discriminate this stimulus from
every other non-target pattern (any spatiotemporal pattern not
identical to the target), and to report any non-target pattern by
licking a different waterspout (Fig. 3b).
 Illuminating Neural Computation Using Precision Optogenetics 373

Fig. 3 Spatial and temporal perturbations of synthetic odors. (a) Schematic of the experimental setup. Dorsal
olfactory bulb was exposed by a chronically implanted 3 mm window. Spatiotemporal stimulation patterns,
created by a digital micromirror device, were projected onto the olfactory bulb of a head-fixed OMP-ChR2
mouse in front of a pressure sensor for sniff monitoring, and lick spouts delivering water. (b) Schematics for
pattern discrimination task. Animals were trained to recognize Target versus Non-target patterns defined on a
stimulation grid. Target patterns comprised six spots, initialized randomly but fixed across subsequent
sessions, activated in an ordered sequence defined in time where 0 marks inhalation onset. Non-target
patterns were six off-Target spots, randomly chosen from trial to trial, with randomized timing within 300 ms
from inhalation (~single sniff). (c) Illustration of spatial perturbations: One or multiple spots in Target patterns
were randomly replaced with Non-target spots. (d) Illustration of temporal perturbations in which one or
multiple spots in Target patterns were temporally shifted. (Adapted from Ref. [30])
374 Jonathan V. Gill et al.

After mice learned to perform this task with high accuracy, we

tested how recognition changed as we systematically manipulated
the activity patterns across several feature dimensions (Fig. 3c, d).
Mice experienced a small proportion of “probe” trials (10% of total
trials), in which the target stimulus was modified, either by repla-
cing spots with non-target spots (Fig. 3c) or by shifting spots in
time (Fig. 3d). We measured the fraction of trials in which mice
reported the modified pattern as being like the “target” pattern.
This proportion reflected the perceptual distance between the
modified and original target pattern.
To determine the relative influence of the spatial and temporal
features on perception, the experiments of Chong et al. 2020 used
precise parameterization of perturbations to the target stimulus.
One key finding was a primacy effect in which perturbations to
earlier activated glomeruli had larger effects on perceptual
responses than later activated glomeruli. Despite the fact that ani-
mals could use any available activity features to solve the
target vs. non-target categorization, such as glomerular identity
or timing, this result suggests that these features do not carry
equal weight for informing an olfactory percept, as all the tested
mice were more strongly influenced by changes to the earliest spots
in the pattern without being explicitly trained to use this feature.
While both spatial and temporal perturbations were effective at
increasing the perceptual distance from the target pattern, the
joint assessment of these features demonstrated that odor identity
representation is nuanced and determined along several dimensions
simultaneously. This study highlights how a fully synthetic
approach can be used to establish basic principles of the neural
code informing perception. Models derived from these findings
could then be tested and further refined by the use of naturalistic
stimuli, ultimately closing the loop between causal manipulation
and ethological observation.

2.4 Technical In the previously described experiments, mice performed a

Implementation of 2-alternative forced choice task in which licking either the left lick
Perceptual Distance spout or the right lick spout signaled a choice of either Target or
Experiments Non-target pattern perceived by the animal. The lick spout
assigned to Target or Non-target should be randomly determined
for each experimental animal to control for any systematic side bias.
Trials consist of a stimulus period, a grace period in which mice can
lick without reward or punishment, and a response period in which
the first lick determines the animal’s choice. The purpose of a grace
period is to reduce the influence of impulsive licking on the trial
outcome and is typically ~0.5 s following the stimulus period. This
period provides time for an animal who may have been licking in
response to the trial initiation to change to an informed licking
pattern after experiencing the stimulus. Initially, mice will often be
biased toward licking one side over the other [53], so it is
 Illuminating Neural Computation Using Precision Optogenetics 375

important during training to perform a de-biasing procedure which

adaptively increases the incidence of trials on the biased-against side
[54]. By doing so, side biases can be eliminated before initiating
critical phases of the experiment.
The first lick during the response period is counted as the
animal’s choice and the trial ends, leading to an inter-trial-interval
before the next trial. Reward will be provided to the animal at
different rates depending on the phase of the experiment. Initially,
during the shaping period, animals are trained to discriminate
between one Target and one Non-target pattern until animals
exceed a threshold of performance (80%). During this phase, ani-
mals are rewarded for correct choices 100% of the time. After the
initial shaping, Non-target patterns are randomly initialized on
each trial, and due to the combinatorics, they never repeat. The
animals are also shaped toward a 70% reinforcement rate. At this
point, the animals experience test sessions in which probe trials
make up 10% of the total trials while Target and Non-target trials
comprise 45% each. These probe trials involve perturbations to the
target pattern along feature dimensions under study (spot identity,
relative timing, shift with respect to respiration, etc.), and are
randomly interleaved with the Target and Non-target trial types.
Critically, probe trials are never rewarded, allowing the animal’s
choices to reflect their internal Target vs Non-target category
boundaries.

3 Validating Precise Manipulation

3.1 Characterizing In order to interpret the results of an experiment combining preci-

the Scale and Timing sion optogenetics with behavior, it is critical that the characteristics
of Response to and specificity of the neural response to the manipulation be
Stimulation known. For example, if one wants to study how the relative order
of neurons activated by 2P photostimulation impacts the perceptual
quality evoked by the manipulation, it is critical that the timing of
cellular responses to stimulation be known, lest the neurons
respond out of order. Similarly, if an experiment relies on compar-
ing how two different groups of neurons are perceived when they
are separately activated, it is critical to determine the specificity with
which the groups can be targeted, as inadvertent activation of
neurons in the wrong group could confound the inferences made
from an animal’s behavior. We will cover three useful methods for
assessing the scale and timing of responses to all-optical targeting
and manipulation in vivo which may aid in the design and interpre-
tation of behavioral experiments.

Targeted Electrophysiology Prior to initiating a study utilizing

all-optical methods such as 2P photostimulation for combination
with behavior, it is strongly advisable to assess the responses of
376 Jonathan V. Gill et al.

A B C
Photostim.

20 ms

Latency
10 ms

5mV

1 Trial

{
5 ms 5 ms Jitter

Fig. 4 Targeted electrophysiology. (a) 2P-guided cell-attached electrophysiological recording in an awake

C57/BL6 mouse mitral cell layer co-expressing ChrimsonR-tdTomato (red) and calcium indicator GCaMP6s
(green). The neuron (white circle) was targeted by a light patch (scale bar, 20 μm). (b) Examples of
electrophysiological recordings during 5, 10, and 20 ms photostimulation. (c) Example raster plot for
10 repetitions of 10 ms photostimulation with 30 mW average power. The response latency is defined as
the time from photostimulation onset to the first spike, and jitter is defined as the standard deviation of the
latency across photostimulation repetitions. (Adapted from Ref. [25])

neurons to a range of photostimulation parameters (power, dura-

tion, frequency, etc.). In this way, the all-optical system can be
properly “tuned” to provide an expected response for a given
opsin, cell-type, tissue depth, etc. (see Note 3). Efficacy and timing
of evoked responses to stimulation using “standard” approaches,
such as holographic patch, or spiral scanning of a focused beam
have been characterized in the literature for only a very limited
range of conditions, and rarely in vivo (though a number of recent
studies have helped to fill this knowledge gap [18–20, 25, 28]. Cur-
rently, single-unit electrophysiology is the standard for measuring
photocurrents and evoked spiking with high temporal precision.
Combining high-impedance electrophysiological recordings per-
formed with a glass pipette and 2-photon imaging, it is possible
to target and record from specific predefined neurons (Fig. 4a).

To perform targeted recordings, one must begin with an

appropriate amplifier, digitizer, and program for recording and
aligning electrophysiological data to photostimulation delivery. As
the main interface to the tissue, borosilicate glass pipettes pulled to
5–11 MΩ (~1–1.5 μm tip size) are appropriate when coupled to an
electrode holder with an outlet for pressure regulation. For juxta-
cellular recordings (cell abutting, but not internal), electrodes can
be filled with a modified current-clamp “external” solution
(130 mM K-gluconate) containing fluorescent dye for visualiza-
tion. In practice, neurons are often labeled with both a calcium
indicator (GCaMP6, jRGeCO1a, etc.) and a fluorophore indicating
the expression of an opsin (ChrimsonR-tdTomato, Chronos-EYFP,
etc., though see Note 1), which makes visualizing the thin tip of a
glass pipette difficult, since both green and red imaging channels
contain information. To solve this, an approach is to use a mixture
 Illuminating Neural Computation Using Precision Optogenetics 377

of green and red dyes (Alexa 488/594 mixture, Milipore) when

preparing the pipette solution, which allows the pipette to be
viewed simultaneously on both the green and red imaging chan-
nels, allowing it to stand out to some degree from the surrounding
tissue.
It is optimal to perform recordings in conditions as similar as
possible to behavioral conditions, so it is recommended to perform
recordings in awake, head-fixed animals. To achieve this, it is
important to have a stable, chronically implanted preparation that
permits repeated electrophysiological access to the targeted area, as
well as optical clarity for 2P imaging. Both of these requirements
can be met by implanting a glass cranial window (Warner) with
pre-drilled holes filled with silicone elastomer (Quik-Sil). If appro-
priately mixed, the elastomer will remain transparent and create an
air-tight seal in the cranial window, allowing it to be implanted
chronically. Prior to the recording session, the silicone plug can be
removed with a pair of fine forceps, exposing the tissue underneath.
The brain should be bathed in sterile saline or artificial cerebral
spinal fluid for the duration of the recordings, after which the
cranial window can be re-sealed using the same technique. This
method permits multiple recording sessions for each animal, and
can drastically increase the yield of stable recordings, since the
implanted cover glass also effectively minimizes brain movement.
For the purpose of measuring the temporal resolution of an
all-optical manipulation, measuring spiking delay from the onset of
photostimulation and the jitter of the timing of the evoked spiking
are essential (Fig. 4b, c). To estimate the spatial resolution of the
manipulation, moving the focus of the stimulation laterally (x,y),
and in depth (z), should be performed by fixing all features of the
photostimulation and moving the objective a fixed amount while
recording the effect on spiking. From this, one can measure how far
from the targeted neuron the photostimulation could evoke spik-
ing and infer how near another neuron would need to be, to be
inadvertently activated by the photostimulation. While this is a
useful estimate, in practice “off-target” activation depends on
many factors, including the geometry of the area under study and
the sparseness of opsin expression, to name a few, meaning the
measured lateral and axial resolution is not the same as the effective
resolution of the system, which can be estimated using methods in
the following sections.

Network Responses to Photostimulation It is highly desirable to

demonstrate that the scale of the manipulation employed in an
experiment combining all-optical manipulation with behavior is
matched to that of the neural representation being probed. For
example, if targeting individual neurons tuned to a particular ori-
entation in visual cortex, and these neurons are interdigitated with
neurons tuned to other orientations in a “salt and pepper”
378 Jonathan V. Gill et al.

organization, it is sensible to demonstrate single-neuron resolution

of photostimulation. A technique to estimate the effective scale of a
manipulation is to target individual neurons composing some rep-
resentation and observe via 2P calcium imaging the effect of stimu-
lation on the non-targeted neurons in the field of view (FOV). For
example, if an experiment involves simultaneously targeting 30 neu-
rons that share the same tuning, it is useful to target each of these
cells individually and measure the effective network response to
each target in addition to targeting the full pattern. In this way,
one can screen for undesirable off-target effects or reject neurons
that do not respond on their own. Both of these factors could be
harder to observe when targeting the full pattern, because a great
degree of network modulation is expected as the number of tar-
geted neurons increases, and targeted neurons that are modulated
by the full pattern may not actually be responding to photostimula-
tion directly, but just modulated along with the rest of the network.

To screen for off-target effects, or inadvertent photostimula-

tion of neighboring neurons, it is useful to look at whether excit-
atory responses to untargeted neurons cluster around
photostimulation sites (Fig. 5). A common way of representing
the spatial extent of the manipulation is to bin the average response
of all neurons in the imaging plane by their centroid distance to the
photostimulation targets (Fig. 5a). Then one can plot the distribu-
tion of responses across a range of radial eccentricities for neurons
that do and do not express the opsin (Fig. 5b, c). To visualize this
distribution, it is useful to compute a spatial heatmap of the average
response to photostimulation across neurons (Fig. 5b). To perform
this analysis, for each single-cell stimulation target, compute the
average response in a brief time window (e.g., 100 ms) following
photostimulation for all the neurons in the field of view expressing
the opsin. Using the spatial footprint of each cell body (outline),
create an image where each cell body spatial footprint is labeled
(colored) with the average response to a photostimulation target,
and repeat the procedure to produce images for all the photosti-
mulation targets. Finally, shift the x–y position of the images to
align the centroids of the photostimulation targets and average
across the images to produce a spatial heatmap of the response to
photostimulation (excluding space between neurons from the aver-
age). From this visualization, one can determine whether there is a
spatial bias of excitatory or inhibitory responses in the area sur-
rounding the targeted neurons. While an idealized version of the
result would be that excitatory responses disappear within one
neuron’s radius away from the photostimulation targets, in practice
this is unrealistic. For example, there may be a large degree of local
excitatory connectivity which falls off with eccentricity, making a
gradually decaying response profile fully expected. How then can
you disambiguate synaptically (network) driven responses in
 Illuminating Neural Computation Using Precision Optogenetics 379

A B C
* Opsin+
Opsin+ Response

Targeted
100 1 Opsin-
250

g
200
150 50 0.75
100

∆ F/F (norm)
50 0 0.05 0.50
Targ.
*

∆ F/F
-50 0.25
0 *

-100 0
-0.05
-100 -50 0 50 100 0 50 100 150 200 250
Distance from target neuron (μm) Distance from target neuron (μm)

Single-Cell photostimulation response predictions

Hypothesis 1: Off-target stimulation Hypothesis 2: Network effects

D dominates population response E dominate population response
Opsin+ Opsin+
Targeted

Targeted
1 Opsin- 1 Opsin-
g

g
0.75 0.75
∆ F/F (norm)

∆ F/F (norm)

0.50 Opsin+ response exceeds 0.50 Opsin+ response does not

Opsin- response exceed Opsin- response

0.25 0.25

0 0

0 50 100 150 200 250 0 50 100 150 200 250

Distance from target neuron (μm) Distance from target neuron (μm)

Fig. 5 Assessing specificity of photostimulation-evoked activity. (a) Example FOV centered on the mitral cell
layer of a Tbet-Cre mouse. One mitral cell is targeted for 2P photostimulation (small white circle, 15 μm
diameter). Red labeling corresponds to FLEX-ChrimsonR-tdTomato expression limited to a subset of mitral
cells, and green labeling corresponds to pan-neuronal GCaMP6s expression. Normalized fluorescence (ΔF/F)
is averaged for every neuron in the 100 ms period following photostimulation and averaged across neurons
occupying the same radial distance from the target (example bin size ¼ 50 μm). (b) A spatial heatmap of
average response vs. ROI position centered on 49 mitral cell targets (n ¼ 2 Tbet-cre mice, 138 total neurons,
3631 photostimulations, 3 pulses per photostimulation: 10 ms on – 10 ms off, at 30 mW, or 0.19 mW/μm2).
Only cells labeled with ChrimsonR-tdTomato were included. The “targeted” bin is outlined by a black circle. (c)
The average radial decay of responses across ChrimsonR-tdTomato positive neurons (Opsin+, red) and
ChrimsonR-tdTomato negative neurons (Opsin
, green). Cell responses radially binned and averaged for
each targeted neuron and bin means were averaged across targets (mean  s.e.m., 49 targeted neurons,
n ¼ 2 Tbet-cre mice). Asterisks indicate a significant difference between the average binned response of
ChrimsonR+ and ChrimsonR
neurons (*p < 0.05, two sample t-test, Holm-Bonferroni corrected for multiple
comparisons). (d) A cartoon of the hypothesis that photostimulation activates neighboring Opsin+ neurons due
to off-target photostimulation: Opsin+ responses will exceed the Opsin
responses, reflecting inadvertent
stimulation of nearby Opsin+ neurons. (e) A cartoon of the hypothesis that single-cell photostimulation leads
to responses of nearby neurons due to network effects: Opsin+ responses do not exceed the responses of
Opsin
neurons. (Adapted from Ref. [25])
380 Jonathan V. Gill et al.

neighboring neurons from “off-target” photostimulation (Fig. 5c–

e)? If opsin expression is sparser than expression of the functional
indicator, this means a subset of the neurons in a FOV will be opsin-
negative, providing an opportunity to compare their responses to
single-cell photostimulation with cells that are opsin-positive
(Fig. 5c). If the non-targeted opsin-positive neurons exceed the
response of the opsin-negative neurons, this is evidence that the
non-targeted opsin-positive neurons are directly activated by the
photostimulation laser (Hypothesis 1: “Off-target stimulation,”
Fig. 5d). If the non-targeted opsin-positive neurons do not
respond in excess of the opsin-negative neurons, this is compelling
evidence that network modulation can be explained by synaptic
effects alone (Hypothesis 2: “Network effects,” Fig. 5e, and the
experimental outcome in Fig. 5c). Confidence in this interpretation
may depend on similar populations of cells being labeled with the
opsin in a random and unbiased way or using a preparation which is
engineered to be sparsely labeled. However, even in densely labeled
samples this approach can be beneficial for identifying
non-responsive targeted neurons to exclude from multi-neuron
patterns and measuring the spatial scale of the influence of individ-
ual neurons in a larger, multiple-neuron manipulation.

Omit-One-Target When targeting many neurons simultaneously,

the probability of inadvertently activating untargeted neurons is
significantly increased. While somatically targeted opsins and axially
confined stimulation techniques like temporal focusing help to
decrease the incidence of inadvertent activation, it is beneficial to
directly test targeting specificity in this more complex regime. A
useful approach is to perform an “omit-one-target” experiment and
analysis (Fig. 6). After defining a set of neurons to target simulta-
neously, one can generate holograms systematically omitting each
spot from the full pattern (Fig. 6a). These holograms can be used
for photostimulation, with multiple repetitions randomly inter-
leaved into blocks of trials. Then it is possible to compare the
response in each neuron when it was targeted vs. when it was
omitted (Fig. 6b). While ideally the response of every
non-targeted neuron would fall to zero, this is unrealistic, especially
if targeting groups of neurons that may compose an interconnected
circuit. However, if target selection was biased toward highly
photostimulation responsive, or sensitive neurons, a general reduc-
tion in response magnitude for the omitted neurons can serve as
strong evidence for cellular specificity. Demonstrating that neurons
within this population can be individually controlled in the pres-
ence of simultaneous photostimulation of a large number of targets
validates that manipulations of neuron number, or specific neuron
identity, can be properly interpreted from behavioral experiments.
 Illuminating Neural Computation Using Precision Optogenetics 381

A All Targeted B Targeted Omitted

0.15

**
0.1

Response (∆F/F)
vs.
0.05
Omit One Target
Target 1 Target 2 Target n

, , ... , -0.05

Fig. 6 Characterizing response in omitted photostimulation targets. (a) Schematic of an omit-one-target

experiment. Top. Pattern of spots targeting many neurons simultaneously. Bottom. Omit one target condition
in which one of the spots is removed from the stimulation pattern. All spots are individually dropped, and the
holograms are randomly presented to the mouse along with the “all targeted” hologram. (b) Average response
to 10 ms photostimulation. The average response per cell when it was targeted vs. when it was omitted with
all other cells targeted. (**p < 0.001, two-sample t-test, targeted: 0.08  0.006, vs. omitted: 0.02  0.007,
mean ΔF/F  s.e.m, 28 targeted cells (19 responsive), n ¼ 2 WT mice, 30 photostimulations per datapoint,
10 ms duration, 20 mW/patch, 0.125 mW/μm2). (Adapted from Ref. [25])

3.2 Registration In addition to measuring the response characteristics of neurons

Between under study in an all-optical behavioral experiment, it is also essen-
Photostimulation and tial to characterize the physical position and scale of the stimulus
Imaging, and In Situ being applied. While extensive characterizations of the point spread
Evaluation of Targeting function (PSF) of the stimulus should be performed prior to
engaging in in vivo experiments, there are several procedures that
can be performed on a routine basis during behavioral experiments
to significantly increase confidence in the experimental outcomes.
These involve registering the photostimulation and imaging arms
of the microscope into alignment and methods for online motion
correction to correct for slow drift in the positions of targeted
neurons. Additionally, we highlight a potentially transformative
approach, holographic optogenetic confocally unraveled sculpting
microscopy (HOCUS), for in situ evaluation of stimulus shape and
position deep in the tissue of awake behaving animals [31].

Calibration It is critical to start by ensuring that the optics of the

2P photostimulation and imaging arms of the microscope have
been appropriately selected and aligned to permit imaging and
photostimulation of the same FOV. Still, small drifts in the align-
ment can occur over time. This might be due to temperature
fluctuations, mechanical stress, vibrations, changes in laser output
382 Jonathan V. Gill et al.

angle, among many other factors. Luckily, small translations, rota-

tions, and shearing of the photostimulation FOV relative to the
imaging FOV can be compensated for with a simple calibration
procedure. A calibration pattern can be burned onto a fluorescent
plate by the photostimulation system. Then the plate can be
imaged, and the calibration pattern can be used to register the
two arms of the microscope by computing the disparity between
the desired pattern and observed pattern.

Using a predefined calibration pattern made of several small

(~1 μm) targets, deliver 1–2 ms of photostimulation while increas-
ing the average power at the sample until small burn marks appear
on the fluorescent plate. The goal is to burn all of the spots in the
calibration pattern evenly at a small size. Then, capture an image of
the burned pattern and compute the inverse rotation matrix and
offset from the desired pattern. This rotation and offset can be
applied when generating holograms to exactly compensate for the
effects of small drifts in alignment between the photostimulation
and imaging arms.

Online Motion Correction For behavioral experiments that

require targeting the same neurons for photostimulation across
days, it is useful to employ an online motion correction method
(see Note 2). In the experiments described by Gill, Lerman et al.,
2020, the FOV was first aligned to a reference image manually, then
the position was fine-tuned automatically using a custom-designed
closed-loop algorithm, implemented as a module within Scan-
Image software [25, 55] (Vidrio Technologies). This algorithm
attempted to minimize the difference between the reference
image and the FOV by iteratively moving the microscope stage
(Sutter 285) to reduce the residual displacement computed using
a rigid motion correction package (NoRMCorre, Flatiron Institute
[56]). The optimization typically converged within 10–15 s once
the residual displacement vector was reduced to <0.5 μm in mag-
nitude. In addition to aligning the FOV across days, we performed
this routine between consecutive blocks (60 trials, 6–9 min) during
each behavioral session to minimize the effect of slow x–y drift due
to brain and microscope motion; therefore, ensuring the photo-
stimulation targets remained consistent throughout each session.
We monitored for drift in the z dimension as well, which was
manually corrected using the reference image between blocks
(60 trials, 6–9 min) if necessary, though displacement was typically
small (~3 μm in a 1.5 h session).

In Situ Evaluation of Holographic Patterns 2-photon precision

optogenetics experiments often rely on holographic wavefront
shaping techniques to generate light patterns targeted to individual
neurons deep in living tissue. Despite careful alignment between
 Illuminating Neural Computation Using Precision Optogenetics 383

the stimulation and imaging fields of view, light propagating

through the brain can undergo significant tissue-induced distor-
tions, mainly through scattering, that lead to a discrepancy between
the desired and actual light patterns reaching the neurons. These
effects can be unpredictable, as distortions will vary as a function of
specific tissue geometry and composition. Distortions are especially
detrimental to experiments combining cellular photostimulation
with behavior, as the shape and position of holographic patches
may be far from desired values, limiting the interpretability of
perceptual judgements.

In order to directly measure the effects of tissue-induced dis-

tortions on projected light patterns, Lerman et al. 2019 described a
new method, holographic optogenetics confocally unraveled
sculpting (HOCUS), for real-time, in situ evaluation of holo-
graphic light patterns (Fig. 7) [31]. This technique involves confo-
cally descanning reflections from light patterns focused into the
brain. Photons emitted from the sample, due to ballistic reflections
of light from the photostimulation laser source, are imaged with a
confocal detection system. This system can be added to most
microscopes combining 2P imaging and photostimulation, making
use of scanning optics typically used for 2P imaging in a reverse
direction to descan reflections from a static light pattern projected
into the tissue (Fig. 7a). Descanned light is focused by an electri-
cally tunable lens to a pinhole to confocally reject out-of-focus
light, permitting an evaluation of reflected light patterns at the
focal plane (Fig. 7a, b). By combining images collected by 2P
imaging and HOCUS (typically averaging frames collected over a
second), targeted neurons and holographic patches as they appear
in the brain can be viewed simultaneously for evaluation. This
technique is capable of measuring tissue-induced distortions
unique to a particular field of view and holographic pattern, and
enables real-time correction of holographic spot position by adjust-
ing the hologram to compensate for any difference from the desired
location. This technique could also be used, in principle, to opti-
mize the generated holograms to compensate for tissue-induced
distortions in the shape of the light patches. Ultimately, directly
viewing and correcting deviations between the desired light pattern
and actual light pattern being used to photostimulate neurons
could significantly improve the reproducibility of experiments com-
bining 2P holographic stimulation with behavior.

3.3 Assessing the While initial measurements of the responses evoked by photosti-
Reliability of mulation as well as calibrations to ensure proper targeting are
Behavioral Readout essential for experiments combining all-optical manipulations with
behavior, they are not the only controls necessary to be confident in
a behavioral result. As an additional precaution, it is extremely
useful to ensure that the observed behavioral effects depend only
384 Jonathan V. Gill et al.

Fig. 7 Visualizing light patterns in vivo with HOCUS. (a) The 2P imaging path is combined with the holographic
2P photostimulation path for in vivo experiments in head-fixed, behaving mice. The reflected stimulation light
passes through the PBS, is descanned by the mirrors, and is then reflected by the dichroic mirror through the
pinhole onto the detector. PBS polarizing beam splitter, PMT1, PMT2 photomultiplier tubes, SLM spatial light
modulator, DET detector. (b) A merged image of GCaMP6s expression (green) and the HOCUS-imaged
reflected stimulation light (red) showing a pattern of four light spots projected onto the brain, 120 μm deep
in the olfactory bulb, and positioned on four respective neurons. Scale bar: 25 μm. (Adapted from Ref. [31])

on manipulation of neural activity and not other features of the

stimulus. We provide two methods for handling this requirement in
the 1P pattern stimulation and 2P photostimulation regimes.

Stimulation Masking When designing a stimulus dependency

control in the 1P regime, a primary factor to consider is that
animals may be able to see the light being used for stimulation.
Even if light is delivered through an insulated optic fiber, prevent-
ing the animal from detecting the light externally, it is possible that
light delivered within the brain can still stimulate the retina.
Humans and mice are capable of visual detection at or near the
level of single photons, so even moderate light powers in highly
scattering media run the risk of inadvertent visual detection
through light traveling within the brain and ultimately to the retina
[38, 57]. This can seriously affect the outcome of an experiment in
which animals are asked to detect photostimulation of groups of
neurons, or discriminate between patterns of activation, as an ani-
mal could potentially learn to solve the tasks partially, or entirely,
using their vision. A common control for this is to perform the
same behavioral experiments on animals that lack a functional opsin
and demonstrate that they do not perform the task above chance
level. This is a useful control for short-term experiments; however,
this does not rule out the possibility of a shifting strategy in opsin-
positive animals over a longer time course. All-optical experiments
exploring the effects of a large photostimulation parameter space
 Illuminating Neural Computation Using Precision Optogenetics 385

on detection or discrimination often take weeks or months to

complete. While animals may initially base their choices on direct
neural activation, if visual cues exist, it is possible for them to later
switch strategies to rely, to some degree, on directly seeing the
stimulus. A solution for this is to use a set of “blanking LEDs”
matched to the wavelength of light used for stimulation. These can
be positioned near the eyes and triggered during behavioral trials. If
the blanking LEDs are significantly brighter than the stimulation,
this removes the possibility of using the stimulation light as a cue, as
the animals will see a strong light of the same wavelength regardless
of the stimulation condition.

Sham-Photostimulation Experiments involving 2P photostimula-

tion provide much less opportunity for inadvertent visual stimula-
tion, since the wavelengths involved are typically near-infrared and
thus invisible to rodents and primates. Still, 2P photostimulation
comes with its own caveats, as the light powers used tend to be a
great deal higher than those used for 1P photostimulation. This
introduces the possibility that animals could sense changes in light-
induced heating within the brain through its effects on neural
activity, or via tactile stimulation of surrounding tissue (see Note
3 and Ref. [58]). Demonstrating an inability of opsin-negative
animals to detect or discriminate photostimulation patterns or
when targeting opsin-negative neurons does not exclude the possi-
bility that opsin-positive animals engaged in extensive behavioral
training could learn to use heat as a cue.

An ideal control would be to provide a version of the stimulus

that reproduces all the features of the original photostimulation,
but does not evoke spiking in the targeted neurons. For this, we can
leverage the non-linear nature of 2P excitation. Since 2P excitation
relies on the peak power of laser pulses reaching the sample, increas-
ing the duration of the laser pulses while fixing the average power
can effectively reduce 2P excitation, and thus activation of the opsin
(Fig. 8a). Many lasers used for 2P photostimulation have an inter-
nal or external compressor for dispersion compensation, or to
maximize peak power of the output pulses. It is possible to change
the pulse width at the sample from a typical value of ~200 fs to
>15 ps by adjusting the laser’s compressor, provided it has the
appropriate range, all while keeping the same average power, or
the amount of light delivered to the tissue, constant. Therefore,
lengthening the pulses provides the opportunity for a “sham”
photostimulation control that can reproduce the heat and possible
indirect sensory effects present during a behavioral task, but is
capable of eliminating spiking induced by 2P photostimulation by
reducing 2P excitation by several orders of magnitude (Fig. 8b, c).
By interleaving blocks of trials identical to the typical behavioral
experiment, but using the sham photostimulation control, it is
386 Jonathan V. Gill et al.

1/f
A B

Control
Stim
Power
th

2P stim
Time W Heat
W


L Indirect
effects

C D
20 trials

Pre-Control Control Post-Control

Detection Accuracy
300 10 ms
2P Stim
Firing rate (spikes/sec)

Control

200 0.75

100
0.5

0
-100 0 100
Time (ms)

Fig. 8 Sham-photostimulation control. (a) A schematic demonstrating the effect of tuning the laser pulse
duration. Time dependence of laser power for pulse trains with the same pulse frequency ( f ) and average
power, but different pulse durations: short pulse, τS (red), and long pulse, τL (gray). To photostimulate a cell,
laser power must exceed a certain threshold, Pth. (b) Left, a table summarizing the differences in effects
evoked by the short and long pulse duration stimuli. Right, a schematic of the behavioral setup for the sham
photostimulation control experiment. (c) Representative example raster plots (top) and peristimulus time
histograms (PSTHs) (bottom) for short pulse photostimulation (~200 fs, red) and long pulse sham photo-
stimulation (control, 15 ps, gray) (20 trials per condition, 30 mW, 10 ms illumination, n ¼ 1 cell in 1 WT
mouse). (d) Detection accuracy as a function of photostimulation condition. During the sham-photostimulation
control blocks, detection accuracy dropped to chance level (0.5  0.003, mean  s.e.m., p ¼ 0.37,
one-sample t-test, 0.06–0.125 mW/μm2, n ¼ 5 mice, 2 WT (filled circles) and 3 Tbet-cre (empty circles)
and was significantly different from both pre- and post-control measurements ( p < 0.001, Fisher’s exact test,
0.06–0.125 mW/μm2, n ¼ 5 mice, 2 WT (filled circles) and 3 Tbet-cre (empty circles). (Adapted from Ref. [25])

possible to confirm to what degree the behavior relies on evoked

spiking, and not on other factors (Fig. 8d). This method could even
be used to calibrate the appropriate average power delivered during
the behavior, as the power could be increased during the sham
photostimulation condition until it is detectable, then reduced
until it is well below the detectable level for the rest of the
experiment.
 Illuminating Neural Computation Using Precision Optogenetics 387

4 Notes

1. Proper co-expression of the opsin and activity indicator is

critical for any study seeking to use precision optogenetics to
probe perception. In our experience, this is the primary factor
limiting the success of experiments. Many combinations of
opsin and calcium indicator simply do not co-express uniformly
following viral injection without some tweaking of the viral
titres, ratio between viruses, and total volume injected. Fur-
ther, serotype can play a role, leading to unpredictable tropism
or competition, where some cells express only the opsin and
others the indicator. In practice, this means it is advisable to
spend considerable time carefully testing the co-expression and
responsiveness of neurons following the injection of different
constructs in a range of titres, ratios, and volumes prior to
conducting behavioral experiments. Even when a suitable com-
bination is found, there can still be dramatic variability in
expression and the proportion of responsive neurons between
experimental animals. This means it is useful to inject and
implant more animals than are predicted to be necessary in
order to choose a cohort suitable for the study. The availability
of transgenic mice expressing either the calcium indicator,
opsin or both may greatly alleviate issues with expression in
the future; however, it is important to note that transgenic
expression is often substantially lower than virally mediated
expression, leading to a significant increase in the laser power
necessary for imaging and/or photostimulation. Constructs
enabling bicistronic expression of both an opsin and calcium
indicator using a single viral vector may also improve outcomes
and have already been used for probing perception [24],
though it is important to note that a 1:1 ratio in expression
may not always be ideal. For example, when an opsin is very
sensitive and a calcium indicator is relatively dim, the high laser
power necessary for imaging could introduce substantial cross-
talk between the imaging laser and opsin.
2. It is extremely important to compensate for the movement of
the brain to ensure that targeted neurons are properly illumi-
nated across each experimental session. The brain is non-rigid
and can experience both fast movements (e.g., artifacts of
licking or animal locomotion) and slow movements (swelling
and contracting on the order of minutes to hours). While trials
containing fast movements can be rejected after the experiment
has been conducted, slow movements can lead to a substantial
difference between the location of the holographic spots and
the targeted neurons that cannot be corrected post-hoc. For
example, one may generate a hologram at the start of a session
to target a group of neurons, only to find at the end of the
388 Jonathan V. Gill et al.

session that the holographic spots no longer align with the

locations of the targets since the brain has shifted relative to
the FOV of the microscope. This may lead to a corresponding
drop in behavioral performance across the session, and signifi-
cantly impact the interpretation of the results. Therefore, it is
best to re-align the microscope position with a reference image
every few minutes either manually or using an online motion
correction procedure (e.g., the method described in
Subheading 3.2).
3. Increasing the light power delivered to targeted neurons does
not always lead to an improvement in photostimulation efficacy
and may be detrimental at high values. The spiking response of
individual neurons will generally increase in rate and consis-
tency across repetitions when the average photostimulation
power is increased, until the response saturates at a range of
values that is particular to each neuron. The point of saturation
has been exceeded when increasing the average power does not
activate significantly more opsin proteins, or when the maxi-
mum firing rate of the cell has been reached. Further increasing
the photostimulation power beyond the minimal required
intensity has several disadvantages including (1) increasing
the likelihood of stimulating untargeted neurons, especially in
the axial dimension, and (2) causing excess tissue heating which
may lead to physiological and perceptual artifacts [58]. At the
limit, high photostimulation power may lead to cellular abla-
tion, and, in practice, the range of effective photostimulation
powers is often near this threshold (though the precise power
limit depends on many factors such as depth, vasculature,
wavelength, etc.). For these reasons, prior to conducting a
behavioral experiment, it is useful to measure the response of
individual neurons to a range of light powers in order to
estimate the minimal light power necessary for
photostimulation.

5 Outlook

The possibility of linking precise coding features to specific beha-

viors now permits questions previously limited to theory and spec-
ulation to be directly addressed. At the coarsest level, determining
the essential building blocks for perception through studies of
detection using both real and synthetic stimuli will define the
boundaries within which to explore the perceptual quality imparted
by specific neurons and patterns of activity. By manipulating sen-
sory circuits at a behaviorally and physiologically relevant spatio-
temporal scale, a relationship can be established between the
perceptual space and the feature space of neural activity. However,
the precision of the inferences that can be drawn about this
 Illuminating Neural Computation Using Precision Optogenetics 389

relationship is jointly determined by the resolution of both the

behavioral metrics and neural manipulations employed. Therefore,
we can expect advances in both capacities as the field continues to
mature.
It is inevitable that the opsins, optics, and technology support-
ing precision optogenetics will continue to improve, ushering in a
host of possibilities for bidirectional modulation of increasingly
large and diverse neural representations. Less apparent is how
behavioral methodologies will adapt to meet the nuance and com-
plexity permitted by these tools. As the dimensionality of the neural
feature space explorable by optical manipulations increases, binary
behavioral readouts (lick vs. no lick), and simple stimulus-reward
associations (stim 1 ¼ rewarded, stim 2 ¼ unrewarded) may be
insufficient, or, at best, inefficient for mapping activity features to
perception.
To meet these changing demands, our research groups, among
others, are exploring new paradigms, as well as adapting existing
methods traditionally overlooked for use in rodent behavior that
increase the information gained about perceptual quality from each
judgement made by an animal. Two examples include continuous
report, in which an animal can smoothly adjust the synthetic stim-
ulus to match or deviate from an internal template (potentially
derived from a real stimulus), or delayed-match-to-sample, in
which two stimuli are directly compared within each trial to deter-
mine if they are perceptually identical or different. These methods
have the advantage of allowing an animal to directly report the
meaningful combination of features composing a percept (contin-
uous report), and allowing many synthetic and natural stimuli to be
compared within one experimental session (delayed-match-to-sam-
ple). The challenge remains to optimize methods for compatibility
with head-fixed behavior and to accelerate training by exploring
more intuitive readouts of choice.
A related, and equally important problem involves determining
which features to manipulate (which neurons, timing, number of
spikes, etc.) to maximize information gained from finite length
experiments. As the number of neurons and features that can be
addressed in a single experiment increases, screening the perceptual
impact of all combinations of features will become prohibitively
time consuming. Therefore, it is important for experiments to be
guided by models implicating which neurons and combinations of
features are likely to have the greatest perceptual impact. One
recent method involves calculating the “intersection information”
of neurons, by using a statistical approach to identify activity fea-
tures carrying stimulus and choice information during a sensory
guided behavior, laying out predictions for how modulation of
features carrying intersection information will affect behavior
[59]. Another approach is to infer the functional connectivity of a
local circuit from the effects of focal stimulation of component
390 Jonathan V. Gill et al.

neurons [28]. Using the inferred structure, one could predict how
the effects of stimulation will propagate through the circuit, and the
stimulation could be designed to activate specific modes of activity,
testing their effect on perception. Ultimately, the emergence of
newly informative behavioral paradigms along with novel concep-
tual frameworks will likely be some of the most exciting outcomes
to come from the application of precision optogenetics and syn-
thetic perception.

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 Chapter 13

Spectrally Focused Stimulated Raman Scattering (sf-SRS)

Microscopy for Label-Free Investigations of Molecular
Mechanisms in Living Organisms
Tamás Váczi, László Himics, Matteo Bruzzone, Miklós Veres,
and Marco dal Maschio

Abstract
Stimulated Raman Scattering (SRS) microscopy is a light-based non-linear imaging method for visualizing a
molecule based on its chemical properties, i.e., the vibrational energy states reflecting the molecule’s
structure and its environment. This technique, relying on the specificity of the molecule’s spectral finger-
print, enables label-free, high-sensitivity, and high-resolution 3D reconstruction of the distribution and the
properties of a molecule within a tissue. Despite its tremendous potentials, the application of SRS is still not
frequent in the field of life science, where it could be applied over an extremely broad investigation range,
from the study of the molecular interactions at subcellular level to the characterization of tissue alterations
in clinical studies. Trying to fill this gap, here, after describing the general principles of SRS, we present the
materials and the methods to integrate spectrally focused Stimulated Raman Spectroscopy (sf-SRS) on
commercial multiphoton microscopes and highlight the critical aspects to consider.

Key words Label-free imaging, Stimulated Raman Spectroscopy, Spectral focusing

1 Introduction

Optical microscopy is currently a fundamental technique in bio-

medical research. The combination of high resolution and low
invasiveness, along with tissue- and cell-labelling methods, allows
the reconstruction of anatomical or functional images with high
contrast and temporal resolution compatible with a large range of
biological processes. Most frequently, the mechanisms leading to
the contrast are taking advantage from the interaction of excitation
light with molecules that hold or are conjugated with fluorescent
moieties (label-based) and result in signals with high signal-to-
noise ratio levels [1].
In parallel with label-based optical imaging methods, there are
techniques, relying on different kinds of light-matter interactions,

Eirini Papagiakoumou (ed.), All-Optical Methods to Study Neuronal Function, Neuromethods, vol. 191,
https://doi.org/10.1007/978-1-0716-2764-8_13, © The Author(s) 2023

393
394 Tamás Váczi et al.

that can reveal the distribution and the properties of a molecule

without the need of a specific labelling (label-free) [2]. Within this
last group, Raman spectroscopy [3–5] represents a powerful tool
for label-free characterization of a sample. In this technique one can
reconstruct the molecule distribution and properties based on the
features of its vibrational states as revealed by the inelastic scattering
mechanisms of the excitation light by the sample [6]. In fact,
following the perturbation imposed by the interaction of the pho-
ton with the molecule, the system reaches a so-called intermediate
state with the molecule in a virtual energy state, from which the
relaxation involves excitation of characteristic molecular vibrations.
As a consequence, the scattered photon emitted during the relaxa-
tion will have energy different from the incident one. The energy
gap characterizing the relaxation transitions toward ground-state
(being equal to the energy of the excited vibrational transition) is
characteristic of the chemical bonds of the molecule and of their
environment. By accumulating these relaxation events to gather
extended statistics, it becomes thus possible to retrieve a Raman
spectrum, i.e., a distribution of the transition probability, returned
by the signal intensity, as the function of the energy difference
associated with the vibrational states, expressed in terms of the
wavenumber or the corresponding Raman shift. Since such type
of spontaneous inelastic scattering emission represents a minimal
component of the total (elastic and inelastic) scattering events and
is statistically not frequent, with typically one Raman photon emit-
ted over 107 incident photons, the detection of these events
becomes possible only with rather long integration times. As alter-
native, the Raman spectrum can be explored adopting a pump-
probe approach [7]. In this scheme of the Raman process, called
stimulated Raman scattering (SRS) [8, 9], the probability of vibra-
tional transitions toward the ground level is enhanced strongly, by
up to a few orders of magnitude, with respect to spontaneous
Raman scattering, taking advantage from a non-linear excitation
process of stimulated emission.

1.1 Stimulated Stimulated Raman scattering (SRS) microscopy represents a label-

Raman Spectroscopy free technique for imaging with high chemical specificity and high
(SRS) acquisition speeds up to video rate [10]. In SRS microscopy, two
pulsed laser beams are temporally and spatially overlapped to coher-
ently excite the sample: a pump beam with the frequency of ωp and
a Stokes beam with the frequency of ωS (Fig. 1a). In the condition
where the frequency Δω ¼ ωp  ωS matches a particular Raman-
active molecular vibration energy level of the sample, the system
enters a resonance regime where SRS signals are generated. These
can be detected either in the form of the decrease of the pump beam
intensity called Stimulated Raman loss (SRL), corresponding to the
annihilation process of the pump photon, or in the increase of the
Stokes beam intensity, known as Stimulated Raman gain (SRG),
 Stimulated Raman Spectroscopy to Unravel Molecular Mechanism in Living Organisms 395

A B
Energy diagram Input Field Intensity Output Field Intensity
Virtual state
Stokes Pump Stokes Pump

Spontaneous
Stokes beam
Pump beam
ΔIs SRG

Raman
ΔIp SRL
ω
Δω Δω

Vibraonal state
Δω
Ground state ωs ωp
ω
ωs ωp
ω

C D E F

Pump ωp
Frequency

δω Stokes ωs
ω0 δω

Time

Δω

Fig. 1 (a) The SRS Jablonsky diagrams showing the energy states and the transitions associated with
Spontaneous Raman and Stimulated Raman signals with the y-axis indicating the energetic level as
represented in terms of the radiation frequency. (b) Changes of the laser intensities during Stimulated
Raman Spectroscopy in the case of Stimulated Raman Gain (SRG) and Stimulated Raman Loss (SRL). (c)
Frequency-time properties of ultrashort Stokes and pump pulses with chirping parameter β ¼ 0 and the
corresponding spectral resolution below. (d) Dispersion of the spectral component in chirped pulse with β 6¼ 0
with matched chirping condition and the corresponding spectral resolution below. (e) Dispersion of the
spectral component in chirped pulse with β > 0 with un-matched chirping condition and the corresponding
spectral resolution below. (f) Dispersion of the spectral component in chirped pulse with β > 0 with matched
chirping condition with different temporal shift and the corresponding spectral window. Time axis corresponds
to the time of arrival of the spectral components at the same point in the space

corresponding to the creation of a photon with ωS frequency

(Fig. 1b). While in the favourable conditions, the existence of a
resonance reveals the presence of a molecule with a vibrational
transition corresponding to the set energy difference Δω, when
the energy difference Δω does not match any Raman active vibra-
tional transition, such resonance does not occur and no SRS signal
is detected. In this way, scanning over the energy interval by
396 Tamás Váczi et al.

changing Δω allows reconstructing the Raman spectrum contained

in the excitation volume. Importantly, with respect to spontaneous
Raman signals, as SRS is a non-linear absorption process based on
the simultaneous interaction of two beams, with frequency ωp and
ωS, the illumination of the sample is capable of revealing the vibra-
tional configuration of the molecules located in a rather small
volume corresponding to the region with high spatial and temporal
density of photons. This property, ensuring a high spatial resolu-
tion, renders SRS a technique suitable for its integration in scan-
ning microscopes designed for the three-dimensional
characterization of the sample properties.

1.2 Spectral A molecule, in general, is characterized by a certain number of

Resolution and the vibrational transitions and properly identifying the molecule of
Role of the Pulse interest through its characteristic vibrational levels requires the
Duration, Chirping and scanning of the energy range across the whole Raman spectrum
Delay in SRS with sufficient spectral resolution. In this regard, one has to con-
sider that the limit in the spectral resolution is related to the spectral
bandwidth of the radiation, δω, and that, in the case of pulsed
sources, it does exist a fundamental relationship between the spec-
tral bandwidth δω and the duration of the pulse, δτ [11]. This
relation is known as time-bandwidth product and dictates that the
product δω·δτ is constant with a value depending on the shape of
the source power spectrum. Under this constrain, given a certain
δω, the shortest pulse duration δτ corresponds to a pulse where all
the spectral components within δω have the same phase. Combin-
ing beams of such ultrashort pulses—with typical pulse duration in
80–180 fs—in the pump-probe scheme of the SRS, results in a
scenario where the range of accessible transitions is large as it
encompasses all the possible energy levels (Fig. 1c). However,
even if the original pulses have the features described above, it is
still possible within the constraint of the time-bandwidth relation,
to reduce the effective δω amplitude and so increase the SRS system
spectral resolution. This approach takes advantage of a technique
called pulse chirping, where a phase delay is introduced between the
different spectral components within δω resulting in a time depen-
dent evolution of the frequency ω(t) ¼ ω0 – 2βt governed by the
chirp parameter β (Fig. 1d) that describes the temporal evolution
and dispersion of the spectral components within the pulse and
where ω0 is the carrier frequency of the envelope [11]. The
obtained temporal dispersion of the spectral components included
in the original δω, along with extending the pulse duration in the ps
range, has the effect in reducing the accessible effective δω band
[12]. If pulse chirping is applied to the pump and the Stokes beams,
the spectral resolution of the SRS acquisition is expected to
improve up to <10 cm1 and to be able to pick transitions with a
narrower bandwidth, as far as the two beams have pulses temporally
overlapping and with a chirp condition properly matched (Fig. 1d).
 Stimulated Raman Spectroscopy to Unravel Molecular Mechanism in Living Organisms 397

Matching the chirp condition basically means that the temporal

evolution of the pulse from the two beams are characterized by
the same chirp parameter β. It is important to keep in mind that
when the condition is matched, the energy difference Δω ¼ ωp  ωS
between the pump and Stokes beam is the same across the region of
the overlap between the pulses. When the chirp condition is not
satisfied, it implies that the effective sensitivity is not optimized, the
δω is not at the minimum, and that also Δω covers a relatively large
range (Fig. 1e). As mentioned above, along with the chirp condi-
tion, also the degree of temporal overlap between the pump and
Stokes pulses holds an important role, not regarding the spectral
sensitivity, but rather for its effect on the range of the spectrum one
can explore. In fact, introducing a temporal delay of one pulse with
respect to the other results in the selection of different subregions
of the chirped pulsed that are temporally overlapping. This means
that it is possible to identify a relation between the relative delay of
the pulses and the corresponding spectral region one can examine
(Fig. 1f). This method of modulating the interpulse delay repre-
sents a fundamental mechanism for scanning over the spectrum
by avoiding the retuning of the beam frequency, which typically
requires a significantly longer time and possibly a fine
re-adjustment of the optical system.

1.3 Spectrally Implementing a system for SRS with spectral focusing capabilities
Focused SRS: The within a microscope for 3D beam scanning typically requires the
General Hardware integration of a set of add-on systems: (1) the optical pathway for
Layout the routing, the temporal control, the high-frequency modulation,
and the conditioning of the pump and the Stokes beams; (2) the
implementation of a dedicated signal detection apparatus; (3) the
arrangement of a signal processing chain based on a lock-in ampli-
fier to return the SRS signal intensity corresponding to the illumi-
nated spot (“pixel”). While these aspects will be described in detail
in the Methods session, here we present the basic principles guiding
the sf-SRS implementation (Fig. 2). SRS relies on a pump-probe
scheme based on the spatial and temporal superposition of the
pulses of two beams characterized by different frequencies, i.e.,
ωp and ωS. As for the spatial overlap, this requires the use of ultrafast
routing mirrors, to ensure the XY (directions transversal with
respect to the light propagation direction) collinear overlap of the
two beams at the sample, and the adoption of beam expanders
(BE), to properly match the diameters and the degree of diver-
gence/convergence of the beams in order to have a good overlap of
the focal spots along the Z direction (longitudinal direction with
respect to the light propagation direction). Along with spatial
overlap of the beams, SRS requires in general a precise control of
the temporal overlap between the pulses of the two beams. It is
then fundamental that the pulses for pump and for Stokes beams
are emitted with a fixed phase delay one from the other. This relies
398 Tamás Váczi et al.

time
Pump beam
time Stokes beam (probe)
BE BDC Stokes beam (balanced detecon)

XY scanner
DM

Delay line
Scan lens

Tube lens
BE AOM BE
time
Main dichroic

PMT

PSA PSA 40x Objecve

Sample

I I
Microscope
DAQ
1.4 NA Condenser

GDD PD Filter
time

PBS

PD

time
Lock-in Differenal
Stokes beam Pump beam
amplifier amplifier

Fig. 2 The hardware layout for the integration of a sf-SRS in a laser scanning microscope. The main
components include: the time-locked pulsed laser sources (pump and Stokes beam), a Group Delay Dispersion
(GDD) system, a laser Intensity control (I) unit, a Pulse Stretching Apparatus (PSA), a set of Beam Expanders
(BE), an Acousto-Optic Modulator (AOM), a delay line, a Balanced Detection Component (BDC), a Dichroic
Mirror (DM), a Photomultiplier Tube (PMT), a Polarization-sensitive Beam Splitter (PBS), a pair of Photodiodes
(PD), a differential amplifier and a Lock-in amplifier. On the upper part, the temporal features of the Stokes
beam pulse and of the pump beams, presenting an orthogonal polarization and a temporal delay in the pulse
replica with respect to the Stokes pulse interacting with pump beam, are presented

on the use of two light sources or a single source with dual output
channels, whose pulse emissions are synchronized. Having syn-
chronized pulses does not imply necessarily that the pulses are
overlapping at the sample and, in general, it is not granted that
the amount of delay between the pulses remains the same when
varying one of the emission frequencies. Considering these aspects
requires a careful design of the optical paths (Fig. 2): first optimiz-
ing the difference in the distance travelled by the two beams in
order to minimize the range of possible delays; second, integrating
a delay line in one of the beam paths for finely tuning the residual
delay excursion range and getting the pulses overlapping at all the
selected frequencies. Along each of the two beam lines, it is practi-
cal to have a system for attenuating the laser intensity (I) and keep
the power for the two beams under control. As discussed in the
previous section, in the case of the sf-SRS the proper tuning of the
Stimulated Raman Spectroscopy to Unravel Molecular Mechanism in Living Organisms 399

pulse temporal properties becomes extremely important. With

respect to SRS using ultrashort pulses, properly tuning the delay
line in sf-SRS not only affects the temporal overlap but also has an
impact on the selection of the proper spectral window when using
chirped pulses (Fig. 1f). The temporal constrains required for the
chirped pulse sf-SRS and the fact that the original pulses from the
two sources could potentially not present the same exact temporal
properties impose a design of the optical path, ensuring a precise
control of the chirp parameter on both beams. Assuming that the
sources generate ultrashort pulses, stretching the pulse to obtain a
sufficient degree of positive chirping is typically achieved by inte-
grating optical elements with dispersive properties (pulse stretching
apparatus, PSA; see Materials section), so that the spectral compo-
nents travel within such materials at different speeds and acquire a
different delay as a function of frequency. As the properties of the
pulse can vary with excitation frequency, it is then mandatory to
insert a component in one of the beams for the fine-tuning of the
chirp parameter β by controlling the group delay dispersion
(GDD). This fine-tuning of the pulse dispersion can be achieved
with an external device (pre-chirper) or by taking the advantage of
the built-in GDD-control unit that is available in certain sources.
Regarding the configuration of the signal detection, one has to
consider that, with respect to other techniques where excitation
and detection channels are spectrally separated, the SRS signal is
represented by a subtle variation in the intensity of one of the
excitation beams (SRL or SRG). This implies that the detection
chain should be able to discriminate such intensity variations asso-
ciated with the Raman process with respect to an average level over
the typical signal fluctuations intrinsic to the sources, even in cases
when system noise is substantially larger. Under these circum-
stances, the limiting factor in the detection is not the sensitivity of
the detector but rather the capability to extract the valid signal from
a very large background. This is achieved by combining a photodi-
ode (PD) as a detector with the detection optics and feeding its
output through a signal amplification chain using a lock-in ampli-
fier. This type of amplifiers implements a phase-sensitive detection
[13], which is capable of amplifying massively the signal at a specific
frequency, suppressing the contribution of the other frequency
components and extracting in this way the contribution due to
Raman interaction with the sample. For this purpose, the lock-in
detection approach requires the insertion of a high-frequency
intensity modulation device (typically an acousto-optic modulator,
AOM), capable of modulating the intensity of one of the excitation
beams at the designed driving frequency. The signal acquired by the
PD, independently by the configuration adopted, will be carrying
the same high-frequency modulation and this will allow for the
detection of subtle changes in the intensity associated with a Raman
400 Tamás Váczi et al.

process. The output of the lock-in amplifier is ultimately sampled

by the acquisition system of the microscope and is synchronized
with the scanning system to form an SRS image.

1.4 Application sf-SRS is a label-free imaging technique that requires a dedicated

Perspectives type of hardware and software to control the acquisition. Moreover,
the optimal tuning of the many critical parameters could be very
time consuming if the system does not envisage some sort of
automatic regulation and re-calibration of the components. It is
then not surprising that the fields of application of this technique
are typically falling within physics or chemistry labs, with applica-
tions that, in most of the cases, deal with chemical or material
sciences. Out of this scenario, the number of the questions that
such techniques could help answering is relevant, especially in the
field of life sciences. This technique can be applied to a very broad
range of investigations, from the mapping of the molecular
mechanisms inside a cell to the characterization of tissues in living
organisms, along with the alterations associated to different patho-
logical conditions [14]. The use of this technique has been
reported for mapping the neuronal activity in a label-free manner
[15, 16] and in its integration with approaches of micro-endoscopy
[17, 18]. Research in human biopsies or human-derived tissues is
becoming more frequent and taking advantage of techniques, like
SRS, for characterizing structural and molecular properties. More-
over, the identification of strategies for having a label-based Raman
signal is opening the scenario for multichannel analysis [19].

2 Materials

Here we report a list of components to assemble a sf-SRS system

and integrate it into a commercial scanning microscope. All com-
ponents not labelled otherwise are from Thorlabs (Newton, New
Jersey, USA).

General Optomechanical Assembly Post holders (PH75E/M,

UPH30/M); posts (TR50/M, TR20/M); clamping forks
(CF038, CF125); kinematic mirror mounts (KM100 or KS1,
KM100DL/M); kinematic mounts for rectangular optics
(KM100S, KM100SL); kinematic platform mounts (KM100B/
M, KM200B/M); kinematic prism mounts (KM100PM/M);
clamps (PM4/M); 30 mm cage cube (C4W) with blank cover
plate (B1C/M), kinematic rotating platform (B4CRP/M) and
dichroic filter holder (FFM1); rotation mount (CRM1); iris dia-
phragms (SM1D12D, ID8, ID15); cage plates (CP33/M,
CP33T/M, CP4S); kinematic, cage-compatible mounts (KC1-T/
M); cage rods (ER1, ER1.5); zoom housings (SM1ZM); thread
adapters (SM1A6, SM1A6T); rotation mounts (DLM1/M,
Stimulated Raman Spectroscopy to Unravel Molecular Mechanism in Living Organisms 401

RSP1D/M), SM2 lens tube coupler (SM2T2); thread adapter

(SM1A2); lens tubes (SML05, SML10, SML15); SM1 lens tube
couplers (SM1T1); SM1 flexure sleeve lens tube couplers
(SM1CPL10); Ø1.5“ mounting post (P150/M); table clamp
(PF175B); Ø1.5” post mounting clamp (C1511/M); compact
5-axis stage (PY005).

Mirrors Low-GDD ultrafast mirrors (UM10-45B in the Stokes

arm, UM10-AG for the pump arm and the common beam path);
ultrafast retroreflecting (hollow roof) mirrors (HRS1015-AG);
knife-edge right-angle prism silver mirrors (MRAK25-P01);
D-shaped silver mirror (PFD10–03-P01); for combining the
pump and the Stokes beams (DM), a 25.2  35.6 mm dichroic
long-pass filter, 1000 nm cut-on (#87–046, Edmund Optics, York,
UK).

Divergence Correction/Focusing Elements Achromatic NIR

lenses (ACN127–020-B, AC254–040-B); variable beam expander
(BE052-B).

Balanced Detection Reference Arm Mounted achromatic half-

wave plates (AHWP05M-980 or AHWP10M-980); mounted
polarization-sensitive beamsplitter cubes (CCM1-PBS255/M).

Balanced Detection Substage Assembly Olympus 1.4 NA oil con-

denser (U-AAC); premium long-pass filters (FELH1000);
mounted polarization-sensitive beamsplitter cube (CCM1-
PBS255/M); achromatic NIR lenses (AC254–035-B); mounted
photodiode detector (SM05PD1A) with bias module (PBM42).

Delay Stage Direct-drive linear translation stage (DDSM100/M)

with programmable controller (KBD101).

Dispersive Glass High-dispersion glass blocks (S-TIH53,

OHARA, Hofheim, Germany), polished on both ends, 1 pc
36  36  200 mm, 1 pc 15  25  75 mm.

Acousto-Optic Modulator MT110-B50A1,5-IR-Hk AOM cell +

MPDS1C RF driver (AA OPTO-ELECTRONIC, Orsay, France).

Sources Chameleon Discovery source with dual output (one fixed

at nominal 1040 nm, one tunable between 680 and 1300 nm),
80 MHz repetition rate, 100–140 fs pulse length, ca. 4 W (Stokes/
probe) and 1.6–1.8 W (pump) power, built-in GDD precompensa-
tion (Coherent, Santa Clara, CA).
402 Tamás Váczi et al.

Microscope FEMTOSmart research-grade two-photon

(2P) galvanometer scanning microscope with one- or
two-channel 2P-epi detection (PMT) (Femtonics, Budapest, Hun-
gary) and custom-made, transmission SRS detection; Nikon NIR
APO 40/0.80 W objective.

Auxiliary Equipment CARPE autocorrelator with external

detector (APE, Berlin, Germany); SPD2122X arbitrary waveform
generator (Siglent, Solon, OH); SR865A lock-in amplifier (Stan-
ford Research Systems, Sunnyvale, CA); laboratory power supply,
24 V (AOM driver) and 32 V (photodiode bias) outputs. MES
control software (Femtonics, Budapest, Hungary) based on
MATLAB (MathWorks, Natick, MA).

3 Methods

3.1 Main Figure 3 shows the schematic of an SRS microscope with single-
Components for channel lock-in detection. It consists of the tunable wavelength
Integrating sf-SRS in a pump (TUN) and fixed wavelength Stokes (FIX) laser beams that,
Multiphoton in case of femtosecond laser sources, are guided along their light
Microscope paths with ultrafast-enhanced mirrors. These optical elements are
designed so that they introduce minimal distortions to the wave-
fronts of the ultrashort laser pulses during their reflection. Mirrors
are used to realize the criteria of SRS excitation described above,
namely to create the extra light path for one of the beams for the
coarse compensation of the relative delay of the pulses, to couple
the beam(s) in and out of the delay line, acousto-optical modulator

Fig. 3 Schematic layout of an SRS microscope system. FIX – fixed wavelength Stokes light source, TUN – tune-
able wavelength pump light source, M – mirrors, PD – photodiode, AUX in – signal input of the microscope for
imaging, AOM – acousto-optical modulator
Stimulated Raman Spectroscopy to Unravel Molecular Mechanism in Living Organisms 403

and other components, as well as to adjust their position and angle

in order to obtain collinearly and concentrically aligned combined
beams delivered to the microscope. Before entering the micro-
scope, the pump and Stokes beams are combined with a dichroic
mirror (optical element with transparent or reflective properties in
certain wavelength regions) reflecting one beam (the pump on
Fig. 3) and transmitting the other (the Stokes on Fig. 3). For the
precise alignment of the two beams, two steering mirrors are
inserted into the light path of the reflected beam before the
dichroic mirror (not shown here). In general, including a pair of
steering mirrors before the main components of the SRS and any
other optical system (delay line, AOM, dichroic mirror, micro-
scope) simplifies the alignment and fine tuning of the beams.
The temporal synchronization of the pulses and the compensa-
tion of its variation with the emission frequency and GDD setting
of the light source is performed by the delay line. Since the delay
between the pulses of the pump and Stokes beams depends on the
wavelength, the delay line is usually realized by micrometer resolu-
tion motorized linear stages. The optical components of this unit
could be mirrors or the combination of a knife-edge and a hollow
roof mirror. In order to avoid the lateral drifting of the beam, the
optical paths to and from the moving mirrors are aligned to be
parallel to the movement of the delay stage.
In the microscope the collinear, divergence-corrected and tem-
porally synchronized pump and Stokes beams are delivered and
moved in lateral direction (scanned) at the sample with the same
optical components (galvo scanners, scan lens, tube lens, and
microscope objective) used for multiphoton imaging. The detec-
tion of the SRS signal is more conveniently performed in the
forward direction (transmission geometry). The light from the
sample is collected with a condenser or an objective lens of high
numerical aperture (NA) (as a rule of thumb, the NA of the light
collection optics has to be equal or larger than that of the excitation
side) and directed to the detector. Since a high laser intensity is
detected during an SRS measurement, in most cases a biased PD is
used to record the signal. An optical filter is placed in front of the
PD allowing only one of the beams to reach the detector surface.
Depending on the configuration, the intensity change in the pump
(SRL detection) or Stokes (SRG detection) beam is recorded.
As mentioned in the previous session, in most cases SRS sys-
tems utilize lock-in detection. Therefore, the intensity of the pump
(for SRG detection, when the Stokes beam is detected) or the
Stokes (for SRL detection, when the pump beam is detected)
beam must be modulated with a driving signal of a given frequency.
This can be realized by using a chopper, an acousto-optic (AOM)
or electro-optic modulator (EOM). For high-speed imaging, the
frequency of the reference signal could be modulated at a few MHz,
404 Tamás Váczi et al.

to operate far from the 1/frequency noise region of the source and
evaluate the SRS signal associated with the pixel over a sufficient
number of modulation cycles.
An SRS measurement starts with the adjustment of the wave-
length of the tunable light source, followed by setting the
corresponding delay line position and turning on the intensity
modulation. Then the intensities of the pump and Stokes beams
have to be adjusted. As a rule of thumb, the intensity of the
modulated beam has to be twice the other, and, in general, the
higher the laser intensity, the stronger the SRS effect. However, in
practice the laser-induced damage of the sample and the appearance
of different artefacts (Kerr effect, cross-phase modulation, etc.)
limit the useful laser intensity levels.

3.2 Methods for The net GDD (chirp) of the pulses comes from three main sources:
Spectral Tuning of the the chirp introduced within the laser, the optical setup including
System and Its the SRS system and the microscope, and the additional element
Optimization: Pulse (s) used to achieve pulse stretching. The contribution of these is
Chirp and Delay difficult to calculate or estimate precisely, therefore, the experimen-
Control tal measurement of the spectral and temporal widths of the laser
beams passing through the SRS system, and the microscope has to
be performed with a spectrometer and an autocorrelator, respec-
tively. The results can be used to determine the amount of chirp to
be introduced into the pump and Stokes beams to achieve the chirp
matching condition.
While the chirp of the emitted pulses cannot be adjusted
directly for some femtosecond lasers, other systems include the
option of compensating the group delay dispersion introduced by
the optical elements. This is a convenient tool to fine-tune the net
GDD and to establish precise chirp matching in spectrally focused
SRS measurements under a variety of laser wavelengths, i.e., Raman
sampling ranges.
The chirp related to the optical components of the SRS micro-
scope is an intrinsic property of the system that has to be taken into
account when adjusting the chirp matching conditions. The main
source is represented by transmissive optical elements—divergence
correctors, filters, beam splitters, objective lens, etc.
The third, and most substantial, component is the pulse
stretching device. In practice, the temporal redistribution of
the spectral components within the pulse can be achieved by
using combinations of gratings, prisms, or multilayered mirrors.
Another approach is to use a dispersive medium, in which the
components of the laser pulse with different wavelengths will travel
with slightly different velocity. The velocity difference per unit
length of the dispersive medium is given as the Group Velocity
Dispersion (GVD), and GDD ¼ GVD∙length of the medium. As
a consequence, a chirp will be introduced into the pulse, the extent
 Stimulated Raman Spectroscopy to Unravel Molecular Mechanism in Living Organisms 405

Table 1
Group velocity dispersion of the Ohara S-TIH53 high density flint glass at different wavelengths [20]

Wavelength, nm 750 800 850 900 950 1000 1050

2
GVD, fs /mm 242.86 219.49 199.89 183.06 168.31 155.13 145.47

of which can be adjusted by the length of the dispersive medium.

With a proper medium of high GVD (for example flint glass), this
solution can be very effective and cost-efficient.
In general, the GVD of dispersive media decreases rapidly with
increasing wavelength and can be very low for the near-infrared
region characteristic for many femtosecond lasers. Therefore, a
longer medium has to be inserted into the beam of higher wave-
length to achieve the same amount of chirp. High-density flint glass
could be a material of choice for pulse chirping providing accept-
able GVD values in a broad spectral range (see Table 1). After
establishing the amounts of the chirp to be introduced into the
pump and Stokes beams, the glass can be cut to the required length
and the cut faces can be polished flat and coated with an anti-
reflective coating in order to minimize the losses and wavefront
distortions of the beam.
Once the chirp introduced by the laser and the optical setup are
known, it is possible to calculate the amount of chirp to be applied
to the two beams for the chirp matching along with the achievable
spectral resolution Δωcc depending on the material length [21–23],
according to the following formulas:
sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
 2
4 ln 2∙GDD
τ ¼ τ0 1 þ ð1Þ
τ20
" 2 #1=2
 2 
Δω 2 ln 2
β¼  ð2Þ
τ πcτ2
" #1=2
ð2 ln 2Þ2 τ2S þ τ2P τ2P τ2S ðβP  βS Þ2
Δωcc ¼ þ ð3Þ
π 2 c 2 τ2P τ2S τ2S þ τ2P

where τ0 is the pulse duration in the case of a transform-limited

Gaussian pulse, c is the speed of the light in vacuum, and τP and τS
the relative length of the Pump and Stokes pulses, respectively.
An important point here is that due to its non-unity refractive
index the optical medium inserted into the beams will affect the
relative delay of the pulses. For the compensation of this, the
additional optical path will have to be calculated by multiplying
the length of the medium with its refractive index (the refractive
index indicates how slower the light will travel in a given medium,
so how much the pulse will be delayed compared to the vacuum).
406 Tamás Váczi et al.

Fig. 4 Chirp matching as a function of glass length (green line; Lp: glass length in pump beam, with additional
300 mm glass in Stokes beam). At the value of βP/βS ¼ 1, the difference between the instantaneous
frequencies of the temporally overlapping pulses is constant. The violet line shows the calculated best
spectral resolution without distortion effects

An example calculation of chirp matching condition for the

Coherent Chameleon Discovery laser with Ohara S-TIH53 high
density flint glass is shown in Fig. 4. The green line represents the
change of the ratio of the βp pump and the βS Stokes chirp para-
meters with the length Lp of the glass the beams travel through.
The chirp matching condition (when the βp/βS ratio of chirp para-
meters of the pump and Stokes beams is 1) is shown around
500 mm glass base length. One can also calculate the spectral
resolution, equivalent to the cross-correlation bandwidth (Δωcc),
which has its minimum close to the chirp matching condition
(purple curve in Fig. 4).
The layout of the optimized spectral focusing unit is shown in
Fig. 5. For the compactness of the system, one 38  38  200 mm
glass rod is used for both the Stokes and pump beams, polished on
both 38  38 mm ends. Combinations of knife edge and hollow
roof mirrors are used to direct the two beams into the glass rod and
make them pass through several times, and then return into the
original beam path of the SRS system. Two hollow roof mirrors are
used for the Stokes beam (length: 800 mm): after the first pass a
 Stimulated Raman Spectroscopy to Unravel Molecular Mechanism in Living Organisms 407

Fig. 5 Schematic layout of the SRS spectral focusing unit

horizontally oriented mirror was used to reflect the beam above the
height of the first pass. At the end of the second pass another, now
vertically oriented hollow roof mirror is used to reflect it in the
height of the second pass, after which the first mirror reflects it onto
the knife edge mirror in the height of the first pass. So, the beam is
passing the 200 mm long glass four times. The tunable beam passes
the 200 mm glass rod only twice. Here a shorter rod of 75 mm
length is also used to achieve the required 550 mm
(2  200 mm + 2  75 mm) of glass length. The distances between
the knife edge and the hollow roof mirrors are adjusted to approxi-
mately maintain the temporal synchronization of the Stokes and
pump pulses.
The knife edge mirrors couple in and out the laser beams to the
spectral focusing unit. If needed, e.g., for femtosecond two-photon
measurements, these mirrors can be mounted on magnetic, detach-
able mounts so the system can be used in both femtosecond and
spectral focusing modes.
A spectral focusing SRS can be used to record vibrational
spectra of the samples. As it was detailed in the introduction, by
tuning the relative delay between the spectral focused pulses, they
will excite different vibrational transitions without tuning the wave-
length of the laser. By recording a calibration Raman spectrum on a
known, suitable sample, the delay line positions can be converted
into Raman shift values after appropriate curve fitting. Figure 6
demonstrates this capability and the high spectral resolution of a
sf-SRS microscope on succinic acid, a compound with intrinsically
narrow Raman lines. The resolution of the system was found to be
on par with the calculated resolution of 8 cm1 for this system
(cf. Figure 4, purple line minimum at 500–550 mm). Note that a
known chirp parameter at the chirp matching condition can also be
408 Tamás Váczi et al.

Delay stage posion [μm]

45700 45800 45900 46000 46100 46200

Intensity [a. u.]

Intensity [a. u.]

2880 2900 2920 2940 2960 2980 3000

Raman shi [cm–1]

Fig. 6 Spontaneous Raman and sf-SRS spectra of succinic acid. The raw SRS spectrum (green) is shown with
respect to the delay line position on the upper X-axis and with respect to the corresponding Raman shift. With
a linear chirp, the stimulated Raman shift changes linearly with delay stage movement. The spontaneous
Raman spectrum (blue) is shown as reference

used to convert the relative delay stage movement to a difference on

Raman shift: the dimensions of β [cm1/fs] can easily be converted
to [cm1/μm].
Multispectral images can also be obtained simply by
performing imaging with different delay line positions. Figure 7
shows the SRS spectrum of a live zebrafish larva corresponding to
the anatomical region of the Tectum Opticum in the C-H finger-
print region. The intensities at 2845, 2931, and 2967 cm1
(marked with colored lines) were found to have good correlation
with lipid, protein, and DNA signatures, respectively [24]. The
corresponding SRS images are shown on the right side.

3.3 Methods for In the Differential Detection (DD), a reference signal is measured
Optimal Signal simultaneously along with the SRS one. This component is sub-
Detection: Differential tracted from the SRS signal in order to suppress the common noise
Detection of the laser light source. DD is an efficient solution to reduce the
noise and increase the sensitivity of the SRS measurements. The
main requirements of the high noise reduction are to minimize the
 Stimulated Raman Spectroscopy to Unravel Molecular Mechanism in Living Organisms 409

Fig. 7 SRS spectrum recorded from the brain of a live zebrafish with the corresponding SRS images (large field
of view, upper line) and zoomed-in insets (lower line) at different vibrational levels. Scale bars are 50 (upper
line) and 10 μm (lower line)

delay between the signals coming from the reference and SRS arms
and to have similar signal levels at the two detectors.
For the SRS with DD, the reference signal can be generated in
several ways. In some configurations, the detected beam is split
before the main dichroic mirror (Fig. 2), and some part of it is
diverted into the reference detector, so the reference beam is differ-
ent from the SRS one, both spatially and temporally. As a conse-
quence of the latter, this solution requires the insertion of a delay—
either into the optical or the electronic path—that compensates for
the longer distance between the beam splitter and the SRS detector
under the stage of the microscope. The main problem with this
approach is that it cannot compensate for the fluctuations of the
signal level at the SRS detector caused by the varying optical density
of the sample during the scanning.
The other approach is to have the reference beam passing the
same optical path as the SRS one and to place the reference detector
also under the sample. This allows for the two components to have
the same intensity change upon passing through the sample of
varying optical density. The reference beam can be realized by
creating a delayed replica of the pulses in the pump or Stokes
beam (the one detected) that will not be synchronized temporarily
with the pulses of the other beam [25, 26]. Then, the only differ-
ence between the pulse and its replica is that the intensity of the
former is affected by the SRS interaction, so their differential
detection will result in efficient common mode noise suppression.
The two arms of the balance detection can be conveniently multi-
plexed and de-multiplexed taking advantage orthogonal linear
polarization states.
410 Tamás Váczi et al.

Fig. 8 Schematic layout of the differential detection. HWP – half-wave plate, M – mirror, PBS – polarization-
dependent beamsplitter, A-B detector – differential detector

Figure 8 shows the schematics of the DD with pulse replica

generation for the Stokes beam. The replica generation unit is
inserted along the beam that is used for the detection of the SRS
signal, in a position upstream the dichroic mirror combining the
pump and Stokes beams (DM in Fig. 2). The combination of a half-
wave plate (HWP) and a polarization-dependent beamsplitter
(PBS) is used to split the beam into two components of orthogonal
polarization (the horizontally polarized beam is transmitted, the
vertically polarized is diverted in Fig. 8). The HWP rotates the
polarization of the laser, then the PBS separates the components
with orthogonal polarizations. The intensity ratio of the two com-
ponents will be determined by the angle of rotation. The diverted
beam is directed into another PBS performing the collinear combi-
nation of the two beams separated earlier. However, due to the
additional distance the diverted beam travelled, the two pulses are
delayed—the horizontally polarized goes ahead of the other. So,
after the replica generation unit, the beam will consist of two,
orthogonally polarized pulse trains, with direct and replica pulses
delayed typically by a few hundred ps. An additional HWP is also
inserted into the replica arm between the two mirrors allowing the
independent control of the intensity of the replica pulses.
The obtained Stokes beam is combined with the pump on the
dichroic mirror (DM) and introduced into the microscope. Both
the original and the replica pulses pass through the same path and
the former undergoes SRS interaction at the sample. Due to the
delay, the latter has no paired pulse for the SRS to occur. A PBS is
used after the sample to separate the orthogonally polarized SRS
and non-SRS beams that are directed to the SRS and reference
arms. Both arms contain identical detectors and filters. The path
length between direct and replica beams can be compensated by the
length of the cabling used in the detection electronics.
The efficient common mode noise suppression requires the
same laser intensities in the two arms. This can be adjusted with
the rotation of the two HWPs. The use of two laser intensity
regulators allows very precise tuning of the intensity ratio.
 Stimulated Raman Spectroscopy to Unravel Molecular Mechanism in Living Organisms 411

Fig. 9 SRS image recorded on 2 micron sized polystyrene microbeads with

(upper part) and without (lower part) differential detection

The effect of DD on the signal-to-noise ratio of the SRS can be

seen in Fig. 9. The upper part of the SRS image on polystyrene
microbeads was recorded in differential detection mode, while the
system was switched to single channel detection in the lower. A
remarkable difference can be seen in the SRS image quality of the
two parts, since the signal-to-noise ratio is significantly reduced due
to higher intensity noise.

4 Notes

4.1 Signal-to-Noise The signal-to-noise (S/N) ratio is a useful parameter to character-

Ratio of SRS Images ize the SRS images. The recording of a good-quality SRS image
requires many parameters of the SRS system to be adjusted to the
optimal setting, including the temporal and spatial overlap of the
pump and Stokes beams, lock-in detection parameters including
the modulation depth, frequency and filtering, pump and Stokes
beam intensities and their ratio, integration time, etc. The measure-
ment of the S/N ratio on a dedicated sample on regular basis or at
the beginning of every session could provide valuable information
on the current status and performance of the SRS system.
Figure 10 below shows an SRS image recorded on polystyrene
microbeads of 5- and 10-micron size. It can be seen that the image
is of good quality and the microparticles are highly visible. This
profile represents the intensity of each pixel belonging to the
selected line and can be used to calculate the S/N ratio. The signal
and noise levels are marked with red lines, in these regions the mean
values and the standard deviations are calculated, and the S/N ratio
is obtained as the difference between the mean values divided by the
standard deviation corresponding to the background noise region.
This particular example (Fig. 10) shows a S/N ratio over 250.
412 Tamás Váczi et al.

x104
4.8

4.6

4.4

Intensity [a.u.]
4.2

Signal
4.0

3.8

3.6 Noise

3.4

3.2
0 5 10 15 20 25 30 35 40
x [μm]

Fig. 10 High quality sf-SRS image recorded on polystyrene microbeads and an intensity profile measured on
the top center microparticle

4.2 Strategies for SRS requires tight focusing of the pump and Stokes beams. This
Ensuring Optimal criterion is very strict; the pulses should be in the very same focal
Spatio-Temporal volume at the same time. Several factors could affect the tight
Overlap Between focusing of the beams (even in an optimally adjusted SRS system)
Pump and Stokes that could have temporary or long-term effect, including the tem-
Beams perature and air density fluctuations, pointing stability of the two
beams, deformation of the mirror surfaces due to heating by the
intense laser beams, etc. On tunable SRS systems, the wavelength
dependence of the depth of focus of many objective lens could be
another issue.
The above problems can be addressed in several ways. The local
temperature and air density fluctuations in the surrounding of the
SRS system can be minimized by using air conditioning with precise
temperature control, proper shielding (closed box) around the
optical setup, and proper cooling of the overheating parts.
The pointing stability of the two beams can be improved by
integrating an automatic beam stabilizer into the optical paths of
the SRS system. The beam stabilizer for a single beam consists of
two steering mirrors, each with high-speed and high-resolution
angular adjustment capabilities in two orthogonal directions (rea-
lized through stepper motors or piezo drives) and two laser beam
position sensors that can detect the lateral misalignment of the laser
beam with high precision (e.g., a camera or a quadrant detector).
The two detectors are placed at a large distance from each other
into an auxiliary arm obtained by placing a beam picker or a beam
splitter into the main beam.
 Stimulated Raman Spectroscopy to Unravel Molecular Mechanism in Living Organisms 413

1.3
Change in Laser divergence

1.2

1.1

1.0

0.9

0.8

0.7
-1 -0.5 0 0.5 1
Distance from ideal posion of the beam expander [mm]

Fig. 11 Dependence of the divergence of a laser beam of 800 nm on the distance of the two lenses of a
divergence corrector telescope

The different depth of focus of laser beams of different wave-

lengths is related to the dispersion of the transmissive optical ele-
ments focusing the light of different color not to the same depth.
This can be corrected by adjusting the divergence of the beams that
will result in changes in the depth of focus. The simplest divergence
corrector is a telescope, the components (lenses or curved surface
mirrors) of which are slightly misaligned from the ideal position
axially. This difference alters the angle of the divergence of the
output beam, which subsequently will affect the depth of the
focus of the beam under the objective lens. The change of the
divergence of the output beam relative to the input for a 1:1
telescope is shown in Fig. 11. As the distance between the optical
components changes by 0.5 cm, the divergence will also be altered
by ca. 15–20%, which clearly indicates the efficiency of this unit for
the adjustment of the focal depths.

4.3 Optimizing the It has been described that detecting the SRS signal relies on lock-in
Beam Modulation amplifiers, which work by driving one of the excitation beams with
Frequency and Depth a high-frequency modulation. This is typically achieved using an
acousto-optical modulator (AOM), a crystal with a piezo actuator
attached to it. It works like a dynamic optical grating, where the
periodic modulation of the refractive index is created by the acous-
tic waves introduced into the crystal via the piezo actuator. As a
result, the crystal will behave as diffractive element and will divert
the collinear incident beam to the diffraction angle. By switching
the acoustic waves on and off, the beam can be switched between
straight and diverted states, so the intensity of both straight and
diverted beams will be modulated. As for optical gratings, the
414 Tamás Váczi et al.

1.0

0.8
Modulaon depth

0.6

0.4

0.2

0
100 101 102 103 104
Frequency [kHz]
Fig. 12 Frequency dependence of poor (blue) and optimized (red) modulation depths

diverting efficiency of the AOM depends on the proper alignment,

diameter, angle of incidence, etc., of the AOM. At kHz modulation
rates the units with relatively large aperture can achieve high mod-
ulation depth even with poor alignment. However, as the modula-
tion frequency increases, the conditions for the efficient
modulation will be stricter and further optimization of the align-
ment will be required. Figure 12 compares the frequency depen-
dence of the modulation depth of a poorly and optimally aligned
AOM module. It can be seen that while the value decreases rapidly
for the poorly aligned setup (<0.8 above 200 kHz, <0.6 above
1 MHz), efficient modulation can be achieved even at frequencies
of few MHz with optimized alignment (>0.9 up to 4 MHz).

Acknowledgments

The authors would like to acknowledge the support of the Depart-

ment of Biomedical Sciences (SID2018, Dal Maschio) and the
Padua Neuroscience Center (ReTurnPD, Dal Maschio) at the Uni-
versity of Padua, the support of EC Research Programs (VISGEN,
Dal Maschio; NEURAM, Veres and Dal Maschio). The authors
would like to thank the colleagues providing help, comments, and
suggestions in drafting the content.
 Stimulated Raman Spectroscopy to Unravel Molecular Mechanism in Living Organisms 415

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 INDEX

A H
Adaptive optics .....................................18, 193, 237, 252, Holographic microscope .............................................. 105
254, 263, 326, 356
All-optical neurophysiology..........................2–19, 21, 32, I
37, 56, 58 Image processing................................... 66, 208–209, 296
Imaging at depth.................................................. 263–282
B
Bessel beam ............................................ 61, 63, 236–238, L
240–242, 244–246, 252, 301, 311
Label-free imaging ........................................................ 400
Brain machine interfacing............................................. 142 Lens system design............................................... 188, 189
Light shaping .............................28, 29, 31, 89, 275, 276
C
Light-sheet microscopy .................................50, 231–258
Calcium imaging ....................................9, 11, 17, 18, 57,
104, 140–142, 144–146, 156, 170, 248, 249, M
284, 308, 309, 334, 336–339, 348, 378
Molecular tools ....................................... 3, 4, 11, 96, 105
Channelrhodopsin......... 2, 4–8, 10–15, 37, 59, 173, 181 Multiphoton microscopy .................................... 196, 239,
ChroME ............... 13, 14, 104, 123, 125–127, 129, 130 264, 265, 295–297
Compressive sensing ........................................... 250, 256,
257, 265, 272–275, 279, 286 N
Computer-generated holography (CGH) .................... 17,
21, 61, 90, 102, 112, 166, 232, 238, 242, 250, Neural imaging..................................................... 293–326
254, 339, 340 Neuronal ensembles................... 331, 332, 337, 347–353
Control of behavior ............................................. 331, 348 Neurophysiology ............................................ 2–19, 49–68
Cross-activation...................................168, 172, 179–181
O
E Olfactory system.......................................... 366, 369, 372
Event-related analysis of functional fluorescence One-photon excitation ........................................... 50, 59,
traces ................................................ 149, 150, 159 61, 141, 144, 148, 322, 333, 335
Opsin ....................................... 3, 5–8, 11–13, 20, 58, 59,
F 64, 78, 101, 104, 105, 112, 122–131, 143, 149,
156, 167, 172–176, 181, 343, 355, 376, 380, 387
Fiber optics .................................................................... 194 Optical microscopy .............................................. 293, 393
Fluorescence microscopy .............................193, 294–325 Optogenetics ................................. 7, 11, 27, 29, 38, 102,
Functional imaging ............................................. 8, 52, 66,
106, 114, 115, 123, 127, 138, 160, 232, 238,
138, 139, 142, 156, 283, 284, 313, 315, 323–325 247, 265, 288, 332–347, 356, 363–414
G P
Generalized phase contrast (GPC)..................... 3, 17, 19, Pattern completion .............................332, 348, 352–354
21–26, 91
Photobiophysics ..................................167, 168, 179–181
Genetically encoded calcium indicators (GECIs) ........ 75, Population vectors ............................................... 348, 350
76, 139 Psychophysics .............................................. 364, 366, 368
genetically encoded voltage indicators (GEVIs) .......... 10,
59, 60, 75

Eirini Papagiakoumou (ed.), All-Optical Methods to Study Neuronal Function, Neuromethods, vol. 191,
https://doi.org/10.1007/978-1-0716-2764-8, © The Editor(s) (if applicable) and The Author(s) 2023

417
 ALL-OPTICAL METHODS TO STUDY NEURONAL FUNCTION
418 Index
S Two-photon imaging......................................14, 63, 102,
118, 122, 146, 172, 179, 197, 198, 208, 337,
Single-pixel imaging................................... 208, 236, 265, 343, 347
271, 272, 275–277, 282 Two-photon optogenetics .................................. 123, 129,
Soma-targeted .....................................7, 13, 14, 124, 177 138, 331, 337, 348
Spectral focusing ......................................... 397, 406, 407
Spectral independency ......................................... 141, 160 U
Stimulated Raman spectroscopy.......................... 394–396
Stimulation artefact avoidance .................................11, 58 Ultrafast pulse propagation ......................................53, 61
Structured illumination microscopy................... 294, 312,
V
314–316, 320
Synapse ........................................................ 294, 306, 313 Voltage imaging ..................................10–12, 16, 57, 325
System integration ..............................138, 140, 149, 158 Volumetric neuronal imaging................................. 16, 63,
212, 236, 295, 326, 336
T
W
Temporal focusing ...........................3, 16, 17, 19, 26–30,
36, 87, 91, 103, 105, 107, 264, 275, 285, 380 Widefield imaging ............................................... 217, 263,
3D holography .............................................................. 111 271, 275, 282, 284, 324
3D photostimulation ................................................88–92 Widefield microscopy...................................192, 293–326
3D-SHOT ........................................29, 31, 90, 102–108,
110–118, 122–124, 126–128, 131 Z
Two-photon excitation fluorescence
Zebrafish ...............................................51, 138, 232, 238,
microscopy ..................................3, 15, 20, 22, 29,
247–250, 254, 307, 408, 409
110, 141, 146, 156, 168, 181, 212, 239, 300,
333, 334, 343