MM 593 Advanced Characterization Techniques

Shanza Rehan
Lecture # Microscopy
Chapter 1, Materials Characterization by Yang Leng, Wiley-VCH
Structural Characterization Techniques of Materials
▪ Structural Characterisation techniques consisting of macro and micro-structural
characterisation by using light or electron microscopes. It consists of:
1. Macroscopy: To observe the structure at low magnification i.e.,<50x.

2. Microscopy: To observe the structure at higher magnification i.e.,>50x.

Microscope
▪ A microscope is an optical system which transforms details of an ‘object’
into an ‘image.
▪ It observes features that are beyond the resolution of the human eye (about 100
microns).

▪ Three branches of Microscopy

▪ Optical
▪ Electron
▪ Scanning Probe

▪ Optical and Electron microscopy measure refraction, diffraction, and

reflection of the source radiation
▪ Optical uses white light, fluorescent light, or lasers
▪ Electron uses electromagnetic radiation/electron beams
▪ Scanning Probe uses a physical probe to interact with the surface of the specimen
Imaging Techniques
Lowest Resolvable Approx Lower
Technique Image Formed By
Unit Limit
1 μm
Optical Microscopy Light Rays Microns (μm)
(monochromatic light)

Coherent Light Source 0.2 μm

Confocal Microscopy Microns (μm)
(Laser) (X-Y Direction)
Transmission
Electron Microscopy 2Ǻ
Electrons Angstroms (Ǻ)
(TEM) (high resolution TEM)

Scanning Electron Nanometers (nm) to 10 nm

Electrons
Microscopy (SEM) Angstroms (Ǻ) (100 Ǻ)

Atomic Force &

Scanning Tunneling
Molecular Mechanical 10-40 Ǻ
Microscopies Angstroms (Ǻ)
(AFM/STM) Probes (theoretical)
Optical Microscopy
▪ Optical Microscopy is a surface investigation technique that relies on
the use of light in the visible spectrum to image the topography of a
sample.

Visible light has the wavelength

of about 400–700 nm

▪ Reflected or transmitted light from the sample enters the eye after
passing through a magnification column.
Optical Microscope
1. Ocular lens
2. Objective turret
3. Objective
4. Coarse Adjustment
5. Fine Adjustment
6. Stage
7. Light source
8. Condenser
9. X-Y Control
Image Formation
▪ Thin lens equation

where u is the 'object distance from the lens’ and v is the 'image distance'.

▪ Magnification M produced by the single lens is given by v/u.

▪ While substitution in the lens equation above gives

▪ where f is the focal length of the lens and v is the distance between the
image and lens.
▪ A higher magnification lens has a shorter focal length.
Image Formation
▪ For high magnification, combinations of lenses are used so that the
total magnification is achieved in two or more stages

▪ Magnification: Total visual magnification of the microscope is derived

by multiplying the magnification values of the objective and the
eyepiece.
▪ Ocular (eyepiece): 5-30 x magnification
▪ Objective: 4-100 x magnification
▪ Total Maximum magnification: 3000X
Magnification and Resolution
▪ We can magnify the image as much as we like in
Optical Microscope (OM) by adding more lenses, but
we cannot see the details clearly.

▪ Consequently, it is not necessary to build a light

microscope with three or more stages of magnification,
since this will not improve the resolution but will
rather degrade it by introducing extra aberrations

▪ The scanning electron microscope however has

inherently better resolution and it makes sense to use it
at higher magnifications.
A series of light and scanning electron micrographs of the
high-temperature superconductor barium-yttrium copper
oxide at increasing magnification
Resolution
▪ Resolution refers to the minimum distance between two points at which
they can be visibly distinguished as two points.

▪ Magnification : Image size/Object size

▪ Resolution : The fineness of detail that can be distinguished in an image.

▪ The resolution of a microscope is theoretically controlled by the

diffraction of light because in microscopes the light must pass through a
series of apertures or the lenses.
Resolution
▪ Wherever light passes through an aperture, diffraction occurs so that a
parallel beam of light (which would be seen as a spot) is transformed
into a series of cones, which are seen as circles and are known as Airy
rings.

▪ The resolving power of a microscope

is defined as the ability to distinguish
between two closely spaced Airy disks
OR
▪ The ability of the microscope to
reveal adjacent structural detail as
distinct and separate.
Resolution
▪ It is the effect of diffraction that limit the ability to
resolve fine details.
▪ The extent and magnitude of the diffraction patterns are
affected by both by the wavelength of light (λ), the
refractive materials used to manufacture the
objective lens and the numerical aperture (NA) of the
objective lens.
Numerical aperture
▪ Numerical aperture is commonly used in microscopy to describe the
acceptance cone of a lens (and hence its light-gathering ability and
resolution)

where μ is refraction index of the material (air μ =1, distilled water μ =1.33,
cedar oil μ =1.51) between the specimen and the lens, and α is the half-
angle of the most oblique light rays that enter the front lens of the
objective.
Numerical aperture
▪ The Light-collecting ability increases with α and setting of the
aperture diaphragm will alter the NA of the condenser and therefore
the NA of the system Objective lenses.

▪ In optics, the numerical aperture (NA) of an optical system is a

dimensionless number that characterizes the range of angles over
which the system can accept or emit light.

▪ The NA value is engraved upon the side of the objective lens piece,
with the typical values ranging from 0.16 to 1.4 in light microscope.
Resolution
▪ There is a finite limit beyond which it is impossible to resolve separate
points in the objective field.

▪ Assuming that optical aberrations in the whole optical set-up are

negligible, the resolution δ, is given by:
Resolution
▪ In order to obtain the best resolution (i.e. the
smallest δ) it is practical to decrease λ using
electromagnetic radiation of smaller
wavelength or increase μ by using lens
material of higher refractive index or α by
reducing the working distance.

▪ With a light microscope λ can be decreased

to 400 nm (or to about 200 nm if it is
possible to use ultraviolet light);
▪ sinα can be increased towards 1 by using as
large an aperture as possible
▪ μ can be increased by using an oil
immersion objective lens.
Resolution
▪ The resolution range that can be obtained in light microscope is 290-
150 nm
▪ To get higher resolution we can use electron beam which has much
lower λ value and improve optical system by using electromagnetic
lenses.
▪ Today’s SEM give resolution 10-1 nm.
Depth of Field
▪ Depth of field is an important concept when photographing an image.
It refers to the range of position for an object in which image
sharpness does not change.

▪ In most microscopes depth of field is rather small and therefore in

order to produce sharp images the object must be very flat.

▪ If a non-flat object is viewed at high magnification using a light

microscope then some out-of-focus regions will be seen.
Depth of Field

OM SEM
Small depth of field Large depth of field
Low resolution High resolution
Depth of Field
▪ This is a useful feature if we wish to accentuate certain parts of the
image at the expense of others for example measurement of a grove
or thread depth but is a grave disadvantage if we would like to see all
parts of a three-dimensional object clearly.

▪ An object image is only accurately in focus when the object lies in a

plane within a certain distance from the objective lens. The image is
out of focus when the object lies either closer to or farther from the
lens.

Figure 1.6 Geometric relation among the depth of field (Df), the half-angle
entering the objective lens (α), and the size of the Airy disk (d).
Depth of Field

▪ Equation indicates that a large depth of field and high

resolution cannot be obtained simultaneously; thus,
a larger Df means a larger R and worse resolution.

Figure 1.6 Geometric relation among the depth of field (Df), the half-angle
entering the objective lens (α), and the size of the Airy disk (d).
Steps to Improve Depth of Field
▪ reduce NA by closing the aperture diaphragm, or use an objective lens
with lower NA;
▪ lower the magnification for a given NA;
▪ use the longest possible wavelength light.
Depth of Focus
▪ Depth of focus refers to the range of image plane positions at which
the image can be viewed without appearing out of focus for a fixed
position of the object.
Depth of Focus
Brightness and Contrast
▪ To make a microscale object in a material specimen visible, high magnification
is not sufficient. A microscope should also generate sufficient brightness and
contrast of light from the object.

▪ Brightness refers to the intensity of light.

▪ In a transmission light microscope the brightness is related to the numerical

aperture (NA) and magnification (M).

▪ In a reflected-light microscope the brightness is more highly dependent on NA.

Brightness and Contrast
▪ Brightness decreases rapidly with increasing magnification, and controlling
NA is not only important for resolution but also for brightness, particularly
in a reflected-light microscope.

▪ Contrast is defined as the relative change in light intensity (I) between an

object and its background.

▪ Visibility requires that the contrast of an object exceeds a critical value

called the contrast threshold. The contrast threshold of an object is not
constant for all images but varies with image brightness.

▪ In bright light, the threshold can be as low as about 3%, while in dim light
the threshold is greater than 200%.
Optimum resolution
Steps for Optimum Resolution
▪ Use an objective lens with the highest NA possible;
▪ Use high magnification;
▪ Use an eyepiece compatible with the chosen objective lens;
▪ Use the shortest possible wavelength light;
▪ Keep the light system properly cantered;
▪ Use oil immersion lenses if available;
▪ Adjust the field diaphragm for maximum contrast and the aperture
diaphragm for maximum resolution and contrast; and
▪ Adjust brightness for best resolution.
Instrumentation
▪ A light microscope includes the following main components:
▪ illumination system;
▪ objective lens;
▪ eyepiece;
▪ photomicrographic system; and
▪ specimen stage.

▪ A light microscope can be divided into two types based on the lights
used for illumination
1. transmitted-light microscopes ➔ used to examine transparent or
semi-transparent materials, such as certain types of polymers
2. Reflected-light microscopes ➔ used for metallography,
Instrumentation
1. Illumination system
▪ The illumination system of a microscope provides visible light by which
a specimen is observed. There are three main types of electric lamps
used in light microscopes:
1. low-voltage tungsten filament bulbs;
2. tungsten–halogen bulbs; and
3. gas-discharge tubes.

▪ In a modern microscope, the illumination system is composed of a

light lamp (commonly a tungsten–halogen bulb), a collector lens and a
condenser lens to provide integral illumination; such a system is
known as the Köhler system.
Instrumentation
▪ Köhler illumination is a method of specimen illumination
used for transmitted and reflected light optical
microscopy.
▪ Köhler illumination acts to generate an extremely even
illumination of the sample and ensures that an image of
the illumination source (for example a halogen lamp
filament) is not visible in the resulting image. (Uneven
illumination is undesirable as it can introduce artefacts
such as glare and shadowing in the image.)
▪ The main feature of the Köhler system is that the light
from the filament of a lamp is first focused at the front
focal plane of the condenser lens by a collector lens.
Then, the condenser lens collects the light diverging from
the source and directs it at a small area of the specimen
be examined. Köhler system illustrated in a
transmitted light microscope
Instrumentation
▪ There are two important controllable
diaphragms in the Köhler system: the
field diaphragm and the aperture
diaphragm.

▪ Field diaphragm
▪ The field diaphragm is placed at a focal
plane for the image-formation rays. Its
function is to alter the diameter of the
illuminated area of the specimen.
▪ The field diaphragm restricts the area of
view and blocks scattering light that could
cause glare and image degradation if they
Figure 1.13 Image of the field diaphragm with
entered the objective lens and eyepiece. an image of the specimen. Magnification 100×.
Instrumentation
▪ Aperture diaphragm

▪ The aperture diaphragm is placed at a focus

plane of the illuminating rays.

▪ Its function is to control α, and thus affect Large aperture

the image resolution and depth of field.

Small aperture
Instrumentation
2. Objective Lens
▪ The objective lens is the most important optical component of a light
microscope.

▪ The magnification of the objective lens determines the total magnification

of the microscope because eyepieces commonly have a fixed
magnification of 10×.

▪ The objective lens generates the primary image of the specimen, and its
resolution determines the final resolution of the image.

▪ The numerical aperture (NA) of the objective lens varies from 0.16 to 1.40,
depending on the type of lens.
Instrumentation
3. Eyepiece
▪ The eyepiece is used to view the real primary image formed
by the objective lens.
▪ The eyepiece allows a glass disc with an etched graticule to
be inserted into the optical path. The graticule serves as a
micrometre (micron marker) for measurement.

▪ The eyepiece has either a helical thread or a sliding mount as

a focusing mechanism which provides a ‘parfocal’ adjustment
of optics (A parfocal lens is a lens that stays in focus when magnification is
changed. There is inevitably some amount of focus error, but small enough to be
Eyepiece graticule
considered insignificant.) Thus, focusing the eyepiece is a
necessary step before photographing images in a microscope
Aberrations
▪ Image distortions are caused by light rays from any point on an object
focus imperfectly by the lens called lens aberrations.
▪ Some aberrations affect the whole field of the image ➔ chromatic and
spherical aberrations

▪ while others affect only off-axis points of the image ➔ astigmatism

▪ The true resolution and depth of field can be severely diminished by

lens aberrations.
Aberrations
▪ Chromatic aberration is a failure of a lens to focus all colors to the
same point. It is caused by dispersion: the refractive index of the lens
elements varies with the wavelength of light. The refractive index of
most transparent materials decreases with increasing wavelength
▪ The refractive index of lens glass is greater for shorter wavelengths (for
example, blue) than for longer wavelengths (for example, red)
Aberrations
▪ Spherical aberration is caused by the spherical curvature of a lens.
▪ Light rays from a point on the object on the optical axis enter a lens at
different angles and cannot be focused at a single point.
▪ The portion of the lens farthest from the optical axis brings the rays to
a focus nearer the lens than does the central portion of the lens.
Aberrations
▪ Astigmatism results when the rays passing through vertical diameters
of the lens are not focused on the same image plane as rays passing
through horizontal diameters.
▪ In this case, the image of a point becomes an elliptical streak.
▪ Astigmatism can be severe in a lens with asymmetric curvature.
Aberrations Correction
▪ Classification of the objective lens is based on its aberration-correction
capabilities, mainly chromatic aberration. The following lenses are shown
from low to high capability of correcting chromatic aberration.
1. achromat➔ corrects chromatic aberration for two wavelengths (red and blue).
▪ materials with differing dispersion are assembled together to form a compound lens (eg crown and
flint glass.

2. Semiachromat➔corrects chromatic aberration for 3 wavelengths (red, blue green).

▪ Its NA is larger than that of an achromatic lens with the same magnification and produces a
brighter image and higher resolution of detail

3. apochromat. ➔lens provides the highest degree of aberration correction. It almost

completely eliminates chromatic aberration by removing aberrations of red, blue,
green and IR. Its NA is even larger than that of a semi-achromatic lens.
▪ Uses lens made up of fluorite glass
Apochromat (APO)

achromat
Imaging Modes
▪ The differences in properties of the light waves reflected/transmitted
from microscopic object enable us to observe these objects by light
microscopy.
▪ The light wave changes in either amplitude or phase when it interacts
with an object as illustrated.
Imaging Modes
▪ The eye can only appreciate amplitude and wavelength differences in
light waves, not their phase difference.

▪ The most commonly used examination modes, bright-field and dark-

field imaging, are based on contrast due to differences in wave
amplitudes.

▪ The wave phase differences have to be converted to amplitude

differences through special optical arrangements such as in the
examination modes of phase contrast, polarized light, and Nomarski
contrast.
Bright-Field and Dark-Field Imaging
▪ Bright-field imaging is the predominant
mode for examining microstructure.
▪ Dark-field imaging is widely used to
obtain an image with higher contrast than
in bright-field imaging.

Figure (a) Bright-field illumination

and (b) dark-field illumination in
transmitted mode. Shaded areas
indicate where the light is blocked.
Bright-Field Microscopy
▪ In bright-field imaging, the specimen is evenly illuminated by a light source. The
typical appearance of a bright-field microscopy image is a dark sample on a bright
background, hence the name.
▪ Bright-field microscopy typically has low contrast with most biological samples, as
few absorb light to a great extent. Staining is often required to increase contrast,
which prevents use on live cells in many situations. Bright-field illumination is useful
for samples that have an intrinsic color, for example chloroplasts in plant cells.

An example bright-
Microscopic view of a field micrograph.
histologic specimen of This image shows a
human lung tissue cross-section of the
stained with vascular tissue in a
hematoxylin and plant stem.
eosin.
Bright-Field Microscopy
Advantages
▪ Simplicity of setup with only basic equipment required.
▪ Living cells can be seen with bright-field microscopes.

Limitations
▪ Very low contrast of most biological samples.
▪ Samples that are naturally colorless and transparent cannot be seen
well, e.g. many types of mammalian cells. These samples often have to
be stained before viewing in contrast to samples that do have their own
color can be seen without preparation
Dark-Field microscopy
▪ Dark-field microscopy (also called dark-ground
microscopy) describes microscopy methods, in both
light and electron microscopy, which exclude the
unscattered beam from the image. As a result, the field
around the specimen (i.e., where there is no specimen
to scatter the beam) is generally dark.

▪ Dark-field imaging requires that the specimen is

illuminated by oblique light rays. There is a central
stop in the light path to block the central portion of
light rays from illuminating the specimen directly.
Dark-Field microscopy
▪ Thus, the angle of the light rays illuminating the
specimen is so large that light from the specimen cannot
enter the objective lens unless it is scattered by
microscopic objects.

▪ In optical microscopy, dark-field describes an

illumination technique used to enhance the contrast in
unstained samples. It works by illuminating the sample
with light that will not be collected by the objective lens
and thus will not form part of the image. This produces
the classic appearance of a dark, almost black,
background with bright objects on it.
Dark-Field microscopy
The light's path
The steps are illustrated in the figure.

▪ Light enters the microscope for illumination of the sample.

▪ A specially sized disc, the central stop blocks some light from the light
source, leaving an outer ring of illumination.
▪ The condenser lens focuses the light towards the sample.
▪ The light enters the sample. Most is directly transmitted, while some is
scattered from sample.
▪ The scattered light enters the objective lens, while the directly
transmitted light simply misses the lens and is not collected due to a
direct-illumination block.
▪ Only scattered light goes on to produce image, while the directly
transmitted light is omitted.
Dark-Field microscopy
Advantages
▪ Dark-field microscopy is a very simple yet effective technique and well suited
for uses involving live and unstained biological samples,
▪ such as a smear from a tissue culture or individual, water-borne, single-celled organisms.
▪ Dark-field microscopy techniques are almost entirely free of artifacts, due to
the nature of the process.

Disadvantages
▪ The main limitation of dark-field microscopy is the low light levels seen in the
final image. This means that the sample must be very strongly illuminated,
which can cause damage to the sample.
▪ Interpretation of dark-field images must be done with great care, as common
dark features of bright-field microscopy images may be invisible
Bright-Field and Dark-Field Imaging
▪ Figure 1.30 shows the comparison between bright- and dark-field
images of an identical field in a high carbon steel specimen under a
reflected-light microscope. Microscopic features such as grain
boundaries and second-phase particles appear self-luminous in the
dark-field image, as shown in Figure 1.30.
Phase-Contrast Microscopy
▪ Phase contrast is a useful technique for specimens such as polymers
that have little inherent contrast in the bright-field mode.
▪ In effect, the phase contrast technique employs an optical mechanism
to translate minute variations in phase into corresponding changes in
amplitude, which can be visualized as differences in image contrast
▪ This conversion in amplitude is based on interference (constructive
and destructive) phenomenon of light waves.
▪ Constructive interference occurs when combining two same-wavelength
waves that do not have a phase difference between them.
▪ However, completely destructive interference occurs when combining two
waves with a phase difference of a half-wavelength λ/2
Phase-Contrast Microscopy
Figure 1.32 Optical arrangement of
phase-contrast microscopy (transmitted-
light microscope). Shading marks the
paths of diffracted light. (Reproduced with
permission from Ref. [1]. © 2001 John
Wiley & Sons Inc.)
Phase-Contrast Microscopy
Phase-Contrast Microscopy
▪ The two most important Optical arrangement of phase-contrast
microscopy includes;
▪ An opaque black plate with a transparent ring, known as condenser annulus,
is placed in the front focal plane of the condenser lens.
▪ A plate of glass with an etched ring of reduced thickness, known as phase
plate, is placed at the back focal plane of the objective lens

▪ The specimen is illuminated by light beams emanating from a ring of

condenser annulus.
Phase-Contrast Microscopy
▪ An incident wavefront present in an illuminating beam of light becomes
divided into two components upon passing through a specimen. The primary
component is an undeviated (or undiffracted) planar wavefront, commonly
referred to as the surround (S) wave, which passes through and around the
specimen, but does not interact with it. In addition, a deviated or diffracted
spherical wavefront (D-wave) is also produced by sample interaction and
becomes scattered over a wide arc (in many directions)

▪ The light beam that passes through a specimen

without diffraction by an object (the straight-through
light beam, S-wave) will pass the ring of a phase
plate. The ring with reduced thickness in the phase
plate enables the waves of the straight-through beam to
be advanced by λ/4.
Phase-Contrast Microscopy
Phase-Contrast Microscopy
▪ The light beam diffracted by the object in the specimen (D-wave)
cannot pass through the ring of the phase plate but only through the
other areas of the phase plate. If the diffracted beam is delayed by
λ/4 while passing through the object, a total λ/2 difference in phase is
generated.

▪ When the straight-through beam and diffracted beam recombine at the

image plane, completely destructive interference occurs. Thus, we
expect a dark image of the object in phase-contrast microscopy.
Variation in phase retardation across the specimen produces variations
in contrast.
Phase-Contrast Microscopy
▪ Figure shows image differences between bright-field and phase-
contrast images of composites in transmitted-light microscopy.
Phase-Contrast Microscopy
Living Cells in (a)Brightfield
and (b)Phase Contrast

Advantages
▪ One of the major advantages of phase contrast microscopy is that
living cells can be examined in their natural state without previously
being killed, fixed, and stained. As a result, the dynamics of ongoing
biological processes can be observed and recorded in high contrast
with sharp clarity of minute specimen detail
Fluorescence Microscopy
▪ Fluorescence microscopy is useful for examining objects that emit
fluorescent light.
▪ Fluorescence is an optical phenomenon; it occurs when an object emits
light of a given wavelength when excited by incident light.
▪ The incident light must have sufficient energy, that is, a shorter
wavelength than that light emitting from the object, to excite
fluorescence.
Fluorescence Microscopy
Basic principle of fluorescence

internal
conversion

Fluorescence
Non-
radiative
decay
Excitation

Jablonski diagram
Fluorescence Microscopy
▪ In the diagram electronic (energy) states are indicated by
bold horizontal lines. The thin horizontal lines above them Jablonski diagram
represent vibrational/rotational sublevels. internal
conversion
▪ In a fluorescent molecule electrons are normally at the
lowest energy state, indicated by S0. When a photon
(indicated by the blue line entering from the left) with
appropriate energy interacts with a molecule the photon

Fluorescence
may be absorbed, causing an electron to jump to one of the
levels of an excited state (S1 or S2 in the diagram).

Excitation
▪ This excitation is most efficient at the optimal excitation
wavelength of the fluorophore, which most often also
corresponds to the absorption maximum. This transition Non-radiative
process is very fast, on the order of 10-15 seconds (a decay
millionth of a billionth of a second).
Fluorescence Microscopy
▪ An excited-state electron rapidly (on the order of 10-12 sec) Jablonski diagram
loses its energy to vibration (heat), a process called
internal conversion, and falls to the lowest level of the first internal
(S1) excited state. From there the electron may fall to one conversion
of the sub-levels of the ground (S0) state, emitting a photon
with energy equivalent to the energy difference of the
transition. This happens on a time scale of nanoseconds
(10-9 – 10-8 seconds) after the initial photon was absorbed.

Fluorescence
▪ Since the emitted photon has less energy than the absorbed

Excitation
photon it is at a longer wavelength. This explains the
magical process of fluorescence that converts light of one
wavelength (color) to another, and leads to the phenomenal Non-radiative
display of highly saturated colors in corals and so many decay
other marine organisms.
Fluorescence Microscopy
▪ The best way to document the fluorescence
properties of a particular specimen is to measure
excitation and emission spectra.
▪ Since energy has been lost during the process
due to the relaxation step, the emitted fluorescent
photon has a lower energy than the excitation
photon which leads to a red shift of the emission
profile relative to the excitation profile.
▪ The emission profile typically resembles a
shifted mirrored version of the absorption profile
Fluorescent Dyes
▪ While only a small number of materials exhibit this capability, certain
types of materials can be stained with fluorescent dyes (fluorochromes
/ flourophores).

▪ The fluorochromes can selectively dye certain constituents in

materials, called fluorescent labelling which is Permanent staining .
Fluorescent labelling is widely used for polymeric and biological
samples.

▪ A fluorophore is covalently attached to a non-fluorescent molecule

such as protein, DNA, or synthetic polymers.
Fluorescent Dyes
▪ A fluorescent dyes can absorb UV/visible light to emit fluorescent
light and are characterised by their absorption and emission spectra.

▪ The absorption and emission wavelength depend on chemical structure

and chemical environment e.g Radium is a radioactive element that
emits a pale blue color as it decays, However, it is best known for its
use in self-luminous paints
Fluorescent Dyes
▪ Choice and selection of appropriate dyes crucial to achieve good
image details.
Fluorescence Microscopy: Construction
▪ Mercury arc lamp or xenon Detector
arc lamp Objective lens

▪ Exciter filter

▪ Dichroic mirror (Beam-

splitter)

▪ Optical lenses
Condenser
▪ Sample stage lens
Fluorescence Microscopy: Photo-bleaching
▪ When a fluorophore lose its ability to fluoresce it is called photo-
bleaching.

▪ It is caused by any photo-induced changes (degradation) in the

chemical structure of a fluorophore due to strong illumination from
source.

▪ The two ways through which photo-bleaching can be countered are;

▪ Reduce power and time of illumination
▪ Use more robust fluorophore
Fluorescence Microscopy
Fluorescence Microscopy
Advantages
▪ High contrast

▪ High specificity

▪ Quantitative

▪ Compatible with living cells

Confocal Microscopy
▪ The principle of confocal imaging was patented in 1957 by Marvin
Minsky and aims to overcome some limitations of traditional wide-
field fluorescence microscopes.

▪ In a conventional (i.e., wide-field) fluorescence microscope, the entire

specimen is flooded evenly in light from a light source. All parts of the
specimen in the optical path are excited at the same time and the
resulting fluorescence is detected by the microscope's photodetector or
camera including a large unfocused background part.
Confocal Microscopy
▪ Confocal microscopy is a new technique that provides three-
dimensional (3D) optical resolution.

▪ Compared with a conventional compound microscope, a modern

confocal microscope has two distinctive features in its structure:
1. a laser light source ➔laser light provides a high-intensity beam to generate image
signals from individual microscopic spots in the specimen

2. a scanning device ➔moves the beam in a rectangular area of specimen to

construct a 3D image on a computer

▪ Thus, the confocal microscope is often referred to as the confocal laser

scanning microscope (CLSM).
Laser Confocal Fluorescence Microscopy
▪ A confocal microscope uses point
illumination and a pinhole in an optically
conjugate plane in front of the detector
to eliminate out-of-focus signal – the
name “Confocal” means “having the
common or same focus” stems from this
configuration

▪ Pinhole controls the focal and non-focal

rays to reach detector

▪ Only the fluorescence very close to the

focal plane can be detected, the image's
optical resolution, particularly in the
sample depth direction, is much better
Confocal Microscopy
▪ Capturing multiple two-dimensional images at different depths in a
sample enables the reconstruction of three-dimensional structures (a
process known as optical sectioning) within an object.
Confocal Microscopy
▪ Effect of confocal and nonconfocal function
Laser Confocal Fluorescence Microscopy
Working Principles
▪ The laser beam is focused as an intense spot on a
certain focal plane of the specimen by a
condenser lens, which also serves as an objective
lens to collect the reflected beam.

▪ A pinhole aperture is situated in a conjugate

plane (confocal) with a scanning point on the
specimen confocal plane in front of the light
detector.
Figure 1.45 Optical path in the confocal
microscope (reflected illumination).
Laser Confocal Fluorescence Microscopy
Working Principles
▪ The reflected beam from the focal plane in a specimen
becomes a focused point at the confocal plane.

▪ The pinhole aperture blocks the reflected light from the out-
of-focal plane from entering the detector.

▪ Since the pinhole aperture can block a large amount of

reflected light, high-intensity laser illumination is necessary
to ensure that sufficient signals are received by the detector.

▪ The detector is commonly a photomultiplier tube (PMT) that

converts light signals to electric signals for image
processing in a computer Figure 1.45 Optical path in the confocal
microscope (reflected illumination).
Laser Confocal Fluorescence Microscopy
▪ To acquire an image of the focal plane, the plane has to be scanned in
its two lateral directions (x–y directions).

▪ To acquire a 3D image of a specimen, the plane images at different

vertical positions should also be recorded.

▪ A scanning device moves the focal laser spot in the x–y directions on
the plane in a regular pattern called a raster.

▪ After finishing one scanning plane, the focal spot is moved in the
vertical direction to scan the next parallel plane.
Methods of scanning in CLSM
1. Specimen scanning
▪ Used in early confocal microscopes.

▪ The specimen moves with respect to the

focal spot and the optical arrangement is
kept stationary

▪ The beam is always located at the optical

axis in the microscope so that optical
aberration is minimized.

▪ The main drawback of this method is the

low scanning speed.
Methods of scanning in CLSM
1. Laser scanning
▪ Laser scanning is realized by two
scanning mirrors rotating along mutually
perpendicular axes.

▪ The scan mirror can move the focal spot

in the specimen by sensitively changing
the reflecting angle of the mirror.

▪ Changing the vertical position of the spot

is still achieved by moving the specimen
in the laser-scanning Method
Optical Sectioning and 3D Imaging
▪ Applicable to UV and fluorescently transparent samples
▪ A three-dimensional (3D) image is obtained by reconstructing a deck
of plane images
Optical Sectioning and 3D Imaging
▪ A biological specimen, pollen grain, was
fluorescently labeled with acridine
orange.

▪ In total, 80 sections of the pollen grain

were imaged for 3D reconstruction.

▪ The vertical distance between sections is

1.1 μm. Figure 1.47 shows 11 of 80 plane
images and the reconstructed 3D image.

Figure 1.47 Confocal fluorescence micrographs of a Spathiphyllum

pollen grain. The optical section sequence is from left to right and top
to bottom. The bottom right image is obtained by 3D reconstruction
Applications of LCFM
▪ Determining natural biopolymers (i.e., proteins, DNA, and RNA),
cytoskeletal filaments and tracing specific cells or structures in a
tissue.

Tri-fluorescent labelling and LCFM imaging of bone osteoblasts and fibroblasts

LCFM: Advantages
▪ Enables deep visualization of both living and fixed cells and tissues,
▪ A laser beam can be focused to any depth from sample surface
▪ Ability to reconstruct 3D images by optical sectioning without sample
destruction
▪ The “confocal” feature allows to obtain high-resolution images
▪ Two-channel illumination (using 2 different lasers) allows to observe
the localizations of two types of fluorophores in the sample
▪ Background subtraction is possible