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OPEN A fast multispectral light

synthesiser based on LEDs
and a diffraction grating
received: 28 June 2016 Gregor Belušič1, Marko Ilić1, Andrej Meglič1 & Primož Pirih2
accepted: 01 August 2016
Published: 25 August 2016
Optical experiments often require fast-switching light sources with adjustable bandwidths and
intensities. We constructed a wavelength combiner based on a reflective planar diffraction grating
and light emitting diodes with emission peaks from 350 to 630 nm that were positioned at the angles
corresponding to the first diffraction order of the reversed beam. The combined output beam was
launched into a fibre. The spacing between 22 equally wide spectral bands was about 15 nm. The
time resolution of the pulse-width modulation drivers was 1 ms. The source was validated with a fast
intracellular measurement of the spectral sensitivity of blowfly photoreceptors. In hyperspectral
imaging of Xenopus skin circulation, the wavelength resolution was adequate to resolve haemoglobin
absorption spectra. The device contains no moving parts, has low stray light and is intrinsically capable
of multi-band output. Possible applications include visual physiology, biomedical optics, microscopy
and spectroscopy.

Many optical experiments require a near-ultraviolet and visible light source with adjustable wavelength and inten-
sity. A classical single-band light source used in e.g. visual physiology experiments consists of a Xenon arc lamp
(XBO), a shutter, a graded neutral density wedge and a monochromator or a filter wheel1,2. Usually, the bandwidth
of the passband is 5~20 nm (full width at half maximum, FWHM), and the wavelength and intensity changes can
be achieved within about a second. In order to achieve a multi-band output and faster changes several beams
have to be combined via beam-splitters. Hyperspectral imaging systems3,4 either use a hyperspectral camera and
a white light source5,6, or a monochrome camera and a hyperspectral source7,8.
An alternative to a monochromator-based system is a multi-band stimulator based on light emitting diodes
(LEDs). Light uniformity can be achieved with an integrating sphere, a light guide with high numerical aperture
or a diffuser9–12. LEDs have typical bandwidths (FWHM) of 10~50 nm, and especially the UV LEDs have tails in
the visible spectrum13, so additional spectral filters may be needed if narrower spectral bands are required.
The light flux cannot be concentrated more than its source radiance due to conservation of étendue, the prod-
uct of the source area and its radiant solid angle. Since the LEDs cannot be in the same location, these sources
either suffer from low spatial uniformity or from large étendue, limiting their usability especially for micros-
copy. It is possible to combine beams of different wavelengths without étendue increase either by using dichroic
beamsplitters or by employing the dispersion innate to refractive or diffractive optical elements. Wavelength
combining by prisms was for the first time put forward by Newton, who proposed in his First Book of Opticks14 to
synthesise white light “By mixing colour’d Lights to compound a beam of Light of the same Colour and Nature with a
beam of the Sun’s direct Light […]”. More recently, gratings are being routinely used in laser optics for wavelength
combining15–17.
Here, we present a light source consisting of a series of LEDs, a planar reflective diffraction grating acting as a
wavelength combiner, a launching lens and a light guide. The source is intrinsically multi-band, contains no mov-
ing parts, and is small and inexpensive in comparison with XBO-based sources. The multi-band light synthesiser
is suitable for applications that require a high time resolution, e.g. time-resolved spectroscopy, or a small étendue,
e.g. in microscopy.

Results
The optical setup. A reflective planar grating with groove density G (e.g. 1200~2400 grooves/mm) diffracts
an input beam of wavelength λ, incident under angle α to the grating normal, into several diffracted orders m,

1
Department of Biology, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia. 2Sokendai (The Graduate
University of Advanced Studies), Hayama, Kanagawa, Japan. Correspondence and requests for materials should be
addressed to P.P. (email: [email protected])

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Figure 1. The optical table layout and principle. (a) Top-view of the optical bench. The LEDs with collimators
are mounted on a profile (left side). G: Grating; L: Launching lens, F: fibre; scale bar: 20 cm. (b) The principle
of wavelength combining with a grating under a constant exit angle. An LED, incident angle α, produces a
specular reflection at the angle β0 =​  −α, and a positive first order diffraction at the angle β+1. The negative first
order angle β−1 may exist for shorter wavelengths. The dashed arc represents the approximate input angle range
for wavelengths 300 to 700 nm for grating density G =​ 1850 mm−1, and the dotted arc indicates the negative
first order beams (UV to blue). UVm, Bm, Rm indicate the positions of diffracted orders m, (c) Input angles
for a grating with G =​ 1850 mm−1, for exit angles β+1 between 30° and 90°. Solid lines represent input angles
α. Dashed lines represent negative order angles β−1. Thick lines represent the configuration with exit angle
β+1 =​ 50°, used in the prototype. Input angle α (dotted), specular reflection β0 (dot-dashed) and negative first
order β−1 (dashed). Shaded area shows the design constraint for small exit angles (β+1 <​50°), where the input
angles may overlap with the exit angle (α =​  β+1) in the wavelength range 500 nm to 700 nm.

exiting under angles βm(λ), according to the grating equation (sinα +​ sinβm =​  mGλ)18. In a classical spectro-
graph, the input angle α is kept constant and the detector spans a range of first order exit angles β+1(λ). In the
wavelength combiner presented here, the input beams span a range of angles α(λ), while the first order exit angle
β+1 is constant.
Figure 1 shows the prototype built around a ruled reflective diffraction grating with groove density
G =​ 1850 mm−1 and a row of LEDs (Fig. 1a). The input angles subtend about 30° and the first order beam exit
angle is set to a constant β+1 =​ 50° (Fig. 1b). The combined beam is launched to a light guide with a single aspheric
lens (Fig. 1a). All inputs are also diffracted to the zero order, while the inputs with wavelengths below ~500 nm are
additionally diffracted to the negative first order β−1 (Fig. 1b,c).
Figure 2 shows the measured bandwidth and efficiency of the prototype. The bandwidths of the installed
LEDs vary substantially (Fig. 2a). However, the combination of grating and fibre strongly narrows and unifies
the bandwidths (FWHM =​ 6–8 nm, Fig. 2b) and the individual output beams have little stray light (Fig. 2b,c).
Using commercially available quasi-monochromatic LEDs, we could cover the wavelength range 350–630 nm in
approximately equally spaced intervals, except for the regions 520–563 and 570–594 nm. We filled these gaps with
phosphorus-based lime green and white LEDs (Fig. 2a,c, dotted and dashed lines).
Due to the restricted aperture of the collecting fibre, only part of the spectral band of each LED is captured.
The coupling efficiency of the LEDs (the ratio of the irradiance at the fibre output and the LED tip) is further
reduced due to vignetting loss, to 0.1%~1.6% (Fig. 2d). Expectedly, the coupling efficiency of the white LED was
the lowest (Fig. 2c arrow, Fig. 2d). To achieve isoquantal (i.e. equal photon flux) output on all channels, the LEDs
with the highest coupling efficiency were attenuated to about 2% (Fig. 2d).
The output of the LEDs was fully linear with the pulse width modulation (PWM) duty cycle and showed good
temporal stability over the whole intensity range. The PWM driver has 1 kHz update frequency and 12-bit resolu-
tion, which allows for a 3.6 decade dynamic range. The remaining quantisation reserve (Fig. 2d, right scale) allows
for at least one decade of general attenuation without serious quantisation errors.

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Figure 2. Bandwidths and efficiency of the wavelength combiner. (a) Spectral irradiances measured at the
tip of the LEDs. Dotted and dashed line represent the white and green phosphorescent LEDs, respectively.
(b) Spectral irradiances of the individual LEDs at the fibre output, adjusted for equal photon flux via PWM.
Thick and thin lines show the spectra of consecutive LEDs. (c) The data of panels (a,b) compared in a semi-
logarithmic plot. The dotted and dashed lines represent the irradiance spectra of a white LED and green
phosphorescent LED, respectively. The arrow shows the spectral narrowing of the white LED. The crosses mark
peak irradiances of the LEDs. (d) A semi-logarithmic plot of the overall coupling efficiency of individual LEDs
(squares), range 0.1~1.6%, and the PWM duty cycle used to achieve equal photon fluxes at the fibre output
(2% for LED 420 nm, 100% for the white LED) (diamonds). The scale on the right side shows the PWM
quantisation levels.

Intracellular spectral sensitivity measurements. We used the LED synthesiser to measure the spectral
sensitivities of R1–6 photoreceptors of the blowfly Calliphora vicina (white-eyed mutant Chalky) by intracellular
recordings (Fig. 3), applying 25 ms monochromatic light pulses. In the calibration run, applying 474 nm light,
we used a geometric series of intensities, corresponding to 0.3 decade steps spanning 3.6 decades. The maximal
response amplitude was about 35 mV. The flash from a high-current green LED (525 nm, 350 mA) launched
through the zero order input angle produced a non-saturating response with 40 mV amplitude (Fig. 3a). To reach
saturation, the LED output would need to be about one decade stronger.
An isoquantal spectral run at 22 wavelengths could be obtained in less than two seconds (Fig. 3b). Due to the
short duration of the sweep, we were able to repeat ten measurements within 30 seconds. The spectral sensitivity
curve of Fig. 3c was obtained by averaging the response amplitudes and transforming them with the inverse
function of the Hill (Naka-Rushton) sigmoid fitted to the amplitudes of the calibration sweep. As a control, we
performed the same spectral sensitivity measurement using a classical photostimulator, yielding very similar
results (Fig. 3c).

Hyperspectral imaging of blood oxygenation. Due to its small étendue, the LED synthesiser is a highly
suitable source for hyperspectral microscopic imaging (Fig. 4). In order to test the wavelength resolution of the
system, we first acquired an image stack of fresh human blood lysed in distilled water, using the LED synthesiser
and a monochrome CMOS camera coupled to a microscope. The blood extinction spectrum obtained from the
hyperspectral stack was in close agreement with that measured with a spectrophotometer, and with the tabu-
lated extinction spectrum of human oxyhaemoglobin19. We also measured the blood spectrum using bare LEDs
(Fig. 2a,c), but this measurement failed to show the fine structure of the oxyhaemoglobin (Fig. 4a), thus demon-
strating the advantage of bandwidth narrowing by the grating and the fibre.
To investigate the blood absorption spectra in situ, we performed hyperspectral imaging of Xenopus abdom-
inal skin, a respiratory tissue where the veins contain oxygenated blood and the arteries contain deoxygenated
blood20. We illuminated the skin from the outside and acquired an image stack of the internal side of the skin
(Fig. 4b). Figure 4c shows the absorbance spectra of an artery, a vein, a capillary and adjacent tissue. We processed

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Figure 3. Intracellular spectral sensitivity measurements from R1-6 photoreceptors of a blowfly.

(a) Membrane potential trace of an R1-6 blowfly photoreceptor upon stimulation with a graded series of
25 ms pulses with 474 nm light. The pulse intensity was graded in a geometric series (PWM 1, 2, 4, …​, 212).
The decadic logarithm of the light intensity is indicated on the top axis. The last response (dot) was evoked
with a 350 mA pulse from a power LED peaking at 525 nm, directed into the zero-order beam. (b) Membrane
potential trace of an R1-6 blowfly photoreceptor upon isoquantal spectral stimulation with 25 ms pulses from
the LED synthesiser. (c) Spectral sensitivity obtained from an average of ten sweeps with the LED synthesiser
(solid circles). For comparison, the spectral sensitivity obtained from a single spectral sweep with a classical
photostimulator is shown (open circles).

the image pixels with a linear unmixing procedure21,22, using six spectral components. The oxyhaemoglobin
(HbO2) and deoxyhaemoglobin (Hb) extinction spectra19 were cut into two long wavelength (LW) and two short
wavelength (SW) components, and additionally we used a spectrally neutral and a red component.
The component contributions are presented in Fig. 4d. The LW oxyhaemoglobin component (Fig. 4d1) reveals
both arteries and veins, while the LW deoxyhaemoglobin component shows the arteries only (Fig. 4d2). The two
SW components (Fig. 4d3,d5) emphasize the thin capillaries. The neutral component (Fig. 4d4) shows melano-
cytes lining the vessels and the mucous glands as dark and bright spots, respectively. The red component, showing
thicker vessels, was necessary to account for the reduced absorbance above 580 nm, which is presumably due to
scattering by erythrocytes (Fig. 4d5).

Discussion
We constructed an LED synthesiser spanning the wavelength range 350–630 nm. With the prototype, based on
inexpensive low-current LEDs, we were able to successfully perform physiological and spectroscopic experiments
that would otherwise require a much more complex and expensive light source. The heart of the system is a planar
reflective diffraction grating, which both combines and cleans (i.e. narrows the bandwidth of) the light inputs.
The main optical components are 5 mm epoxy-mould LEDs, a grating, an aspheric lens and a light guide. The
custom-made parts are the LED holders with collimator lenses. The size of the holders and the grating density
determine the optical bench length, currently about 1 m. The electronics consists of an Arduino Due microcon-
troller and a 12-bit PWM driver. The overall cost of the components is about €700 (€100 for the LEDs, €100 for
the electronics, €500 for the optical components without the breadboard).
Compared with systems based upon a white light source and a monochromator, the LED synthesiser has
a strongly reduced stray light component, because the LED channels are switched on only when their out-
put is needed. Moreover, while the crosstalk from the second diffraction order (e.g. UV crosstalk in the range
580–800 nm) is problematic with XBO-monochromator sources, this is not an issue in the case of LEDs, because
they have little output at half the main peak wavelength.
We have tested the capability of the prototype in a fast measurement of the spectral sensitivity of blowfly
photoreceptors by intracellular recording. The LEDs were attenuated to be isoquantal via PWM. A single spec-
tral run, obtained within seconds, was sufficient for measuring an accurate sensitivity spectrum. The responses
of blowfly photoreceptors to maximal intensity pulses were non-saturating, but this is not a serious limitation,
since reaching saturation is not a necessity for many measurement paradigms. Also, because the 1 kHz switching
frequency is sufficiently above the 60 Hz corner frequency of the blowfly R1–6 photoreceptors23, the measured
responses did not show significant ripple. The LED wavelength spacing of about 15 nm is adequate for photore-
ceptor measurements, with the rare exceptions of the spectral sensitivity of fly photoreceptors in the UV24 or of
the narrow-peaked red photoreceptors of some butterflies25.
Several measurement paradigms for visual physiology could be implemented autonomously in the micro-
controller: selective adaptation26, lock-in signal extraction27, various versions of voltage clamp by light28–30, or
random spectral stimulation with maximal length binary sequence (m-sequence) stimulation31 in the wavelength
domain. The LEDs could also be connected to analogue LED drivers if white-noise stimulation10,32 is desired.
Another attractive option for wide field stimulation33 would be to couple the LED synthesiser to a digital mirror
device (DMD) projector. The effective intensity of the LED synthesiser coupled to a DMD projector could be
increased using the extended Maxwellian view34.
We have used the LED synthesiser as a light source for microscopic hyperspectral imaging of blood oxy-
genation. A simple unmixing procedure with six components revealed arteries, veins and capillaries. Absolute
haemoglobin concentrations and tissue oxygen saturation levels can be estimated with the unmixing, provided
that sufficiently rigorous calibration is performed. For oxymetry of the retinal fundus, the LED synthesiser and a
monochrome camera could be easily coupled to an ophthalmoscope7.

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Figure 4. Imaging spectroscopy of lysed human blood and of Xenopus skin in situ. (a) Comparison of
tabulated molar extinction spectra of human oxyhaemoglobin (HbO2, dotted line) and deoxyhaemoglobin
(Hb, dashed line) with scaled absorbance measurements of diluted fresh human blood. The spectrophotometric
measurement (solid line) closely follows the HbO2 spectrum above 440 nm; at wavelengths below 440 nm, the
measurements are unreliable (shaded area). The absorbance spectrum obtained with imaging spectroscopy
using the LED synthesiser (solid circles) closely follows the HbO2 spectrum above 420 nm. The absorbance
spectrum obtained with bare LEDs (open circles) fails to reproduce the fine structure of the HbO2 absorbance
spectrum at 530–580 nm. (b) Absorbance spectra from the artery (A), vein (V), capillary (C), skin background
and bright spots. (c) Images of the frog skin obtained with the LED synthesiser. The thin capillaries are best seen
in the images between 390 and 430 nm. At 625 nm, the thick vessels show a bright lumen and are surrounded
by dark pigmented cells. (d) Images obtained by unmixing with six absorbance components: HbO2 and Hb
above (1, 2) and below 455 nm (3, 6), a spectrally neutral (4) and a red component (5). The LW Hb component
discriminates the vein from the artery. The SW Hb component shows an image of the capillaries. (e) False-color
image of frog skin. Components 1–3 from (d) were allocated to RGB channels (Red, LW HbO2; Green, SW Hb;
Blue: LW Hb). Inset: colour photo of the analysed patch. Scale bar in (d): 1 mm.

When using white light illumination for spectrophotometric absorbance measurements, stray light and the
limited dynamic range of diode-array spectrophotometers often limit the measurable absorbance range to about
three decades (Fig. 4a). Although this dynamic range can be expanded by using a filter that selectively blocks the

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light in the wavelength range where the sample has low absorbance, the LED synthesiser offers the same function-
ality, but with added flexibility: the spectrometer reading can be optimized by dynamically adjusting the source
composition via a similar procedure as being used for component unmixing. Due to high linearity and stability of
the LEDs, repeated reference measurements are not required. As the LEDs have temporal bandwidths in excess of
1 MHz, the LED synthesiser can be used as the source for high speed spectroscopic measurements35 by employing
faster driver electronics.
In colour science, the LED synthesiser can be used as the light source for BRDF (bidirectional reflectance
distribution) measurements with an imaging hemispherical scatterometer36, or with an imaging angular reflec-
tometer37. If larger scenes need to be illuminated from several angles, the LEDs and output fibres can be stacked
in vertical columns around a single grating, or several LED synthesisers can be operated in synchrony.
The multi-band light synthesis by means of wavelength combining presented here brings about important
advantages over solutions with broad-band light sources and dichroic mirrors or monochromators. The reflective
diffraction grating transforms the often complex spectra of the various LEDs into bands with uniform band-
widths. Furthermore, the LEDs have a long operational life and the optical system has no moving parts. The
LED synthesiser has adequate light output for transmission and reflection light microscopy, and it may become a
standard tool for visual physiologists and a rig in advanced neurophysiology lab courses38. Particularly appealing
are possible applications in biomedical imaging and in clinical electrophysiology of vision. With brighter LEDs,
the synthesiser will become also attractive for fluorescence microscopy, optogenetics, and as a general hyperspec-
tral illumination source.

Methods section
Theory of wavelength combining. A grating with groove density G diffracts a light beam of wavelength λ,
incident at an angle α to the grating normal, into diffraction orders (m =​  …​ −​1, 0, 1, …​), exiting at angles βm,
according to the grating equation18:
mGλ = sin α + sin β m (1)
The zero order (m =​ 0) represents specular reflection: α =​  −​β0. Non-zero orders are diffracted when |mGλ| <​  2
and are evanescent otherwise. Solving Eq. 1 for the limiting condition (grazing exit angle, sin β+1 =​ 1) yields the
relation between the grating density and the highest passable wavelength: λ+1 =​  2/G. For the UV-visible range
(300–700 nm) it follows that G <​ 2700 mm−1.
The light synthesiser works as a beam combiner. The beams are incident upon the grating at input angles α(λ)
and exit under a constant exit angle of the positive first order diffraction, β+1:
α (λ) = arcsin(Gλ − sin β +1) (2)
A design constraint of the wavelength combiner is that the exit angle β+1 should not overlap with the input
angles. The overlap is avoided when:
β +1(λ) > arcsin(0.5Gλ) (3)
For a grating with G =​ 1850 mm (λ+1 =​ 1081 nm), the exit angle β+1 should be larger than 47° to avoid the
−1

overlap below λ =​ 800 nm. Another point of consideration is the existence of the negative first order. The grating
equation (Eq. 1) yields as the limiting case for orders m =​  (+​1, −​1):

λ−1 = 0.5G−1 (sin β +1 − sin β−1) (4)

The wavelength limit for which the negative order exists for the particular design λ−1(G, β+1) is found by
setting sin β−1 =​  −​1. By further setting the exit angle to β+1 =​  90° (sin β+1 =​ 1), the maximal wavelength of the
negative order is λ−1, max =​  G−1. For the grating density 1850 mm−1, the maximal negative first order wavelength
is 540 nm.

Optical setup. We used a blazed grating with density G =​ 1850 mm−1 (Thorlabs GR50-1850, nominal blaze
wavelength 500 nm). The exit angle was set to β+1 =​ 50° (Fig. 1c). The exit beam was launched with an aspheric
lens (f =​ 32 mm, ACL50832U, Thorlabs) onto an optical fibre, either an 0.4 NA, high OH, 1 mm fibre (M35L01,
Thorlabs) or an 0.22 NA, fused silica, 200 μ​m fibre (P200-2-UV-VIS, Ocean Optics).
The span of the input angles for the wavelength range 350 to 630 nm was ~30°. Because the illumination cones
of the LEDs were larger than the grating, the projection efficiency was approximately proportional to the projec-
tion of the input angle, cos α(λ), i.e. it is half-maximal at 60° incidence. For G =​ 1850 mm−1 and β+1 =​  50°, the
projection efficiency is down to 0.8 (80%) at 700 nm. The negative first order existed up to λ−1 =​ 472 nm (Eq. 4).

LEDs. LEDs (epoxy mould, diameter 5 mm, maximal current 20 mA) with peak wavelengths between 350 nm
and 630 nm were purchased from an on-line reseller (www.roithner-laser.com). The LEDs have been selected for
small emission angles (Δ​ρ1/2 =​ 8–15°). The spectral range was covered in steps of approximately 15 nm. 22 LEDs
were mounted into aluminium T-shaped profiles (30 ×​  30 ×​ 13 mm, thickness 3 mm) with collimating lenses (LT-
0454, www.led-tech.de). The LEDs were secured with M3 screws along a 15 ×​ 15 mm aluminium beam with
T-slots (Open beam, www.openbeamusa.com) and mounted on the holders about a meter away from the grating
(Fig. 1a). The input and output peak wavelengths differed maximally ~3 nm.

Electronics. An Arduino Due microcontroller with ARM Cortex M3 processor (www.arduino.cc) controlled
a TLC5947 chip-based 24-channel PWM LED driver (12-bit resolution, 4 MHz clock, update frequency 1 kHz,

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www.adafruit.com) via the SPI bus. The software that controlled the driver and communicated with the computer
over the USB was written in the Arduino IDE environment, using the provided libraries.

Calibration procedure. For calibration measurements, we used a radiometrically calibrated spectropho-

tometer (Flame, Ocean Optics, USA) and a photodiode with a linear amplifier (OPT-101, Texas Instruments,
USA). When necessary, we used the same fibre as the one for stimulation and a collimator (74-UV, Ocean Optics,
USA). The positions for mounting individual LEDs were first empirically determined by illuminating the dif-
fraction grating with white light from the XBO lamp in reverse, via the optical fibre and the launching lens. The
diffracted light was projected onto the mounting beam and the approximate positions corresponding to LED
emission peaks were determined with the spectrophotometer.
To monitor the LED light, the XBO lamp was substituted with the spectrophotometer, and the LEDs were
precisely positioned so that their light output around the declared emission peak was maximised. Most of the
LEDs were additionally collimated with the LED holder’s collimating lens. For some LEDs, the collimating lens
degraded the light output and was removed.
Light fluxes of the LEDs in the array were measured with the spectrophotometer. The efficiency of individual
LEDs was calculated by dividing the output intensity from the optical fibre with the intensity measured by collect-
ing the light with the same fibre, equipped with a collimating lens, positioned next to the LED on its optical axis.
We integrated the photon flux of each channel in a 20 nm bin around the emission peak, using the radiometri-
cally calibrated spectrometer, which measured the light reflected from a magnesium oxide block positioned 15 cm
in front of the fibre. In order to generate isoquantal stimuli, we adjusted the PWM for each channel until the irra-
diance integral was inversely proportional to the wavelength. Consequently, the LEDs with narrower bandwidths
had somewhat higher peaks. The most effective LED had to be attenuated to a 2% duty cycle and the white LED
was running at the full duty cycle (Fig. 2d).

Classical photostimulator. The classical photostimulator was constructed from a stabilised XBO lamp
(66475-150XV-R22, Newport USA), a monochromator (model 77250, Oriel/Newport) and a motorised, graded
neutral density filter on a fused silica substrate (NDL-25C-4, Thorlabs). We operated the photostimulator in the
wavelength range 300 nm to 700 nm with 1 nm or 5 nm steps in isoquantal mode.

Electrophysiology. The white-eyed blowflies Calliphora vicina Chalky were reared in a lab culture.
Experimental animals were immobilised and put into a motorised goniometric positioner stage (Standa,
Lithuania), which directed a collimated fibre output towards the centre of rotation of the goniometer. The pho-
toreceptor cells were impaled with a sharp borosilicate microelectrode (resistance ~80 MΩ) and illuminated
with a series of isoquantal flashes of 25 ms duration, which were produced either with the LED synthesiser or the
classical photostimulator. The photoreceptor membrane voltage was amplified with a high impedance amplifier
(SEC10LX, NPI electronic, Germany). The signals were acquired using a 16-bit laboratory interface (1401micro
Mk2 with Signal2 software, Cambridge Electronic Design, UK) and Stratchlyde WinWCP software39,40.
The stimulus-response relation was estimated from the calibration run (Fig. 3a) by fitting the response ampli-
tudes V(I) to the Hill (Naka-Rushton) function:

V (I ) = V0 I n (Rn + I n)−1 (5)

here I is the light intensity, V0 the maximal response, R the intensity needed for a half-maximal response, and n
the slope of the sigmoid. Figure 3b presents the raw responses to the isoquantal stimuli. The intensity Ic(λ) neces-
sary to create a criterion response Vc then is:

Ic (λ ) = R [V0/Vc − 1]−1/n (6)

The spectral sensitivity is subsequently calculated as the normalised inverse criterion intensity .
25,41,42

Hyperspectral imaging. We performed spectroscopy of a human blood sample. The sample (200 μ​l) was
collected from a finger and lysed in 5 ml distilled water. The absorbance spectra of the diluted blood were acquired
on a Leitz Orthoplan microscope in transmission mode with a Flame-S spectrophotometer (Ocean Optics, USA),
using the XBO source. A hyperspectral image stack was taken with a monochrome CMOS camera (Grasshopper
2.3 MP USB3, Point Grey Research, Canada), using the light output of the LED synthesiser.
We also performed hyperspectral imaging of the skin of adult African clawed frogs, Xenopus laevis,
obtained from a commercial supplier (Xenopus Express, France). The frog was euthanised by brain concus-
sion and the brain and spinal cord were destroyed with a steel probe inserted through the palate. The experi-
ment was performed in a cadaver with a preserved functioning circulatory system. A euthanised frog does not
breathe with its lungs, so the skin is its only functional respiratory surface. Therefore, the skin arteries carry
dark red deoxygenated blood, while the veins carry light red oxygenated blood. The identity of both types of
blood vessels can be determined with the application of norepinephrine, which constricts the arteries, but not
the veins20.
An incision was made on the abdomen, exposing the inner surface of the abdominal skin of about 2 by 3 cm.
The skin’s outer surface was illuminated with the LED synthesiser through a 200 μ​m diameter light fibre focussed
with a fused silica collimator from about 2 cm distance from the skin. A monochrome camera was attached to a
stereomicroscope. The individual LED stimuli were set to maximal output to obtain a hyperspectral image stack.
Due to the small aperture of the stereomicroscope objective and scattering loss, skin images had to be taken with

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a relatively long exposure of 1 s and gain 10 dB. The images of the reference measurement were taken with 5 ms
exposure and 0 dB gain.
The stacks with 20 images were preprocessed with Fiji (www.fiji.sc). Hot pixels were replaced with the median
value of neighbouring pixels. The images with low signal amplitudes (UV and green wavelength ranges) were
additionally filtered with a Gaussian low pass filter. The stacks were scaled down to images with 576 ×​  512 pixels,
exported to Octave (www.octave.org) and transformed to decadic absorbance, using a reference stack. The pixel
absorbance spectra were unmixed21 as the sum of six component spectra (short and long wavelength parts of
haemoglobin and deoxyhaemoglobin extinction spectra, cut at 455 nm; neutral component and red component).
The unmixing procedure was optimised to a single matrix-vector multiplication per pixel and fully vectorised
to reduce the stack processing time in Octave to ~0.1 s. A detailed description of the analysis will be published
elsewhere.

Animal experiment clearance. Experiments in euthanized animals were performed at the Department
of Biology of the University of Ljubljana in accordance with the national guidelines (ZZZiv-UPB 23, Ur. list RS
38/13) and approved by the Administration of the Republic of Slovenia for Food Safety, Veterinary Sector and
Plant Protection (Permission no. U34401-32/2014/4).

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Acknowledgements
The authors dedicate this work to the memory of prof. Matti Weckstrom. The authors thank drs. Kentaro Arikawa,
Doekele Stavenga, Trevor Wardill and Bodo Wilts for commenting on the manuscript. Financial support was
provided by the AFOSR-EOARD (grant FA9550-15-1-0068 to GB and AM), ARRS (grant P3-0333 to GB, young
researcher grant to MI) and JSPS (Kaken-hi 26.04764 to PP).

Author Contributions
G.B. and P.P. conceived the instrument. P.P. assembled the proof-of-principle setup, and performed theoretical
calculations. M.I. programmed the microcontroller and contributed a droplet of his blood. A.M. and M.I. set
and wired the prototype. A.M., G.B. and M.I. performed calibrations and animal experiments. G.B. analysed the
intracellular recordings. P.P. analysed data and prepared the figures. G.B. and P.P. wrote the paper. All authors
contributed to the final draft.

Additional Information
Competing financial interests: The authors declare no competing financial interests.
How to cite this article: Belušič, G. et al. A fast multispectral light synthesiser based on LEDs and a diffraction
grating. Sci. Rep. 6, 32012; doi: 10.1038/srep32012 (2016).
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Scientific Reports | 6:32012 | DOI: 10.1038/srep32012 9