This article has been accepted for publication in a future issue of this journal, but has not been

fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/TMI.2019.2937760, IEEE
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Comparison of NIR versus SWIR fluorescence

image device performance using working
standards calibrated with SI units
Banghe Zhu, Sunkuk Kwon, John C. Rasmussen, Maritoni Litorja, and Eva M. Sevick-Muraca*

Abstract— Recently, fluorescence imaging using shortwave

infrared light (SWIR, 1,000-2,000 nm) has been proposed as I. INTRODUCTION
having advantage over conventional near-infrared fluorescence
(NIRF) imaging due to the reduced tissue scattering, negligible
autofluorescence, comparable tissue absorption, and the discovery
that indocyanine green (ICG), used clinically as a NIRF contrast
O ver the past two decades, techniques of near-infrared
fluorescence (NIRF) imaging have been developed and
translated into clinical use [1-3]. The technique utilizes incident
agent, also has fluorescence emission in SWIR regime. Images of light within the wavelength range of 760 - 900 nm, within which
ICG in small animals acquired by commercial Si-based and the hemoglobin and water in tissues exhibit low absorption such
InGaAs-based imaging cameras have been qualitatively that incident near-infrared (NIR) light penetrates deeply into the
compared, however the lack of working standards to quantify tissue, activates exogenous NIRF agents, and allows imaging of
performance of these imaging systems limits quantitative large tissue volumes from the emitted fluorescent light. ICG
comparison. Without quantification using a traceable in vitro test,
clinical adoption of rapidly evolving advances in both NIRF and
was originally approved by the US Food and Drug
SWIR imaging devices will become limited. In this work, we Administration (FDA) in 1956 as a reagent for clinical use for
developed an ICG based fluorescent solid working standard determining cardiac output, hepatic function and ophthalmic
calibrated with SI units (mW · cm-2· sr-1) for quantification of angiography following i.v. administration and has an excellent
measurement sensitivity of Si, GaAs-intensified Si, and InGaAs record of safety. Today there are now approximately 25
based camera systems, their signal-to-noise ratio (SNR), and commercially available NIRF imaging devices including open-
contrast in non-clinical tests. In addition, we present small animal field surgery camera systems, endoscopes, and laparoscopes
and large animal imaging with ICG for qualitative comparison of with the majority designed for intraoperative detection of ICG
the same SWIR fluorescence and NIRF imaging systems. Results to assess perfusion of skin flaps [3]. These devices employ 700
suggest that SWIR fluorescence imaging of ICG may have
superior resolution in small animal imaging compared to NIRF
– 806 nm excitation light and collect fluorescent emission
imaging, but lack of measurement sensitivity, SNR, contrast, as between 800 nm and 900 nm. Yet unlike all other conventional
well as water absorption limits deep penetration in large animals. imaging modalities, these NIRF imaging devices lack IEC
(International Electrotechnical Commission) traceable
Index Terms— Molecular and cellular imaging, Optical measurements of performance in International System (SI) of
imaging/OCT/DOT, Evaluation and performance, System design, units that enable accurate predictions of sensitivity/resolution
Validation. for detecting ICG or other NIRF imaging agents with non-
Manuscript received May 30, 2019; accepted August 21, 2019. This work was clinical tests. The seven base SI units are defined using the
supported in parts by the Translational Research Institute through NASA physical constants of the universe, lending confidence that all
Cooperative Agreement NX16AO69A. scientific and commercial measurements that are SI-traceable
B. Zhu is with the Brown Foundation Institute of Molecular Medicine, The
University of Texas Health Science Center, Houston, Texas, 77030 USA (e- are comparable across instrumentation, methods, time and
mail: Banghe.Zhu@uth.tmc.edu). location. The lack of non-clinical, metrologically-traceable test
S. Kwon is with the Brown Foundation Institute of Molecular Medicine, The standards complicate the efficient adaptation and efficient
University of Texas Health Science Center, Houston, Texas, 77030 USA (e- translation of ongoing, rapid technological advancements of
mail: Sunkuk.Kwon@uth.tmc.edu).
J. C. Rasmussen is with the Brown Foundation Institute of Molecular optical components driven primarily by the military and
Medicine, The University of Texas Health Science Center, Houston, Texas, communications industries [4, 5]. One recent example of
77030 USA (e-mail: John.Rasmussen@uth.tmc.edu). technological advancement is the recent advent of InGaAs-
M. Litorja is with the National Institute of Standards and Technology, based devices that expand biomedical fluorescence imaging
Gaithersburg, MD 20899 USA (e-mail: maritoni.litorja@nist.gov).
*E. M. Sevick-Muraca is with the Brown Foundation Institute of Molecular devices into the shortwave infrared (SWIR, 1,000 –2,000 nm)
Medicine, The University of Texas Health Science Center, Houston, Texas, wavelength regime [6]. Because the degree of tissue scattering
77030 USA (e-mail: Eva.Sevick@uth.tmc.edu). is inversely proportional to wavelength λ-α (α=0.2~4 for
JCR, BZ and EMS-M are listed as inventors on patents related to near- biological tissues), fluorescence at longer SWIR wavelengths
infrared fluorescence lymphatic imaging and may receive future financial
benefit from its commercialization. JCR, BZ, EMS-M and The University of experiences reduced tissue light scattering and less
Texas Health Science Center at Houston have research-related financial autofluorescence than at NIRF wavelengths, albeit at the
interests in Lymphatics Technologies, Inc. The remaining authors have no expense of somewhat increased water absorption [7]. The
financial relationships relevant to this article to disclose.

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benefits of reduced photon scattering, negligible autofluores- II. MATERIALS AND METHODS
cence, and comparably low absorbance in SWIR window have A. ICG based working standards calibrated with SI units
been hypothesized to enable high-resolution imaging at greater Previously we have used stable, working standards based upon
penetration depths than achievable in NIR window [8-12]. the small fluorescent yield of QDots800 to assess performance
Moreover, ICG has recently been found to have SWIR of various custom and commercial NIRF devices that operate
fluorescence emission (>1064 nm) following excitation at 808 within the same or similar wavelength regimes. In this work,
nm and its SWIR emission spectra can be found in [13]. ICG detection of ICG at NIR and SWIR wavelengths defines the
has been successfully used in small animals to demonstrate task for which device performance is assessed. For this reason,
SWIR intravital microscopy, noninvasive SWIR imaging in we constructed working standards similar to that of our prior
blood and lymph vessels and SWIR assessment of hepatobiliary work [4, 5], using ICG as opposed to QDots800. The standard
clearance using emerging commercial InGaAs array detectors consists of 6 wells machined from a 96 well plate and filled with
[14]. Despite the small fluorescent yield of ICG at 1064 nm a mixture of polyurethane, TiO2 (1.5 mg/mL) and increasing
following the NIR excitation, the large 200 nm Stokes shift may concentrations of ICG diluted in water or in ethanol.
be particularly advantageous for SWIR fluorescence imaging. Polyurethane was chosen as the base material for the phantom
Even with a comparatively larger fluorescent yield, the due to its optical clarity, fast curing time, and affordability;
incomplete rejection of excitation light due to the small (10 – TiO2 was added to the phantom to exaggerate the optical
60 nm) Stokes shift experienced in NIRF systems limits their scattering properties of tissue that lead to incomplete excitation
performance [15], a problem potentially avoided by the large rejection; and 6 wells of the phantom had varying
Stokes shift probed by SWIR imaging. As a result, SWIR has concentrations of ICG. A low radiance phantom was
thus been proposed as having advantage over NIRF imaging for constructed to have similar fluorescent radiances to our prior
clinical applications [13, 14], yet no quantitative comparison work with ICG concentrations of 0 (acting as a non-fluorescent
using a non-clinical or in vitro test assessment has been background), 20, 40, 60, 80, and 100 nM and a high radiance
presented. Non-clinical or in vitro testing provides an phantom was constructed with ICG concentrations of 200, 400,
inexpensive, repeatable, and when reported in SI units, 600, 800 and 1000 nM. To calibrate the radiance from the ICG
traceable measurement that can hasten direct comparison and based fluorescent solid phantoms with SI units in NIR window,
enable evaluation of improvement in device advancements such we used the same methodology described previously for
as potentially offered by SWIR detectors. Figure 1 is a calibration of QDots 800 based luminescent solid tissue
schematic depicting the typical spectral responses of the phantoms with SI units [5]. Briefly, a NIST calibrated power
systems currently deployed in clinical imaging of ICG. meter is used to assign a radiance scale to an 830 nm diffuse
source (830 nm laser diode and integrating sphere, known as
the “reference source”) with output matching emission from the
fluorescent tissue phantoms. Both reference source and tissue
phantoms are viewed by the camera system under the same
radiometric conditions. Thus, the reference source is used to
calibrate the arbitrary unit (AU) output of the camera system
configured under specific radiometric conditions and
transferring the radiance scale (mW ∙ cm−2 ∙ sr −1 ) to the tissue
phantoms illuminated at a calibrated irradiance at 785 nm. The
tissue phantoms can then be used as working standards at this
wavelength to evaluate camera systems at any other radiometric
configuration. To calibrate SI units in SWIR window, we
replaced 785 nm excitation source with 808 nm excitation
source, and the 830 nm reference source with 1064 nm
reference source with all other configurations held constant.
Fig. 1. The typical spectral response curves of detectors made of Si, GaAs, Radiance from the integrating sphere reference sources were
and InGaAs materials. measured by NIST-calibrated InGaAs- and Si-photodiode
power meters at the wavelengths of 1064 nm and 830 nm
The main objective of this work is to evaluate performance (NIST15, Gaithersburg, MD; S2281-04, Hamamatsu, Japan).
of Si-, InGaAs- and GaAs intensified- based camera systems
for detecting ICG using an in vitro, fluorescent solid phantom B. NIR and SWIR fluorescence imaging systems
calibrated with SI-traceable units. A secondary objective is to In NIRF imaging set-ups shown in Fig. 2(a), a 785 nm laser
relate performance assessed from the quantitative in vitro test diode (HPD1005-9mm-78503 model, High Power Devices
to qualitative images from large and small animals. It should Inc., NJ) that was typically employed in our investigational
be noted that the comparisons made herein are specific to the devices was used to illuminates the tissue/standard surfaces of
devices compared, and that the methodology for assessing interest through a diffuser. The collection of fluorescence
performance in SI-traceable units could be used to drive signals were implemented using two 830 nm band pass filters
technological advancements that improve existing and future (830FS10, optical density > 5 at 785 nm, Andover, Salem, NH)
NIRF and SWIR fluorescent devices. separated with the Nikon focus lens (AF NIKKOR 28 mm
f/2.8D, Nikon, NY). The Nikon lens introduced a small amount

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Fig. 2. Schematics of NIR (a) and SWIR (b) fluorescence imaging systems installed with Si, GaAs coupled Si, and InGaAs based cameras.

of loss between two filters to efficiently cancel multiple-path SWIR fluorescence imaging systems. To calculate the SNR and
interference effects and increase the combined optical density. contrast, the ICG-based fluorescent solid phantom with SI units
This reduced the leakage of excitation light through the filters. was first positioned under the FOV and the irradiance measured
The collected fluorescence signals were then registered on a Si- on the phantom surface was 3.7 mW/cm2 for 785 nm excitation
based commercial electron-multiplying couple-charged device and 4.2 mW/cm2 for 808 nm excitation. Fluorescence images
(EMCCD) camera (Photon Max 512, Princeton Instruments, were acquired at two different integrating times of 33 and 200
Trenton, NJ) or a GaAs-based intensified scientific ms with the gain adjusted such that an individual pixel value
complementary metal–oxide–semiconductor (IsCMOS) within the phantom well with the highest ICG concentration
camera (a customized fiber optic coupled Zyla 5.5 sCMOS, was just less than the full well capacity of the camera. From the
ANDOR, Concord, MA) as developed in our past works [5]. In acquired fluorescence images, the SNR corresponding to each
the SWIR fluorescence imaging system shown in Fig. 2(b), a well in the calibrated ICG-based fluorescent solid phantom was
laser source operating at 808 nm (L808P1000MM model, calculated using the following equation:
Thorlabs, NJ) was utilized to illuminate the tissue/standards.
S t C   S b 0
The choice of 808 nm as the excitation wavelength was based SNR  (1)
upon prior literature reports of SWIR fluorescence detection of 
ICG. The generated fluorescence signals were collected with a where S t C  and S b 0 represent the averaged arbitrary units
1064 nm long-pass filter (BLP01-1064R, optical density >5 at
808 nm, Semrock, Rochester, NY) and then focused through a (AU) values for the pixels corresponding to the entire well
SWIR lens (SR2343-A01 model, StingRay Optics, Keene, NH) containing C concentration of ICG and to the well without ICG
onto an InGaAs-based PIN Photodiode camera (Ninox SWIR respectively, and σ represents the standard deviation of AU
640, Raptor Photonics, kindly loaned by Phoenix Engineering, values in the well without ICG. The calculated SNR was then
Carson, CA). The working distances and hence radiometric plotted as a function of radiance (mW ∙ cm−2 ∙ sr −1 ) of each
conditions were identical for all systems. All cameras were well in ICG based fluorescent solid phantom. The contrast was
cooled to their corresponding temperatures suggested by the computed by the following relationship:
manufacturers and the field of views (FOVs) of both NIR and S t C 
Contrast  1 (2)
S b 0
SWIR fluorescence imaging systems were adjusted to 10 cm in
diameter.
The computed contrast was then plotted as a function of
C. Figures of merit for fluorescence imaging radiance (mW ∙ cm−2 ∙ sr −1 ) of each well of the working
Signal-to-noise ratio (SNR) and contrast were used to compare standard.
and quantify the measurement sensitivity of both NIR and

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D. Small and large animal imaging with NIRF and SWIR emission radiance to excitation irradiance for both NIR and
imaging systems SWIR fluorescence channels is proportional to the ICG
Animal studies were conducted under protocols approved by concentration, although at the lower and upper ranges of
the University of Texas Health Science Center Animal Welfare radiance, the radiometric-specific function curves slightly
Committee. Dynamic imaging was performed using the deviate from linearity, demonstrating the dark noise of the
imaging systems described above on anesthetized athymic mice detector and maximum measurable radiance prior to camera
after 100 µL of 323 µM ICG was injected into the tail vein. saturation. The slope of the lines refer to the fluorescent
Images were acquired dynamically with acquisition times of 50 characteristic of the standard, which for our past work with
ms using the EMCCD system and 200 ms using the GaAs-based inorganic QDots800 is stable for years, but for ICG deteriorates
IsCMOS and InGaAs systems. The irradiance measured on the with light exposure and time slowly [5]. As a result, these
animal surfaces has a spatial variation of 3.0~4.4 mW/cm2 for standards were prepared and used in a darkroom and exposure
785 nm excitation and 3.1-5.1 mW/cm2 for 808 nm excitation. to illumination for no more than a few seconds over which time
In ancillary studies of swine, dynamic imaging was conducted their output appeared to be stable. It is noteworthy, that despite
using the IsCMOS and InGaAs systems following the small fluorescence yield at SWIR over NIR wavelengths,
intramuscular injection of 1-2 mL of 62.5 µM using an 18- the radiance in SWIR fluorescence channel is higher than that
gauge needle. in NIRF channel. We believe that the narrow bandwidth of 830
nm bandpass filter limited the amount of NIR fluorescence
collected as compared to the SWIR fluorescence collected
III. RESULTS using the 1064 nm long pass filter. We did not evaluate whether
A. Radiance of ICG based fluorescent solid phantom in SI enhancement of optical effects that occur due to the low
units absorption and high refractive index of TiO2 scattering particles
When reconstituted in water prior to incorporation with in the presence of a fluorophore could be responsible the large
polyurethane and TiO2, or when diluted in ethanol at differences between the NIR and SWIR measurement of
concentrations lower than 200 nM, ICG emission was too weak fluorescent characteristics of the working standard.
for detection or quantification using the SWIR system. Figure
3(a) shows that ICG in ethanol exhibited higher emission at B. Measurement sensitivity of both NIR and SWIR
>1064 nm than ICG in water, which has been attributed to its fluorescence imaging systems
higher absorption cross section in ethanol [13], but could also Figure 5 shows that the SNR of NIRF imaging systems are
be impacted by water absorption at SWIR wavelengths. much higher than that of SWIR system despite that there is an
Because SWIR fluorescence imaging systems can detect 2-3 order of magnitude greater radiance in SWIR fluorescence
emission of the lowest ethanol-diluted ICG concentration (200 channel. At longer integrating times of 200 ms, the SNR is
nM) as shown in Fig. 3(b), working standards prepared with improved as compared to that at 33 ms for both the Si-based
ethanol dilution of ICG at concentrations of 200 to 1000 nM EMCCD and InGaAs systems as depicted in Fig. 5(a) and (c).
were adopted. Figure 4 illustrates the ratio of emission radiance For the GaAs-based IsCMOS imager (shown in Fig. 5(b))
at 830 nm to the incident 785 nm excitation irradiance (ratio shorter integrating time is compensated by the increased gain of
uncertainty = 0.10) as well as the ratio of emission radiance at the intensifier. Figure 6 shows that the Si-based EMCCD NIRF
1064 nm to the incident 808 nm excitation irradiance (ratio imaging system provides approximately twice the contrast as
uncertainty = 0.11) for each of the wells containing different that of SWIR system. In comparison to both, GaAs-based-
concentrations of ICG. It can be observed that the ratio of IsCMOS NIRF imaging system gives greater contrast as shown

Fig. 3. (a) The acquired SWIR fluorescence image of Eppedorf tubes filled Fig. 4. Emission radiance normalized to excitation irradiance of ICG
with ICG diluted in water and ethanol, demonstrating higher fluorescence based working standards in NIR (Left axis) and SWIR (Right axis)
signals in ethanol. (b) Three acquired fluorescence images from ICG based fluorescence imaging channels, where arrows to show which y-axis
fluorescent solid phantom using NIR and SWIR fluorescence imaging corresponds to each dataset.
systems.

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Fig. 5. The plots of signal-to-noise ratio as a function of radiance at 33 ms and 200 ms exposure times using EMCCD (Si) (a) or IsCMOS (GaAs intensified)
(b) camera based NIRF imaging system and InGaAs (c) camera based SWIR fluorescence imaging system.

Fig. 6. The plots of contrast as a function of radiance at 33 ms and 200 ms exposure times using EMCCD (Si) (a) or IsCMOS (GaAs Intensified) (b) camera
based NIRF imaging system and InGaAs (c) camera based SWIR fluorescence imaging system.

in Fig. 6(b). Using working standards with lower NIR

radiances that challenge device sensitivity, we previously
showed that the GaAs-based intensification improved SNR and
contrast compared to non-intensified CCD and sCMOS NIRF
systems [4, 5]. Fig. 7 shows a comparison of SWIR and GaAs-
based IsCMOS performance at low radiances and 200 ms
exposure times as provided by standards made with ICG diluted
with water up to 100 nM. The results suggest that SWIR does
not have as much sensitivity as NIRF-based systems to measure
clinically relevant concentrations of ICG for medical imaging.
Typically, a USAF target is generally used to assess device
resolution which will be a function of irradiance, radiometry,
and pixilation of the detector. However, one advantage of
SWIR over NIR may be the reduced scattering by tissues that
results in the improved resolution of fluorescent targets. To Fig. 7. Signal-to-noise ratio versus contrast for low radiance set of working
demonstrate this potential impact on target detection, we standards that included 0 – 100 nM of ICG diluted in water as measured
by GaAs-IsCMOS NIRF system and InGaAs SWIR system. As contained
determined the full-width half maximum of a (27.5 mm long, in the insert, radiance values at SWIR were not sufficiently detected as in
4.7 mm diameter) rod filled with ICG diluted with ethanol to a the high radiance set of working standards that included > 200 nM of ICG
concentration of 322 μM concentration as it was immersed in diluted in ethanol (Figures 5, 6) and therefore could not be calibrated with
1.0% liposyn, to depths of 0, 2 and 4 mm from the surface. SI units. As a result, SNR is plotted as a function of contrast to show
imaging at SWIR had lower contrast with lower SNR than NIRF imaging.
Figure 8 (a) shows the acquired NIR and SWIR fluorescence

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Fig. 8. (a) Fluorescence images in NIR and SWIR fluorescence imaging channels of a rod consisting ICG (322 μM) at depths of 0, 2, and 4 mm in 1.0%
Liposyn. (b) Full-width-half-maximum (FWHM) of fluorescent rod as a function of depth in 1.0% Liposyn, demonstrating improved spatial-resolution at
SWIR as opposed to NIRF wavelengths.

images, from which the full-width-half-maximum (FWHM) of fluorescence imaging of ICG or any other potential fluorescent
feature width was calculated, as shown in Fig. 8(b). At this contrast agent [16] that could be translated clinically for
high concentration of ICG, the FWHM of the SWIR ICG signal molecularly targeted detection of diseased tissues. With more
deteriorates less with increasing depth than does the NIRF ICG SWIR sensitive array technologies made of other materials such
signal. as InSb and HgCdTe, or perhaps more impactful, the advent of
To relate performance characterized by the working SWIR sensitive intensifiers that couple to conventional Si-
standards and in vitro tests to actual imaging, we also evaluate based detectors, it may be possible that sensitivity could be
the devices with small and large animal tests. Dynamic in vivo improved beyond that possible with NIRF imaging systems.
imaging of mice injected i.v. with 100 L of 323 M ICG was Interestingly, a recent work by Carr et al [17] demonstrates that
performed using NIRF (EMCCD and GaAs-based IsCMOS) the SWIR system has highest image contrast with a higher
and SWIR fluorescence demonstrate comparable performance imaging penetration depth at the peak absorbance of water
with potentially less scatter and greater clarity with the SWIR (~1450 nm). The SWIR system evaluated herein was less
system, as shown in supplemental videos 1- 3 and Fig. 9. This sensitive in detecting ICG than NIRF systems as depicted in
result may be expected in the context of the results in Figure 8. Figures 5-7. Future comparisons of imaging systems need to
However, when using the larger swine animal, i.m. injection be based on quantitative characterization using traceable
with 2 mL of 62.5 µM can be seen using the GaAs-based working standards that are calibrated in SI units and that are
IsCMOS NIRF system, but not with the InGaAs-based SWIR appropriately used to objectively address performance in
system, as shown in Fig. 10. This latter result is likely due to clearly defined tasks through receiver operator curve analysis.
the relatively strong absorption of water at SWIR wavelengths Before task-based assessments are performed within expensive
that limits penetration depth and is expected with the results in vivo or clinical tests, an in vitro or non-clinical test can
obtained in the ethanol and water-based working standards. provide a simple means of assessing and comparing
performance of different devices, their operation, and
radiometric geometries in which they are used. Subsequent
IV. DISCUSSION evaluation of task-based performance within in vivo or clinical
testing will require calibration with SI units to be effective.
Herein we used an ICG-based working standard calibrated with
Previously, we developed a QDots 800 based working
SI-traceable units to report the quantitative performance of two
standard that, unlike the ICG working standard described
NIR and a single SWIR fluorescence imaging system and then
herein, possessed stable emission over a period of a year or
followed with a qualitative comparison in large and small
greater, but did not possess emission into the SWIR wavelength
animal studies. The advantages of SWIR, namely reduced
regimes. The broad emission spectra of ICG ranging from NIR
tissue scattering, a large Stokes shift that minimizes
to SWIR spectra region allows performance comparisons of
contributions from autofluorescence and excitation light
both NIR and SWIR fluorescence imaging systems possible.
leakage, and the larger bandwidth for fluorescent collection
On the downside, these ICG based working standards
bode well for its deployment in biomedical optical imaging.
photodecay with exposure to light and therefore are not as
However increased water absorption and the smaller
desirable as inorganic ones. Future work to develop stable
fluorescent yield at SWIR wavelengths may offset these
working standards with durable stability and spanning NIR and
advantages, especially for clinical applications that use ICG.
SWIR wavelength ranges could help spur the development of
Because this study was confined to the three separate systems
successful fluorescent imaging devices and the imaging agents
studied, we would like to emphasize that we do not broadly
they are designed to detect.
conclude that NIRF imaging is advantageous over SWIR

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Fig. 9. Example of ICG imaging of small animals using EMCCD (Si) (a) or IsCMOS (GaAs) (b) camera based NIRF imaging system and InGaAs (c) camera
based SWIR fluorescence imaging system.

Fig. 10. Example ICG imaging in large animals using IsCMOS (GaAs) (a) camera based NIRF imaging system and InGaAs (b) camera based SWIR
fluorescence imaging system.

*
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