Characterizing short-wave infrared

fluorescence of conventional near-

infrared fluorophores

Brook K. Byrd
Margaret R. Folaron
Joseph P. Leonor
Rendall R. Strawbridge
Xu Cao
Petr Bruza
Scott C. Davis

Brook K. Byrd, Margaret R. Folaron, Joseph P. Leonor, Rendall R. Strawbridge, Xu Cao, Petr Bruza, Scott
C. Davis, “Characterizing short-wave infrared fluorescence of conventional near-infrared fluorophores,” J.
Biomed. Opt. 24(3), 035004 (2019), doi: 10.1117/1.JBO.24.3.035004.
Journal of Biomedical Optics 24(3), 035004 (March 2019)

Characterizing short-wave infrared fluorescence

of conventional near-infrared fluorophores
Brook K. Byrd,a,† Margaret R. Folaron,a,† Joseph P. Leonor,a Rendall R. Strawbridge,a Xu Cao,a,b Petr Bruza,a
and Scott C. Davisa,c,*
a
Dartmouth College, Thayer School of Engineering, Hanover, New Hampshire, United States
b
Xidian University, Engineering Research Center of Molecular and Neuro Imaging, School of Life Science and Technology, Ministry of Education,
Xi’an, Shaanxi, China
c
Norris Cotton Cancer Center, Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire, United States

Abstract. The observed behavior of short-wave infrared (SWIR) light in tissue, characterized by relatively low
scatter and subdiffuse photon transport, has generated considerable interest for the potential of SWIR imaging to
produce high-resolution, subsurface images of fluorescence activity in vivo. These properties have important
implications for fluorescence-guided surgery and preclinical biomedical research. Until recently, translational
efforts have been impeded by the conventional understanding that fluorescence molecular imaging in the SWIR
regime requires custom molecular probes that do not yet have proven safety profiles in humans. However, recent
studies have shown that two readily available near-infrared (NIR-I) fluorophores produce measurable SWIR
fluorescence, implying that other conventional fluorophores produce detectable fluorescence in the SWIR win-
dow. Using SWIR spectroscopy and wide-field SWIR imaging with tissue-simulating phantoms, we characterize
and compare the SWIR emission properties of eight commercially available red/NIR-I fluorophores commonly
used in preclinical and clinical research, in addition to a SWIR-specific fluorophore. All fluorophores produce
measurable fluorescence emission in the SWIR, including shorter wavelength dyes such as Alexa Fluor 633 and
methylene blue. This study is the first to report SWIR fluorescence from six of the eight conventional fluorophores
and establishes an important comparative reference for developing and evaluating SWIR imaging strategies for
biomedical applications. © The Authors. Published by SPIE under a Creative Commons Attribution 4.0 Unported License. Distribution or repro-
duction of this work in whole or in part requires full attribution of the original publication, including its DOI. [DOI: 10.1117/1.JBO.24.3.035004]
Keywords: short-wave infrared; near-infrared II window; fluorescence spectroscopy; cancer imaging; medical imaging; fluorescence-
guided surgery.
Paper 180553RR received Sep. 19, 2018; accepted for publication Feb. 22, 2019; published online Mar. 8, 2019.

1 Introduction in the field. First, the high price point of specialized SWIR cam-
Early reports of fluorescence imaging in the short-wave infrared eras remains a significant, though not insurmountable, barrier for
(SWIR) regime,1–3 also known as the near-infrared II (NIR-II) many research labs. A more significant barrier arose from the
window, have generated substantial interest in the biomedical conventional assumption that specialized molecular constructs
optics community. Fluorescence imaging in this regime, gener- were required for SWIR fluorescence imaging. These molecular
ally considered to extend from 1000 to 2000 nm, is character- probes can be difficult to obtain, usually manufactured in indi-
ized by reduced photon scatter, minimal tissue autofluorescence, vidual research labs (with a few commercial exceptions), and,
and wavelength-dependent absorption, which can be exploited importantly, do not yet have pharmacokinetic and safety profiles
to optimize image resolution and depth sensitivity.4,5 Broad necessary for mid-to-late-stage translational research. These bar-
efforts to leverage these favorable properties for in vivo imaging riers have generally limited SWIR imaging to animal research in
have facilitated the development of a wide array of SWIR- labs with appropriate expertise.
specific molecular probes used for imaging tumor biomarkers6,7 In this context, recent investigations showing robust fluores-
and vascular dynamics2,3,6,8 in preclinical models. Although a cence emission signals in the SWIR regime from a handful of
complete understanding of these properties and their potential conventional NIR-I dyes [indocyanine green (ICG), LI-COR
implications for biomedical imaging applications has yet to IRDye 800CW, and IR-12N3] represent a major development
be fully realized, these early studies suggest that SWIR imaging for the field.9–11 ICG is an extensively studied fluorophore
may be highly advantageous for visualizing subsurface struc- approved for human use in several clinical indications, and
tures during fluorescence-guided surgery (FGS), noninvasive IRDye 800CW has been used widely in preclinical models and
monitoring of disease, as well as a wide array of preclinical is under clinical investigation for a variety of indications.12 The
applications requiring information from molecular reporters. observation that the tails of the fluorescence emission peaks
Until recently, two characteristics of SWIR imaging presented of these fluorophores are readily detectable well into the SWIR
significant barriers to accelerating translational research efforts window suggests that other common NIR fluorophores likely
produce measurable fluorescence emission in this regime.
The availability of SWIR reporters that are accessible, well-
*Address all correspondence to Scott C. Davis, E-mail: Scott.C.Davis@
characterized, and have historic use in biological and human
Dartmouth.edu systems could further accelerate development and evaluation
†
The authors contributed equally.
of this promising new imaging modality.

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 Byrd et al.: Characterizing short-wave infrared fluorescence. . .

To that end, this study aims to characterize and compare

the fluorescence emission profiles in the SWIR regime of
eight commercially available red/NIR fluorophores as well as
a SWIR-specific fluorophore. Several of these fluorophores are
either approved for clinical use or currently in clinical trials,
whereas others are commonly used for preclinical in vivo stud-
ies. Using a SWIR spectrometer, fluorescence emission spectra
for each fluorophore were examined to establish a comparative
reference of SWIR fluorescence efficiency for different excita-
tion wavelengths. Additionally, a wide-field imaging system
was used to report SWIR fluorescence as a function of concen-
tration in tissue-simulating phantoms. These data will be valu-
able in guiding the development of SWIR fluorescence imaging
strategies for biomedical applications.

2 Methods and Materials

The red/NIR-I fluorophores under investigation in this study
were selected for their general accessibility and historical use
in preclinical and/or clinical studies. ICG (Chem-Impex Int’l.
Inc., Wood Dale, Illinois) and methylene blue (Sigma-Aldrich, Fig. 1 Experimental configuration for (a) SWIR fluorescence spec-
St Louis, Missouri) are both approved for human use, LI-COR’s troscopy and (b) SWIR phantom imaging. (c) Normalized absorbance
spectra of each fluorophore with overlays indicating excitation laser
IRDye 800CW (LI-COR Biosciences, Inc., Lincoln, Nebraska)
wavelengths used to measure SWIR fluorescence.
has undergone clinical investigation for FGS in multiple tumor
sites,12 and LI-COR’s IRDye 680RD and IRDye 700DX, as well
as the Alexa Fluor fluorophores (AF 633, AF 647, and AF 750, For each excitation source–fluorophore combination, 10
ThermoFisher Scientific, Waltham, Massachusetts), are used SWIR fluorescence spectra frames were acquired sequentially
extensively as laboratory standards for in vitro and in vivo im- and the median of these 10 frames was computed, a common
aging. Finally, we included IR-m1050 (Nirmidas Biotech, Inc., acquisition/processing strategy for cameras with high dark
Palo Alto, California), one of the few commercially available noise. Background subtraction was performed using dark count
SWIR-specific fluorophores. spectra without laser illumination. Integrated fluorescence was
With the exception of ICG, which was dissolved in deionized computed between 1130 and 1200 nm and normalized to the
water, all fluorophores were diluted in phosphate-buffered saline maximum integrated intensity value measured for all fluoro-
(PBS). The absorbance spectrum for each fluorophore, acquired phores (ICG excited at 785 nm). Signal-to-noise-ratios (SNRs)
using a Varian Cary® 50 Bio UV–VIS spectrophotometer were calculated as the mean/(standard deviation) of the inte-
(Walnut Creek, California), was used to calculate fluorophore grated intensity values of 10 frames. All measurements were
dilutions. All fluorophores were diluted to a concentration of acquired using the same camera settings, laser power, and
1 μM for SWIR fluorescence spectroscopy. sample preparation and thus were comparable.
A diagram of the SWIR spectroscopy system used to quan- To establish the feasibility of imaging SWIR fluorescence of
tify SWIR fluorescence is shown in Fig. 1(a). The liquid these fluorophores in more realistic conditions, a concentration
samples were prepared in standard 1-cm (3 mL) spectroscopy dilution study in tissue-simulating phantoms was completed
cuvettes and placed in the sample interface, which positioned using a wide-field SWIR imaging system. This system, depicted
illumination and collection fibers perpendicular to one another in Fig. 1(b), consists of a chosen laser source fiber coupled to a
on adjacent cuvette faces. The samples were excited with each condenser lens for illumination, and a Nirvana 640 InGaAs cam-
of the following five lasers sequentially: 635, 730, 760, 785, and era (Princeton Instruments, Trenton, New Jersey) with an 1100-
670 nm (World Star Tech, Markham, Ontario, Canada) through nm longpass dichroic filter for detection. Tissue-simulating
a 200-μm illumination fiber. All laser power outputs were tuned phantoms consisted of 5-mL liquid wells containing 1% intra-
to 21 mW∕cm2 to ensure fluorescence intensity values were lipid and 1% whole blood in PBS to approximate photon scatter
comparable. Emitted light was collected using a spectroscopy and absorption of tissue, respectively, and the dye of interest.
fiber bundle terminating in a linear array at the entrance of the The concentration of each dye was varied from 5 μM to 19 nM
spectrograph. The detection system consisted of a Spectra Pro in 50% serial dilutions. The excitation lasers used for each phan-
SP2150 (Princeton Instruments, Acton, Massachusetts) spectro- tom were as follows: 635 nm for AF 633 and AF 647; 670 nm
graph with a 300-g∕mm grating blazed at 1200 nm, coupled to for methylene blue, IRDye 680RD, and IRDye 700DX; 760 nm
an InstaSpec IR spectroscopy camera (Newport Corporations, for IRDye 800CW, AF 750, IR-m1050, and ICG. The excitation
Franklin, Massachusetts). This camera is a 1024 × 256 pixel power densities measured at the sample surface ranged between
InGaAs transferred-electron electron-bombarded charged- 25 and 74 mW∕cm2 depending on laser source. Five 200-ms
coupled device cooled to −50°C. The detected light passed images were acquired for each dye concentration. Images were
through an 1100-nm dichroic longpass filter (Thorlabs, Newton, scaled to the excitation intensity and baselined using the inten-
New Jersey) positioned in a filter wheel at the entrance of the sities measured from phantoms containing no fluorophore for
spectrometer. All measurements were acquired with a camera each laser, though were not adjusted based on dye absorbance.
gain setting of 800- and 40-ms exposure time. Data from the Mean values of a region of interest in each phantom were used to
sensor were binned vertically during acquisition. report the measured intensity as a function of dye concentration,

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 Byrd et al.: Characterizing short-wave infrared fluorescence. . .

and SNR was calculated as the mean/(standard deviation) of the Inspection of Fig. 2 indicates that in spectroscopy mode
five frames. For each dye, linear fits and Pearson’s coefficient (dilute, nonturbid solutions), all fluorophores under investiga-
were determined using measurements with SNR > 5. tion generated measurable fluorescence in the SWIR window
under proper illumination conditions, and the intensity of the
3 Results emission matched expectations based on the characteristic
absorbance spectrum of each fluorophore. As expected, fluoro-
The absorbance spectra for all fluorophores under investigation
phores with absorbance and emission peaks at shorter wave-
are plotted with the overlapping laser diode excitation wave-
lengths in Fig. 1(c). Representative SWIR fluorescence spectra lengths—AF 633, AF 647, methylene blue, IRDye 680RD,
and corresponding integrated signal values are shown in and IRDye 700DX—generated the lowest signal intensities in
Figs. 2(a)–2(f), respectively. The characteristic longpass filter the SWIR window, and the highest fluorescence intensities were
cut-on is observable at 1100 nm in the spectral plots, and the observed for the longer wavelength fluorophores. The strongest
spectral features of these measurements are consistent with the fluorescence emission signal of all fluorophores was observed
tails of the fluorophores’ emission peaks. The complete set of from ICG when illuminated with 785-nm excitation light,
integrated signal values was normalized to the highest signal though IRDye 800CW and IR-m1050 also showed robust fluo-
value (ICG excited at 785 nm) and plotted in Fig. 2(g) for every rescence emission. These results are consistent with the absorb-
excitation source–fluorophore pairing. For these measurements, ance spectra for each fluorophore.
the maximum SNR for each dye was as follows: AF 633 = 5, AF Images and quantitative analysis of the tissue-simulating
647 = 10, methylene blue = 6, IRDye 680RD = 22, IRDye phantoms, acquired with the wide-filed imaging system as
700DX = 4, AF 750 = 37, IRDye 800CW = 49, IR-m1050 = shown in Fig. 1(c), are presented in Fig. 3. Figure 3(a) shows
38, and ICG = 38. representative images of dye dilutions for AF 633, IRDye

Fig. 2 (a)–(c) Normalized fluorescence emission spectra and (d)–(f) corresponding integrated fluo-
rescence signals for three excitation sources. (g) Normalized integrated SWIR fluorescence for each
fluorophore/excitation source investigated.

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 Byrd et al.: Characterizing short-wave infrared fluorescence. . .

Fig. 3 (a) Representative images of SWIR fluorescence in tissue-simulating phantoms containing AF

633, IRDye 680RD, and AF 750. Each image is the median of five acquisitions. (b)–(d) Fluorescence
intensities of phantom images as a function of fluorophore concentration (error bars are standard
deviation) with linear fits and Pearson’s coefficients.

680RD, and AF 750 over a selected range of concentrations. SWIR imaging system. The results from the imaging study are
Qualitatively, these images show responses to changing dye particularly striking and showed a robust linear response down
concentration indicative of sensitivity to the dyes. A quantitative to the lowest concentrations imaged (20 nM) for nearly all fluo-
analysis of all dyes are presented in Figs. 3(b)–3(d) and confirm rophores. Although many of these dyes will likely be detectable
the qualitative observations. These panels show the mean fluo- at even lower concentrations, this degree of sensitivity is already
rescence intensity values of all dyes as a function of dye well within the range commonly encountered during in vivo
concentration for 635-, 670-, and 760-nm excitation sources, molecular imaging.
respectively, with linear fits to the data. For reference, the base- The relatively robust SWIR fluorescence for the shorter
line values for phantoms without fluorophores were 1560, 820, wavelength fluorophores (AF 633, AF 647, methylene blue,
and 6030 counts∕s for the 635-, 670-, and 760-nm lasers, IRDye 680RD, and IRDye 700DX) is particularly noteworthy.
respectively (these values were subtracted from the data shown Although using these shorter wavelength fluorophores to image
in Fig. 3 before plotting). The SNRs for all dyes at 156 nM were in the SWIR window will have inherent limitations, namely,
as follows: AF 633 = 150, AF 647 = 8, methylene blue = 13, lower fluorescence intensity and shorter wavelength excitation
IRDye 680RD = 22, IRDye 700DX = 16, AF 750 = 281, IRDye sources that may not penetrate as deeply in tissue, there are
800CW = 150, IR-m1050 = 221, and ICG = 119. likely specific instances for which these fluorophores offer
Examination of Fig. 3 confirms that SWIR fluorescence unique benefits. For example, methylene blue is commonly used
from all dyes studied is readily detectable in imaging mode over for sentinel lymph node mapping during cancer resection and
a large range of biologically relevant concentrations using a has been under clinical investigation for other indications.13
simple imaging system. With the exception of AF647, for which Thus, clinical evaluation of SWIR fluorescence for this indica-
the minimum detectable concentration was between 100 and tion could be undertaken without the need for an investigational
200 nM, all dyes showed a linear response down to the lowest drug designation.
concentration measured (20 nM). The fluorescence intensity heat map shown in Fig. 2 can be
used as a reference for comparing SWIR fluorescence efficiency
4 Discussion between the fluorophores tested; however, it should be noted
This study is the first to report SWIR fluorescence from AF 633, that some dyes were not excited at their optimal wavelength.
AF 647, methylene blue, IRDye 680RD, IRDye 700DX, and This can be accommodated by scaling the results in the figure
AF 750, and provides a comprehensive, quantitative comparison to the normalized absorbance spectra of each dye.
of SWIR fluorescence efficiency among a catalog of commonly The general trends observed in the spectroscopy measure-
used fluorophores. SWIR fluorescence was readily detectable in ments and the imaging experiments were comparable, although
nonturbid samples measured using spectroscopy and in turbid the relative intensities between some dyes changed. This is
tissue-simulating phantoms imaged with an epi-illumination most likely due to the well-known effects of changing chemical

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 Byrd et al.: Characterizing short-wave infrared fluorescence. . .

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