APPLICATION NOTE
© 2014 Princeton Instruments, Inc. All rights reserved.

Deeply Cooled, Scientific InGaAs Cameras Facilitate NIR-II /SWIR Imaging for Drug Discovery / Small-Animal Research“The utilization ofmaterials such as SWNTs,
rare-earth–doped phosphors,and quantum dots in concert
with deeply cooled, scientific
InGaAs cameras holds
great promise for the futureof in vivo optical imaging
applications in the NIR-II /SWIR range.”

Introduction
For decades, x-ray and UV-vis-NIR detection methods have been used in various scientific,
military, and medical applications. Although generally employed to good success, thesesystems nonetheless have some limitations when utilized for such types of work.
Effectively leveraging the good sample penetration afforded by x-rays, for example, can provedifficult for personnel in the field owing to the need for a compact, high-power x-ray tube aswell as the lack of system portability. Alternatively, UV and visible wavelengths are quite easily
detectable using silicon-based CCD technologies but are unable to penetrate samples due toreflection and scattering of light.
CCD cameras can detect NIR-I wavelengths between 750 and 1060 nm, which provide slight
penetration into samples, but above this threshold the silicon itself is transparent to light. SuchCCD camera systems have therefore found limited use in military (e.g., surveillance), drug
discovery, and commercial (e.g., inspection) applications.
The NIR-II window / short-wavelength infrared (SWIR) range, which extends to 1700 nm,
achieves much deeper penetration. Spurred on by the development of SWIR-sensitive
InGaAs and InSb detectors, it has opened up a new world of scientific, military, and medical
applications.
Unfortunately, early NIR-II / SWIR detection systems still had key limitations for scientificresearch. Insufficiencies associated with camera system linearity, noise performance, andsynchronization with external equipment, as well as the lack of sufficient flexibility to control
exposure times all presented critical obstacles.
This application note will discuss newly developed, deeply cooled, scientific InGaAs cameras(see Figure 1) from Princeton Instruments that facilitate leading-edge methods of small-animal
imaging for preclinical research in addition to other advanced research applications.

Figure 1.

Princeton Instruments’ NIRvana®
scientific cameras feature athermoelectrically cooled InGaAs
detector and can be operated at rates
up to 110 full frames per second.

1

SWIR for Small-Animal Imaging
Figure 2 shows the difference between NIR-I and NIR-II / SWIR in terms of resolution andpenetration depth. Notice that NIR-I wavelengths scatter, resulting in ‘fuzzier’ images. By
comparison, the longer wavelengths of NIR-II / SWIR do not scatter, yielding ‘crisper’ images.

Figure 2.

NIR-I vs. NIR-II / SWIR: resolution andpenetration depth. Images and graphcourtesy of Dr. Dominik J. Naczynski,
Stanford University, adapted from Lim,Y.T. et al. Mol Imaging. (2003) 2, 50.

Signal detection (% of max)

100
NIR-I(808 nm)

SWIR(1525 nm)
3 mm

101SWIRNIR-I
0246810Depth (mm)In recent years, research groups around the world have been developing different agents1-7 tobe utilized with the NIR-II / SWIR window in small-animal imaging for preclinical research, withthe ultimate goal of earlier disease detection in human patients. Most of these approaches arebased on one of three primary technologies: single-walled carbon nanotubes (SWNTs), rareearth–doped phosphors, or quantum dots.
SWNTs, the most mature of the three approaches, demonstrate good emissivity in the NIR-II/ SWIR range and can be PEGylated to reduce toxicity1,2. Low-toxicity, rare-earth–doped
phosphors have also shown great promise, providing emissions that are tunable to different
wavelengths, dependant on the size of the nanoparticle’s undoped shell3. Similarly, quantum
dots have demonstrated low toxicity and excellent emissivity that is tunable via particle size.
(This application note focuses on the SWNT approach.)

Figure 3.

Absorption chart. Courtesy of Dr.
Dominik J. Naczynski, Stanford
University, adapted from Smith, A.M.
et al. Nat Nanotechnol. (2009) 4, 710.

Absorption coefficient, µa (cm-1)

Each approach utilizes a similar experimental setup, whose main elements are NIR-I
illumination, a specimen (e.g., a mouse), and a camera system to detect emitted light in thewavelength range from ~1100 to ~1650 nm. See Figure 3.

1000

100

101200600

1000
Wavelength (nm)

APPLICATION NOTE

2

www.princetoninstruments.com info@princetoninstruments.com
TOLL-FREE +1.877.474.2286 | PHONE +1.609.587.9797
Visit the Princeton Instruments website to find a dealer in your area.

14001800

Figure 4.

(a) Upon excitation by a 785 nm laser,
the SWNT-IRDye-800 conjugate emits
at ~800 nm (NIR-I region) from the
IRDye-800 dye and between 1100
and 1400 nm (NIR-II region) fromthe SWNT backbone. (b) Absorption
spectrum of the SWNT–IRDye-800conjugate (black dashed line), emission
spectrum of the IRDye-800 dye (greenline), and emission spectrum of theSWNTs (red line). (c) Imaging setup for
simultaneous detection of both NIR-I
and NIR-II / SWIR photons using silicon
and InGaAs cameras, respectively.
A zoomable lens set was used for
adjustable magnifications. Schematics1and spectra1 courtesy of Prof. Hongjie
Dai, Stanford University.

Experimental Results
Prof. Hongjie Dai at Stanford University has performed NIR-I as well as NIR-II / SWIR
fluorescence imaging of blood vessels in mice1,2. Biocompatible SWNT-IRDye-800 conjugateswere utilized as dual-color imaging agents, where IRDye-800 was a commercial NIR-I
fluorophore and high-pressure carbon monoxide conversion SWNTs were stably suspended
by biocompatible surfactants1. See Figure 4 for experimental schematics and spectra.

Figure 5 presents NIR-I and NIR-II / SWIR images acquired via the experimental setup
shown in Figure 4c. The NIR-I image shown in Figure 5a was acquired using a scientific,
silicon-based CCD camera. The NIR-II / SWIR image shown in Figure 5b was acquired usinga scientific InGaAs camera. All images acquired in the NIR-I region employing IRDye-800
fluorescence showed indistinct vascular anatomy within the mouse; however, the detectionof SWNT fluorescence in the NIR-II / SWIR window provided substantially improved spatial
resolution of vessels in the same mouse at all magnifications1.

abNIR-I IRdye 800

NIR-II SWNT

1.0
0.8
0.6
0.4
0.2
246810Position (mm)

121.0
0.8
0.6
0.4
0.2246810Position (mm)New Technology
The NIRvana® family of InGaAs cameras from Princeton Instruments differentiates itself fromother InGaAs cameras, which are typically designed for night vision applications, via a numberof scientific performance features, including deep cooling, low dark noise, high linearity, lowread noise, high frame rates, intelligent software, and precision control over integration times.

APPLICATION NOTE

3

Normalized intensity(a) NIR-I fluorescence image (top) andcross-sectional fluorescence intensity
profile (bottom) along red dashed lineof a mouse injected with the SWNTIRDye-800 conjugates. Gaussian fit
to the profile is shown in red dashedcurve. (b) NIR-II / SWIR fluorescence
image (top) and cross-sectional
fluorescence intensity profile (bottom)
along red dashed line of a mouse
injected with the SWNT-IRDye-800
conjugates. Gaussian fit to the profileis shown in red dashed curve. Data1
courtesy of Prof. Hongjie Dai, Stanford
University.

Normalized intensity

Figure 5.

www.princetoninstruments.com info@princetoninstruments.com
TOLL-FREE +1.877.474.2286 | PHONE +1.609.587.9797
Visit the Princeton Instruments website to find a dealer in your area.

First and foremost, maintenance-free thermoelectric cooling (not liquid nitrogen) chills theNIRvana camera’s InGaAs detector down as low as -85°C. This deep cooling is coupled witha proprietary cold shield design and vacuum technology to facilitate the lowest possible darknoise, which helps increase sensitivity as well as preserve signal-to-noise ratio (SNR) for longexposure times.
The NIRvana camera has the ability to expose for as short as 2 µs, up to many minutes. Ultralow-noise readout electronics help ensure good SNR even when the camera is operated at its
maximum rate of 110 full frames per second. Furthermore, excellent camera linearity meansthat the NIRvana is highly reliable for scientific research.
Princeton Instruments’ 64-bit LightField® software, available as an option, provides a powerful
yet easy-to-use interface that puts real-time online processing capabilities at the researcher’sfingertips. The NIRvana camera can also be integrated into larger experiments using anavailable National Instruments LabVIEW® toolkit. Full triggering support is provided forsynchronization with external equipment.

SummaryThe extension of in vivo optical imaging for disease screening and image-guided surgicalinterventions requires brightly emitting, tissue-specific materials that optically transmit throughliving tissue and can be imaged with portable systems that display data in real-time3. Workperformed by a number of research groups around the world has begun to demonstrate thatfluorescence imaging in the NIR-II / SWIR range can provide appreciably greater in vivo
sensitivity compared to fluorescence imaging in the NIR-I region1–7.
The utilization of materials such as SWNTs, rare-earth–doped phosphors, and quantum dots
in concert with deeply cooled, scientific InGaAs cameras like the new Princeton Instruments
NIRvana holds great promise for the future of in vivo optical imaging applications in theNIR-II / SWIR range.

Resources
To learn more about the NIR-II / SWIR imaging techniques with SWNTs being developed byHongjie Dai, please visit: http://dailab.stanford.edu/
To learn more about the NIR-II / SWIR imaging techniques with rare-earth–doped phosphorsbeing developed by Dominik Naczynski, please visit:https://med.stanford.edu/profiles/dominik-naczynski
For additional information about Princeton Instruments NIRvana cameras, please refer to:www.princetoninstruments.com/products/imcam/nirvana

APPLICATION NOTE

4

www.princetoninstruments.com info@princetoninstruments.com
TOLL-FREE +1.877.474.2286 | PHONE +1.609.587.9797
Visit the Princeton Instruments website to find a dealer in your area.

References
1.

Hong G., Lee J.C., Robinson J.T., Raaz U., Xie L., Huang N.F., Cooke J.P., and Dai, H.
Multifunctional in vivo vascular imaging using near-infrared II fluorescence. Nat. Med. 18,
1841–1846 (2012).

2.

Hong G., Lee J.C., Jha A., Diao S., Nakayama K.H., Hou L., Doyle T.C., Robinson
J.T., Antaris A.L., Dai H., Cooke J.P., and Huang N.F. Near-infrared II fluorescence for
imaging hindlimb vessel regeneration with dynamic tissue perfusion measurement. Circ.
Cardiovasc. Imaging (2014).

3.

Naczynski D.J., Tan M.-C., Zevon M., Wall, B., Kohl J., Kulesa A., Chen S., RothC.M., Riman R.E., and Moghe P.V. Rare-earth–doped biological composites as in vivo
shortwave infrared reporters. Nat. Commun. 4, (2013).

4.

Welsher K., Liu Z., Sherlock S.P., Robinson J.T., Chen Z., Daranciang D., and Dai H.
A route to brightly fluorescent carbon nanotubes for near-infrared imaging in mice. Nat.
Nanotechnol. 4, 773–780 (2009).

5.

Welsher K., Sherlock S.P., and Dai H. Deep-tissue anatomical imaging of mice using
carbon nanotube fluorophores in the second near-infrared window. PNAS 108, 8943–8948(2011).

6.

Hong G., Robinson J.T., Zhang Y., Diao S., Antaris A.L., Wang Q., and Dai H. In vivo
fluorescence imaging with Ag2S quantum dots in the second near-infrared region. Angew.
Chem. 124, 9956–9959 (2012).

7.

Tao Z., Hong G., Shinji C., Chen C., Diao S., Antaris A.L., Zhang B., Zou Y., and Dai H.
Biological imaging using nanoparticles of small organic molecules with fluorescenceemission at wavelengths longer than 1000 nm. Angew. Chem. Int. Ed. 52, 13002–13006(2013).

APPLICATION NOTE

5

www.princetoninstruments.com info@princetoninstruments.com
TOLL-FREE +1.877.474.2286 | PHONE +1.609.587.9797
Visit the Princeton Instruments website to find a dealer in your area.