Introduction

Infrared astronomy started about three decades after infrared (IR) radiation was discovered by Sir William Herschel in 1800. Work in the IR range has been an important facet of astronomical investigation since the 1950s and 1960s, when technological advances in IR detectors began being leveraged to augment the success of contemporary radio astronomy. By the mid-1980s, array detectors engineered to provide photonic sensitivity in the near-infrared (NIR) range — at wavelengths between 700 and 3000 nm — were letting astronomers explore the universe in new and exciting ways.

Since then, the development of InGaAs, InSb, and HgCdTe detectors has facilitated ground-based observations in the J and H astronomical bands of the NIR-II / short-wavelength infrared (SWIR) range extending from 1050 nm to beyond 1700 nm. The J and H bands, which are depicted in Figure 1, correspond to 1100–1400 nm and 1500–1800 nm, respectively.

These two astronomical bands are among several “infrared windows” (wavelengths) at which the Earth’s atmosphere is essentially transparent to infrared light. Although most infrared light is absorbed by water vapor in the Earth’s atmosphere, atmospheric transmission of light within the so-called infrared windows is much better.

To optimize ground-based observations, most IR-capable telescopes on the Earth’s surface are located in dry geographical regions and at rather extreme elevations that are higher than most of the water vapor in the atmosphere.

Today, infrared astronomy remains a critical subfield of astronomy, a fact underscored by a 2015 NRC report that recommends improvements in the US ground-based optical and infrared astronomy system so as to meet specific long-term scientific goals pertaining to astronomy, planetary science, and astrophysics [1].

The use of infrared radiation for astronomy is crucial because unlike light at visible wavelengths, infrared light is not blocked by interstellar dust. Ground-based observations via the “infrared windows” described above can therefore aid in the ongoing search for planets (such as M dwarfs) that may possess the potential to support life. Furthermore, for astrophysics investigations, the red-shifted light originating from objects in the early universe can only be detected within the IR spectral region.

J & H Band Applications

Over the past three decades, myriad investigations have relied upon ground-based observations in the J and H astronomical bands. In the 1990s, for instance, such observations helped astronomers produce a new grid of faint NIR standard stars as well as a secondary list of red stars suitable for determining color transformations between photometric systems [2].

At the dawn of the twenty-first century, the advent of silicon immersion grating technology coupled with adaptive optics at large ground-based telescopes started to revolutionize high-resolution infrared spectroscopy [3]. And earlier this decade, studies of integrated J- and H-band spectra of globular clusters in the LMC [4] as well as spectroscopic investigations of M dwarfs [5,6] using data collected from the J and H bands have been published.

Applications utilizing the J and H astronomical bands may also include ground-based observations of phenomena associated with planets in the Solar System, such as volcanic activity on Jupiter’s moon Io and storms on Saturn.

New Data: Sunspot Images

An example of the utility of the latest generation of deeply cooled, scientific InGaAs detector technology from Princeton Instruments for ground-based observations in the J and H astronomical bands is presented in Figure 2. These two images of sunspots were acquired in 2014 using a Teledyne Princeton Instruments NIRvana:640 InGaAs camera at Yunnan Observatories’ Fuxian Lake Solar Observing Station (China).

The main instrument hosted by the station is the New Vacuum Solar Telescope (NVST), a 1 meter infrared solar observation telescope. The primary scientific mission of NVST astronomers consists of obtaining high-resolution images and spectra of the sun at wavelengths ranging from 0.3 to 2.5 microns for the purposes of studying the fine structures of solar magnetic fields, as well as evolutionary processes in high temporal and spatial resolution.

Enabling Technology

Ground-based observations in the J and H astronomical bands of the NIR-II / SWIR can be improved by utilizing newly developed, deeply cooled, scientific InGaAs cameras (see Figure 3) from Princeton Instruments.

The NIRvana® family of InGaAs cameras from Princeton Instruments differentiates itself from other InGaAs cameras via a number of scientific performance features, including deep cooling, low dark noise, high linearity, low read noise, high frame rates, intelligent software, and precision control over integration times.

First and foremost, either maintenance-free thermoelectric cooling or liquid nitrogen can be employed to chill the NIRvana’s two-dimensional 640 x 512 InGaAs focal plane array down as low as –85°C (188 K) or –190°C (83 K), respectively. A proprietary cold shield design and sealed vacuum technology facilitates the lowest possible dark noise, which helps increase sensitivity as well as preserve signal-to-noise ratio (SNR) for long exposure times. It should be noted that all infrared detectors used by modern telescopes (i.e., detectors designed for NIR sensitivity as well as detectors designed for mid-IR or far-IR sensitivity) require deep cooling to minimize thermally generated dark noise that would otherwise swamp the desired
photonic signal.

Thermoelectrically cooled NIRvana cameras have the ability to expose up to many minutes, whereas LN-cooled NIRvana camera exposure times can reach 1 hour. Ultra-low-noise readout electronics help ensure good SNR even when the cameras are operated at their maximum full-frame readout rates (i.e., 110 full frames per second for thermoelectrically cooled cameras; 2.77 full frames per second for LN-cooled cameras). Furthermore, excellent system linearity means that NIRvana cameras collect highly reliable astronomical data.

Princeton Instruments’ 64-bit LightField® software, available as an option, provides a powerful yet simple-to-use interface that puts real-time online processing capabilities at astronomers’ fingertips. NIRvana cameras are easy to integrate with telescopes. Full triggering support is provided for precise synchronization with external equipment.

Deeply cooled, ultra-high-sensitivity NIRvana InGaAs cameras are ideal for making ground-based observations in the J and H astronomical bands.

References

Introduction

One of the main areas of research at the Memorial Sloan Kettering Cancer Center in New York City is the development of nanoscale sensors to detect cancer at its earliest stages. The research group led by Dr. Daniel Heller uses novel nanomaterials with unique optical properties, making it easier to identify disease biomarkers within the body and thus permit detection before symptoms arise. These nanotechnologies also allow the measurement of important molecules within live cells and tissues, offering new tools that accelerate biomedical research [1].

Working in the near-infrared (NIR) region of the spectrum affords several advantages, such as the abilities to circumvent unwanted fluorescence backgrounds and to probe more deeply into sample surfaces. Over the past few years, the advent of deep-cooled cameras that employ indium gallium arsenide (InGaAs) focal plane arrays (FPAs) has increased the utility of various NIR spectroscopy and imaging techniques for low-light-level scientific applications [2].

This type of scientific camera can be even more helpful to researchers when used in concert with a new class of dispersive spectrographs that feature an innovative Schmidt-CzernyTurner (SCT) design. High-precision SCT spectrographs greatly reduce optical aberrations, providing sharp images with superb spatial resolution across the entire focal plane and enabling researchers to utilize the full two dimensions of the FPA sensor to acquire images [3].

Dr. Heller’s research group in New York has successfully used such light-dispersion and -detection tools to perform novel experiments investigating properties of photoluminescent (PL) single-walled carbon nanotubes (SWCNTs), which could ultimately result in the development of new optical probes and sensors for biophysical measurements and biomedical applications [4-6]. This application note will present some of the highlights of the group’s work as well as the integral role played by advanced SCT spectrographs and deep-cooled InGaAs FPA cameras.

Example of Experimental Setup

Dr. Heller’s research group has performed several PL spectroscopy studies on SWCNTs in recent years. Three of these studies will be highlighted in this note.

The first study, reported in 2014, involved the encapsulation of SWCNTs with a diverse set of functional coatings (polymers) that exhibited ordered surface coverage on the carbon nanotubes and allowed systematic modulation of nanotube optical properties [4]. In the second study, the researchers used the intrinsic NIR emission of semiconducting SWCNTs to optically reconstruct the carbon nanotubes’ localization within a three-dimensional volume and thus resolve the relative permeability of two different multicellular tumor spheroids [5]. Finally, in the third study, SWCNT emission energy response to solution ionic potentials was investigated and it was observed that nanotubes respond to cell surface electrostatic potentials that are mediated by membrane proteins [6].

Although each of these SWCNT studies necessitated the implementation of distinct experimental protocols, all three used a next-generation Princeton Instruments IsoPlane®-320 spectrograph and a deep-cooled Princeton Instruments NIRvana® InGaAs FPA camera to perform near-infrared photoluminescence spectroscopy. For example, in Study #3, the researchers integrated the IsoPlane and the NIRvana into their own custom-built apparatus to enable PL excitation/emission spectroscopy of nanotubes on live eukaryotic cells (see Figure 1).

When investigating SWCNT emission energy response to solution ionic potentials (Study #3) with the experimental setup shown in Figure 1, the sample was excited using a supercontinuum light source coupled to a variable bandpass filter in order to tune the excitation from 500 to 827 nm with a 20 nm bandwidth. The light was injected into an inverted fluorescence microscope through a 50X objective. The same objective was utilized to collect the resultant NIR emission and direct it into the IsoPlane spectrograph, which was coupled to the NIRvana camera (thermoelectrically cooled InGaAs array: 640 x 512 pixels; pixel size: 20 x 20 μm; quantum efficiency: >85% in the 0.9–1.7 μm range) [6].

To conduct excitation/emission measurements for Study #3, the excitation was varied from 500 to 827 nm in steps of 3 nm. At each excitation wavelength, with an exposure time of 0.5–3.0 sec, the emission from 930 to 1370 nm was dispersed using a ruled grating (86 grooves/mm). Corrections for wavelength-dependent variations in excitation power (5–30 mW measured at the sample), as well as grating and detector efficiencies, were applied. The system was automated to illuminate the sample with 109 different excitation bands and collected spectra from carbon nanotubes in solution or in contact with a cell monolayer to produce a full photoluminescence plot in 0.5–5 min [6].

Data & Results

Study #1 (Polymer-Coated SWCNTs): In this study, the astigmatism-free IsoPlane spectrograph and the deep-cooled InGaAs camera were utilized to perform PL excitation/emission measurements on polycarbodiimide−SWCNTs. The excitation wavelength was varied from 491 to 824 nm and the emission was recorded from 915 to 1354 nm (see Figure 2).

The researchers reported noncovalent functionalization of SWCNTs via encapsulation in helical polycarbodiimides to form water-soluble, well-dispersed, polymer–nanotube complexes with NIR emission that were stable under ambient conditions. The polymers facilitated the intensity modulation of nanotube fluorescence and enabled inter-nanotube exciton energy transfer between individually encapsulated nanotubes. This was the first instance of exciton energy transfer produced spontaneously between nanotubes due to Coulombic attraction between the encapsulating polymers and it displayed directed reversibility. The finding augurs the measurement of dynamic processes and a potential mechanism for switchable molecular probes and sensors [4].

Study #2 (Tumor Permeability): Here, the intrinsic NIR fluorescence of SWCNTs was leveraged to interrogate the permeability of multicellular tumor spheroids. The Teledyne Princeton Instruments IsoPlane spectrograph and InGaAs camera were employed to perform excitation/emission measurements on surfactant-sodium-deoxycholate−SWCNTs (see Figure 3).

The research group reported developing a tumor spheroid model of murine liver cancer cells. These tumor spheroids were compared with a breast-cancer cell line that forms spheroids under low-adhesion conditions. Widefield NIR fluorescence microscopy in live cells spatially resolved the locations of nanotubes associated with multicellular tumor spheroids. The
researchers found that the nanotubes showed little penetration in one type of spheroids (the liver cancer), but penetrated to the center of the other (the breast cancer). Therefore, the group effectively presented the use of NIR-fluorescent SWCNTs as a validated and qualitative method to interrogate the permeability of live tumor spheroids [5],

Study #3 (Live Cell Membranes): In this study, the spectrograph and InGaAs camera were utilized to perform excitation/emission spectroscopy of SWCNTs associated with the membranes of live cells (see Figure 4). As mentioned in the “Example of Experimental Setup” section, the excitation was varied from 500 to 827 nm in steps of 3 nm. At each excitation wavelength, with an exposure time of 0.5–3.0 sec, the emission from 930 to 1370 nm was dispersed using a ruled grating with 86 grooves/mm.

The researchers found that the nanotube photon emission energy responded to charge accumulation mediated by cell surface proteins and that nanotube photon emission energy correlated with both the degree to which a cell adheres to a substrate, as well as the whole-cell zeta potential. They asserted that the photoluminescence responses on the cell surface could be recapitulated in vitro by introducing ionic charge into the local environment of the nanotube. The research group also proposed a mechanism in which the nanotube photoluminescence is modulated by the charge density on live cell surfaces. This study suggested that the nanotube optical bandgap modulation can be mediated by ionic or polyelectrolyte charge accumulation on the nanotube surface. The findings portend a nanoscale tool for the optical quantification of electrostatic charge accumulation on live cell membranes for biomedical applications [6].

Enabling Technology

The aforementioned research relied on the award-winning IsoPlane-320 spectrograph (see Figure 5) from Teledyne Princeton Instruments. This high-precision instrument’s unique optical design completely eliminates field astigmatism at all wavelengths and at all points across the focal plane. Coma is reduced to negligible levels. Reduced optical aberrations result in significantly improved signal-to-noise ratio (SNR) and exceptional image quality. IsoPlane spectrographs feature a 320 or 160 mm focal length and a three-position, on-axis grating turret.

In addition to an IsoPlane-320 spectrograph, Dr. Heller’s group utilized an NIR-sensitive InGaAs FPA camera from Princeton Instruments to conduct the research highlighted herein. This camera, the NIRvana:640 (see Figure 6), differentiates itself from other InGaAs cameras via a number of scientific performance features, including deep cooling, low dark noise, high linearity, low read noise, high frame rates, intelligent software, and precision control over integration times.

First and foremost, either maintenance-free thermoelectric cooling or liquid nitrogen can be employed to chill the NIRvana’s two-dimensional 640 x 512 InGaAs FPA detector down as low as -85°C or -190°C, respectively. A proprietary cold shield design and vacuum technology facilitates the lowest possible dark noise, which helps increase sensitivity as well as preserve SNR for long exposure times.

A thermoelectrically cooled NIRvana camera offers the ability to expose from 2 μsec up to many minutes, whereas the exposure time of an LN-cooled NIRvana camera can range from 100 μsec up to 1 hour. Ultra-low-noise readout electronics help ensure good SNR even when the camera is operated at its maximum full-frame readout rate (i.e., 110 full fps for a thermoelectrically cooled NIRvana; 2.77 full fps for an LN-cooled NIRvana). Excellent system linearity means that every NIRvana camera is highly reliable for scientific research.

Furthermore, Princeton Instruments’ 64-bit LightField® data acquisition software, available as an option, provides a powerful yet easy-to-use interface that puts real-time online processing capabilities at the researcher’s fingertips. NIRvana cameras can be integrated into larger experiments using an available National Instruments LabVIEW® toolkit. Full triggering support is provided for synchronization with external equipment.

Acknowledgements

Princeton Instruments would like to thank Dr. Daniel Heller, Memorial Sloan Kettering Cancer Center, for his invaluable contributions to this application note.

Resources

Additional details about the work being performed by Dr. Daniel Heller’s research group can be found by visiting: https://www.mskcc.org/research-areas/labs/daniel-heller

References

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, these systems nonetheless have some limitations when utilized for such types of work.

Effectively leveraging the good sample penetration afforded by x-rays, for example, can prove difficult for personnel in the field owing to the need for a compact, high-power x-ray tube as well 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 to reflection 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. Such CCD 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 scientific research. Insufficiencies associated with camera system linearity, noise performance, and synchronization 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.

SWIR for Small-Animal Imaging

Figure 2 shows the difference between NIR-I and NIR-II / SWIR in terms of resolution and penetration 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.

In recent years, research groups around the world have been developing different agents [1-7] to be utilized with the NIR-II / SWIR window in small-animal imaging for preclinical research, with the ultimate goal of earlier disease detection in human patients. Most of these approaches are based on one of three primary technologies: single-walled carbon nanotubes (SWNTs), rare earth–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 toxicity [1,2]. 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 shell [3]. Similarly, quantum dots have demonstrated low toxicity and excellent emissivity that is tunable via particle size. (This application note focuses on the SWNT approach – for other approaches see NIR-II Probes For In Vivo Imaging.)

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 the wavelength range from ~1100 to ~1650 nm. See Figure 3.

Experimental Results

Prof. Hongjie Dai at Stanford University has performed NIR-I as well as NIR-II / SWIR fluorescence imaging of blood vessels in mice [1,2]. Biocompatible SWNT-IRDye-800 conjugates were 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 surfactants [1]. 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 using a scientific InGaAs camera. All images acquired in the NIR-I region employing IRDye-800 fluorescence showed indistinct vascular anatomy within the mouse; however, the detection of SWNT fluorescence in the NIR-II / SWIR window provided substantially improved spatial resolution of vessels in the same mouse at all magnifications [1].

New Technology

The NIRvana® family of InGaAs cameras from Princeton Instruments differentiates itself from other InGaAs cameras, which are typically designed for night vision applications, via a number of scientific performance features, including deep cooling, low dark noise, high linearity, low read noise, high frame rates, intelligent software, and precision control over integration times.

First and foremost, maintenance-free thermoelectric cooling (not liquid nitrogen) chills the NIRvana camera’s InGaAs detector down as low as -85°C. This deep cooling is coupled with a proprietary cold shield design and vacuum technology to facilitate the lowest possible dark noise, which helps increase sensitivity as well as preserve signal-to-noise ratio (SNR) for long exposure 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 means
that 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’s fingertips. The NIRvana camera can also be integrated into larger experiments using an available National Instruments LabVIEW® toolkit. Full triggering support is provided for
synchronization with external equipment.

Summary

The extension of in vivo optical imaging for disease screening and image-guided surgical interventions requires brightly emitting, tissue-specific materials that optically transmit through living tissue and can be imaged with portable systems that display data in real-time [3]. Work
performed by a number of research groups around the world has begun to demonstrate that fluorescence imaging in the NIR-II / SWIR range can provide appreciably greater in vivo sensitivity compared to fluorescence imaging in the NIR-I region [1–7].

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 the NIR-II / SWIR range.

Resources

To learn more about the NIR-II / SWIR imaging techniques with SWNTs being developed by Hongjie Dai, please visit: http://dailab.stanford.edu/

References

Overview

Working in the shortwave-infrared (SWIR) region of the spectrum affords researchers several advantages, including the abilities to circumvent unwanted fluorescence backgrounds and to probe more deeply into sample surfaces. The advent of deep-cooled camera systems that employ indium gallium arsenide (InGaAs) focal plane arrays (FPAs) has further increased the utility of various SWIR imaging and spectroscopy techniques for low-light-level scientific and industrial applications [1].

Recently, researchers at the Massachusetts Institute of Technology (MIT) published results of a study in which they leveraged the most advanced deep-cooled InGaAs FPA camera technology commercially available (see Figure 1) to evaluate novel InAs-based SWIR quantum dots. Working under the auspices of principal investigator Dr. Moungi G. Bawendi, lead authors Dr. Oliver T. Bruns and Dr. Thomas S. Bischof demonstrated that these new SWIR-emitting core/shell quantum dots (QDs) hold great promise for the next generation of in vivo SWIR imaging [2].

Dr. Bawendi’s laboratory, which is in the university’s Department of Chemistry, focuses on the fundamental science and leading-edge application of nanocrystals, especially semiconductor nanocrystals (i.e., quantum dots). The lab collaborates with a number of biology and medical groups to design nanocrystal probes that meet specific challenges, including utilization as molecular imaging agents in vivo [3].

This application note will summarize the lab’s use of a Princeton Instruments NIRvana scientific camera to perform the aforementioned evaluation of novel InAs-based SWIR emitting quantum dots as in vivo imaging agents.

Macroscopic & Microscopic SWIR Imaging

For macroscopic imaging of the vasculature of the subject’s (i.e., the mouse’s) brain through skin and skull, the MIT researchers built a custom experimental setup to direct the light emitted through various filters to a NIRvana camera equipped with various C-mount lenses. To eliminate excess light whilst enabling manipulation of the field-of-view during operation, the entire macroscopic imaging assembly was surrounded by a partial enclosure.

Microscopic imaging was performed using the NIRvana camera attached to the sideport of an inverted microscope, and either a 10x or a 2x objective. A fiber-coupled 808 nm laser diode was utilized for illumination, with a speckle remover employed. A dichroic filter directed the excitation light to the sample. Then a 1000 nm long-pass filter selected the emission light

The camera’s InGaAs array was cooled to -80°C for microscopic imaging, the analog-to-digital conversion rate was set to 10 MHz, and the gain was set to high. The use of different exposure times resulted in different frame rates.

Data and Results

The MIT researchers call attention to the fact that even though low levels of light absorption by blood and tissue, reduced scattering, and a general lack of autofluorescence can render a mouse translucent when imaged in the SWIR region, the sheer want of a versatile emitter platform has prevented widespread adoption of in vivo SWIR imaging by the biomedical research community. Their new InAs-based core/shell quantum dots (see Figure 2), however, exhibit a dramatically higher emission quantum yield (QY) than previously described SWIR probes, as well as a narrow and size-tunable emission that allows multiplexing in the SWIR region.

To demonstrate a few of the key capabilities of these quantum dots, SWIR imaging was utilized to measure the heartbeat and breathing rates in awake and unrestrained mice, as well as to quantify the lipoprotein turnover rates of several organs simultaneously in real-time in the mice.

The researchers also generated detailed three-dimensional quantitative flow maps of brain vasculature by intravital microscopy, visualizing the differences between healthy tissue and a tumor in the brain. These newly designed SWIR QDs enable biological optical imaging with an unprecedented combination of deep penetration, high spatial resolution, and fast acquisition speed.

For more data, as well as detailed methodologies and in-depth discussions of results, please refer to Bruns et al. Next-generation in vivo optical imaging with short-wave infrared quantum dots. Nature Biomedical Engineering. 2017, 1.

Although the MIT researchers did not observe toxic effects in the mice during their short-term studies, they do acknowledge that the chemical composition of their SWIR QDs might prohibit use in humans. Recently, however, they also published work showing the suitability of indocyanine green (ICG) and other substances that are clinically approved or in advanced clinical trials for SWIR bio imaging [4].

Enabling Technology

The NIRvana family of InGaAs FPA cameras from Princeton Instruments differentiates itself from other InGaAs cameras, which are typically designed for night vision applications, via a number of scientific performance features, including deep cooling, low dark noise, high linearity, low read noise, high frame rates, intelligent software, and precision control over integration times.

First and foremost, maintenance-free thermoelectric cooling chills the NIRvana 640 camera’s InGaAs detector down as low as -85°C. This deep cooling is coupled with a proprietary cold shield design and vacuum technology to facilitate the lowest possible dark noise, which helps increase sensitivity as well as preserve signal-to-noise ratio (SNR) for long exposure times when necessary

The NIRvana 640 camera has the ability to expose for as short as 2 μs, up to many minutes. Ultra-low-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 means that it is highly reliable for scientific research.

Complete control over the NIRvana camera is simple thanks to the latest version of Princeton Instruments’ 64-bit LightField® data acquisition software, available as an option. Myriad functions are provided for the easy capture and export of imaging and spectral data via the exceptionally intuitive LightField user interface. A built-in math engine can analyze the collected data in real-time. LightField also permits direct data acquisition into LabVIEW® (National Instruments) and MATLAB® (MathWorks).

Acknowledgments

Princeton Instruments would like to thank Dr. Oliver T. Bruns, Massachusetts Institute of Technology, for his invaluable contributions to this application note.

Resources

To learn more about the research being conducted by the Bawendi Lab at MIT, please visit: http://nanocluster.mit.edu/index.php

A video is also available on the Teledyne Princeton Instruments YouTube Channel, courtesy of the Bawendi Lab and MIT.

References

Introduction

UV, VIS, and NIR-I detection methods have been used in various scientific and medical applications for decades. Each of these approaches, however, has its limitations. For instance, light at UV and visible wavelengths is quite easily detectable using silicon-based CCD technologies but is unable to penetrate samples due to reflection and scattering.

New CCD cameras can detect NIR-I wavelengths from 750 nm up to almost 1100 nm and thus provide slight penetration into samples. Unfortunately, above this threshold the silicon is transparent to light. Such CCD cameras have found limited use in drug discovery applications.

The NIR-II window, whose wavelength range extends to 1700 nm, achieves much deeper sample penetration (see Figure 1). Spurred on by the development of InGaAs and InSb detectors, the NIR-II, also known as the shortwave-infrared or SWIR, has opened up a new world of scientific and medical applications.

As many research groups around the world are developing and evaluating new NIR-II probes appropriate for small-animal imaging in preclinical research, scientists now begin to focus more on the clinical applications for disease detection, morphology, and drug detection in human patients

NIR-II Fluorescence Endoscopy for Imaging of Colorectal Cancer

Recently, a multidisciplinary collaborative research team in China and the United States designed, constructed, and characterized an endoscopic system to perform targeted NIR-II fluorescence imaging of colorectal cancer [1]. The team used the new NIR-II endoscopy imaging system to evaluate a synthesized fluorescent molecular probe, indocyanine green (ICG) conjugated bevacizumab (Bev-ICG), which targets vascular endothelial growth factor (VEGF), an important biomarker that is overexpressed in most colorectal cancers.

The study was spearheaded by the research group of Dr. Hongguang Liu at the Institute of Molecular Medicine Joint Laboratory for Molecular Medicine at Northeastern University in Shenyang, Liaoning, and supported in part by the Natural Science Foundation of China, the Clinical Capability Construction Project for Liaoning Provincial Hospitals, and a China postdoctoral science foundation grant.

The researchers note that while endoscopy is a clinical gold standard for examining the interior of a hollow organ or body cavity, they are the first to report the use of NIR-II fluorescence endoscopy for the targeted detection of colorectal cancer. Their innovative endoscopic imaging system, which enables the simultaneous acquisition and display of white light (WL) and NIR-II fluorescence, delivers subcellular resolution of 20 μm for sharp images in the NIR-II region. A scientific InGaAs camera from Teledyne Princeton Instruments was used for the realtime, noninvasive, in vivo NIR-II imaging of colorectal tumor biomarkers (see Figure 2).

Unlike NIR-I fluorescence imaging, which uses visible light for excitation, the excitation wavelength for NIR-II fluorescence imaging is typically in the (invisible) NIR-I range. Therefore, the conventional endoscopy imaging process does not need to be interrupted to collect the NIR-II fluorescent data. The NIR-II fluorescence image can be generated and displayed simultaneously with the WL image to afford researchers real-time visualization of tissue morphology and molecular characteristics from fluorescent probes. The future utilization of molecular probes with stronger fluorescence emission in the NIR-II region is expected to further improve the sensitivity of the NIR-II endoscopy system for tissue characterization.

By design, the new system can be easily adapted to endoscopy systems that are equipped with a standard coupling. The researchers anticipate similar hardware upgrades will greatly promote the application of NIR-II fluorescence imaging in clinical settings.

NIR-II Fluorescence Imaging of Circulatory and Skeletal Systems

Dr. Zhen Cheng, an associate professor of radiology at Stanford University, is a member of the collaborative research team responsible for the NIR-II endoscopy system discussed above. Dr. Cheng’s laboratory at Stanford seeks to develop novel molecular imaging probes and techniques for noninvasive detection of cancer and its metastasis at the earliest stage.

The techniques developed via Dr. Cheng’s research enable a close examination of the molecular, metabolic, and physiological characteristics of cancers and their responses to therapy. His lab identifies novel cancer biomarkers with significant clinical relevance, develops new chemistry for probes preparation, and validates new strategies for probes high-throughput screening using NIR-II fluorescence imaging [2–6].

In a recent project, Dr. Cheng and his colleagues performed in vivo imaging of the circulatory system comparing various NIR-II organic small-molecule probes, CQ-T (CQ-1-4T), that were designed in the lab and loaded with biocompatible human serum albumin (HSA) to improve quantum yield [2]. The experiments recorded emission spectra, quantum yield, and in vivo NIR-II images using an InGaAs camera from Teledyne Princeton Instruments (see Figure 3).

Among the series of probes evaluated, the researchers found one, CQL (CQ-4T/HSA), that demonstrates superior optical properties and a 6.65x increase in fluorescence compared to the small molecule alone. Utilized in concert with the noninvasive, nonradiation NIR-II imaging modality, CQL enables multifunctional imaging for visualizing and monitoring circulatory system-related physiological and pathological processes in vivo, including thrombosis, peripheral arterial disease, tumor angiogenesis, and lymphatic drainage.

During NIR-II imagenavigated surgery, CQL is capable of helping physicians implement precise intervention of tumor and sentinel lymph node biopsy more accurately with less side effects. The researchers conclude that high resolution, excellent biocompatibility, and favorable in vivo performance make CQL a promising candidate for further preclinical applications and future translations into clinical practices.

Another recent study from Dr. Cheng’s lab investigated a NIR-II fluorophore based on DSPEmPEG encapsulated rare-earth–doped nanoparticles, RENPs@DSPE-mPEG, that shows inherent affinity to bone without linking any targeting ligands (see Figure 4) [3].

This noninvasive, nonradiation strategy for skeletal system mapping and bone disease diagnoses is also applicable to blood vessels and lymph nodes. Significantly, RENPs@DSPEmPEG can be internalized by circulating white blood cells, which may increase efficient nanoparticle delivery for immunotherapy as well as improve the diagnostic and therapeutic efficacy of cancer-targeted nanoparticles in clinical applications.

Enabling Technology

The NIRvana® family of InGaAs cameras from Teledyne Princeton Instruments differentiates itself from other InGaAs cameras via a number of scientific performance features, including deep cooling, low dark noise, high linearity, low read noise, high frame rates, intelligent software, and precision control over integration times.

First and foremost, maintenance-free thermoelectric cooling (not liquid nitrogen) chills the NIRvana 640 camera’s InGaAs detector down as low as -85°C. This deep cooling is coupled with a proprietary cold shield design and vacuum technology to facilitate the lowest possible dark noise, which helps increase sensitivity as well as preserve signal-to-noise ratio (SNR) for
long exposure times.

The NIRvana 640 camera has the ability to expose for as short as 2 μs, up to many minutes. Ultra-low-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 means that it is highly reliable for medical research.

Teledyne 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’s fingertips. NIRvana cameras can also be integrated into larger experiments using an available National Instruments LabVIEW® toolkit. Full triggering support is provided for synchronization with external equipment.

Acknowledgments

Teledyne Princeton Instruments would like to thank Dr. Hongguang Liu and Dr. Zhen Cheng for their invaluable contributions to this application note.

Resources

To learn more about the work of Dr. Zhen Cheng’s laboratory at Stanford University, please visit: https://profiles.stanford.edu/zhen-cheng?tab=bio

References

Introduction

Near-infrared (NIR) fluorescent is a technique used widely within biological and medical research due to ability for NIR light to penetrate deeply in biological specimens, the high spatiotemporal resolution that it offers, and the capability to image quickly (Fan 2019).

The second NIR window (NIR-II, 900 – 1700 nm) is of recent interest due to its superior penetration depth, reduced tissue absorption and decreased photon scattering compared to the visible and shorter NIR wavelength range (Fan 2019, Carr 2018, Diao 2015, Hong 2014). This is because photon scattering is scaled within biological tissues via λ^-α (where α = 0.2–4 dependent on tissue type) (Fan 2019, Diao 2015). NIR-II wavelengths also reduce background autofluorescence, with background autofluorescence becoming negligible with wavelengths above 1500 nm.

However, the absorbance of water increases above 1400 nm, therefore, the optimal region for NIR imaging in biological tissues is between 800 and 1400 nm, resulting in better spatial resolution and penetration depths (Fan 2019, Zhu 2019).

Specialized fluorescent probes emitting within the NIR-II range have been developed by researchers in the last few years. Those encompass quantum dots (Bruns 2017), organic dyes (Carr 2018), and single-walled carbon nanotubes (SWCNT) (Takeuchi 2019).

Imaging of these fluorophores requires cameras sensitive to NIR-II wavelengths. Due to the size of the bandgap, silicon-based camera sensors are not sensitive to light above 1100 nm. Scientific cameras based on InGaAs FPAs have a high quantum efficiency in the 900 – 1700 nm wavelength range, therefore, they are optimal for NIR fluorescence imaging (Smith 2009). 

Over the past decade, new NIR-II probes are being developed, with more techniques being explored from in vivo imaging to optical microscopy. A research group at École Polytechnique Fédérale de Lausanne (EPFL), led by Prof. Ardemis A. Boghossian, has published an article showcasing the first spinning-disc confocal light microscopy (SDCLM) in the NIR-II optical window (Zubkovs 2018).

The first author of this work, Dr. Vitalijs Zubkovs, has compared optical resolution of the SDCLM with a wide-field NIR-II microscope, allowing for fast, real-time image acquisition. Through the use of a cooled InGaAs FPA camera coupled to a NIR-II optimized spinning-disc module (SDCLM-InGaAs), they demonstrated the benefits of the device in three applications:

SDCLM

SDCLM is a confocal microscope with additional spinning disc with multiple pinholes etched onto the surface, as shown by the schematic in Figure 1. When spun at high speeds, these pinholes allow for images to be acquired across a much larger surface area. This increases the speed of acquisition and reduces any photo damage of the sample. By varying pinhole size, disc speed and pinhole spacing alterations in image contrast, brightness and quality can be controlled.

To learn more about the different types of SDCLM please see the Educational Resource by Teledyne Photometrics.

The EPFL team measured lateral and axial resolutions of the setups and compared it to the theoretical values which were calculated in Equations 1 and 2.

where r_x and r_z are the resolution in the lateral (x-axis) and axial (z-axis) direction respectively, F_x and F_z are the FWHM pre-factor in the lateral and axial direction (determined by the diameter of the pinholes on the spinning-discs),  is the wavelength, and λ_EM is the numerical aperture.

SDCLM experimental set up with NIR-II

The EPFL researchers coupled an InGaAs camera (NIRvana-ST, Teledyne Princeton Instruments) to a spinning-disc module (CrestOptics) fixed on a microscope body (Nikon Eclipse), shown in Figure 2. Traditional light microscopes typically do not have anti-reflective optics in the NIR-II region; therefore, the throughput of photons is minimized due to absorption from the optical components. Hence, coating of the lens within the spinning-disc confocal was imperative to maximize photon throughput.

SDCLM systems are advantageous as they enable fast image acquisition with higher lateral and axial resolution than wide-field imaging. The spinning-disc within the system is designed to achieve a maximum theoretical acquisition speed of approximately 18000 fps (~50 ms exposure time).This requires the disc to rotate at a speed of 15000 RPM. By using an InGaAs camera with a frame rate of 110 fps (9 ms), the researchers were able to establish an optimal acquisition time of 20 fps (50 ms) to image NIR beads with acceptable signal-to-noise ratio.

Data and Results

Using this system, the researchers at EPFL used a wide-field microscope with an InGaAs camera, to image two neighboring NIR fluorescent beads immersed within oil. When the distance between the beads was less than 600 µm, they were not optically resolved in the wide-field system. However, the SDCLM-InGaAs system provided an improvement in contrast of the obtained image, improving the separation between the beads, as shown in Figure 3. Measured lateral and axial resolutions in the SDCLM microscope at 980 nm were 0.5±0.1 µm and 0.6±0.1 µm respectively. Compared to the wide-field system obtained improvement in the resolution was 17% in lateral and 45% in axial direction.  

Enhancement in spatiotemporal resolution in the NIR region could expand new horizons in studies which involve in vitro and in vivo imaging. In the recent decades, many biological and medical studies focus on understanding living cell and nanoparticle interaction better. Some cells are able to uptake functionalized particles that penetrate organelles inside the cell (Feng 2019, Kodiha 2015, Chen 2019). Organelles which are present is plant cells are chloroplasts, which are interesting subject to study because of their light energy harvesting bio-function.

As chloroplasts absorb over a wide range of visible light, and also autofluoresce, it becomes challenging to image any chloroplast-internalized particles labeled with visible dyes. Therefore, NIR-II fluorophores, such as SWCNTs, can be employed in nanoparticle uptake studies to draw more accurate conclusions on how nanoparticle surface functionalization promotes or inhibits their uptake in organelles. The SDCLM-InGaAs microscope allowed for clearer localization of SWCNTs within chloroplasts through improved resolution and contrast – indicating that a DNA coating was optimal for SWCNT uptake.

The EPFL team pioneered the demonstration of the first spatiotemporal glucose nanosensor, which allowed the tracking of axial diffusion of glucose in agarose gel at the microscopic level. In the future, such sensors could be immobilized within a gel matrix and implanted in vivo for continuous analyte monitoring (Marchetti 2020, Zhang 2019, Kamanina 2019).

For example, glucose oxidase (GOx) enzyme-functionalized SWCNTs can be used as glucose-specific sensors (Zubkovs 2017, Kamanina 2019). Changes in the fluorescence intensity of the GOx-SWCNT nanosensors is proportional to the change in glucose concentration. During the experiment, as the glucose diffused and interacted with the GOx-SWCNTs dispersed within the gel matrix, the SDCLM-InGaAs microscope monitored the sensor response at different axial sections of the gel, as shown in (Figure 4). Although this can also be achieved via wide-field imaging, SDCLM imaging allowed for smaller axial segments to be imaged giving a more representative diffusion gradient.

Conclusion

The researchers at EPFL have clearly demonstrated that the use of a SDCLM in combination with an InGaAs camera will be highly beneficial to biological and medical research, especially when investigating in vitro and in vivo biosensors. NIR-II is the perfect wavelength range to obtain high-resolution images of biological tissue with reduced photon scattering, autofluorescence and increased penetration depth. Merging optical microscope techniques, such as SDCLM to increase image acquisition times and scanning surface area, and sensitive cameras, such as InGaAs, high resolution real-time imaging of biological tissues and bioprocesses can be achieved.

As InGaAs cameras are highly sensitive within the 900 – 1700 nm range they are ideal for NIR-II imaging. InGaAs cameras are also advantageous as they are deep-cooled to reduce dark noise. Dark noise is the noise produced from the electric current flowing through photosensitive devices. Further information regarding InGaAs FPA cameras and their advantages can be found in the Technical Note by Teledyne Princeton Instruments.

References