Visualizing structures deep within biological tissue is a central challenge in biomedical imaging, with both preclinical implications and clinical relevance. Using shortwave infrared (SWIR) light enables imaging with high resolution, high sensitivity, and sufficient penetration depth to noninvasively interrogate sub-surface tissue features. However, the clinical potential of this approach has been largely unexplored. Until recently, suitable detectors have been either unavailable or cost-prohibitive. Additionally, clinical adoption of SWIR imaging has been inhibited by a poor understanding of its advantages over conventional techniques. For fluorescence imaging in particular, there has further been a perceived need for clinically-approved contrast agents. Here, taking advantage of newly available detector technology, we investigate a variety of biomedical applications with SWIR-based imaging devices. We describe the development of a medical otoscope and our clinical observations using this device to evaluate middle ear pathologies in both adult and pediatric populations, showing that SWIR otoscopy could provide diagnostic information complementary to that provided by conventional visible otoscopy. We further describe fluorescence detection of an endogenous disease biomarker in animal models including nonalcoholic fatty liver disease and cirrhotic liver models and models of a neurodegenerative disease pathway. While this biomarker has been known for decades, we describe a method for its noninvasive detection in living animals using near infrared and SWIR light, as opposed to its conventional ex vivo detection. Furthermore, we show that SWIR image contrast and penetration depth are primarily mediated by the absorptivity of tissue, and can be tuned through deliberate selection of imaging wavelength. This understanding is crucial for rationally determining the optimal imaging window for a given application, and is a prerequisite for understanding which clinical applications could benefit from SWIR imaging. Finally, we show that commercially-available near infrared dyes, including the FDA-approved contrast agent indocyanine green, exhibit optical properties suitable for in vivo SWIR fluorescence imaging, including intravital microscopy, noninvasive, real-time imaging in blood and lymph vessels, and tumor-targeted imaging with IRDye 800CW, a dye being tested in clinical trials. Thus, we suggest that there is significant potential for SWIR imaging to be implemented alongside existing imaging modalities in the clinic.

Our understanding of the fundamental processes that drive biology and medicine is, in large part, based on our ability to visualize biological structures and monitor their transformations over time. Fluorescence imaging is one of the most transformative technologies of modern biomedical imaging as it provides a low cost, high sensitivity method for real-time molecular imaging in vivo. As the scattering and absorption of light through biological tissue impose significant restrictions on imaging penetration depth, acquisition speed, and spatial resolution, the development of novel optical imaging technologies has increasingly shifted toward the use of light of longer wavelengths. Fluorescence imaging in the shortwave infrared (SWIR, 1000 - 2000 nm) spectral region mitigates the negative effects of light attenuation and benefits from a general lack of tissue autofluorescence. As a result, SWIR imaging promises higher contrast, sensitivity, and penetration depths compared to conventional visible and near-infrared (NIR) fluorescence imaging. However, the lack of versatile and functional SWIR emitters has prevented the general adoption of SWIR imaging both in academic and clinical settings. Here, we will present progress toward the synthesis of a new generation of SWIR-emissive materials and discuss their use in enabling biomedical imaging applications. In the first part of this thesis, we will examine the synthesis of SWIR-emissive indium arsenide (InAs) quantum dots (QDs). To address existing challenges in the synthesis of these semiconductor nanocrystals, we will investigate the processes that govern nanoparticle formation and growth. Combining experimental and theoretical methods, we demonstrate that the synthesis of large nanocrystals is hindered by slow growth rates for large particles, as well as the formation and persistence of small cluster intermediates throughout nanocrystal growth. Based on these insights, we design a novel, rational synthesis for large InAs QDs with high brightness across the SWIR spectral region. Second, we will discuss the use of InAs-based QDs in functional SWIR imaging applications in pre-clinical settings. We will present three QD surface functionalizations that enable the non-invasive real-time imaging of hemorrhagic stroke, the quantification of metabolic activity in genetically-engineered animals, and the measurement of hemodynamics in the brain vasculature of mice. In addition, we will present preliminary results for the synthesis of SWIR-emissive QD probes for the molecular targeting of biological entities and for advanced particle tracking applications. Using a QD-based broadband SWIR emitter, we will further investigate the e↵ect of SWIR imaging wavelength on image contrast and tissue penetration depth. While it was previously assumed that reduced scattering of light at longer wavelengths is the primary cause for increased image contrast, our results indicate that for imaging scenarios with strong fluorescent background signals, image contrast and penetration depth correlate closely with the absorptive properties of biological tissue. As a result, deliberate selection of imaging wavelengths at which biological tissue is highly absorptive can help to overcome contrast-limited imaging scenarios. In the last part of this thesis, we will take a closer look at SWIR emitters with the potential for translation into clinical settings. We will demonstrate that the FDA-approved NIR dye indocyanine green (ICG) exhibits an unexpectedly high SWIR brightness that arises from a large absorption cross-section and a vibronic shoulder in its fluorescence spectrum that extends well into the SWIR spectral region. We expand on this finding by showing that ICG outperforms commercial SWIR dyes during in vivo imaging, and additionally by demonstrating a variety of high-contrast and high-speed imaging applications in small animals. These results suggest that ICG enables the direct translation of SWIR imaging into the clinic. In summary, this thesis will paint a comprehensive picture of the current state of SWIR-emissive materials, present the synthesis of novel versatile SWIR probes, and show their application in unprecedented functional SWIR imaging applications.

The evolution of technological advances in infrared sensor technology, image processing, "smart" algorithms, knowledge-based databases, and their overall system integration has resulted in new methods of research and use in medical infrared imaging. The development of infrared cameras with focal plane arrays no longer requiring cooling, added a new dimension to this modality. Medical Infrared Imaging: Principles and Practices covers new ideas, concepts, and technologies along with historical background and clinical applications. The book begins by exploring worldwide advances in the medical applications of thermal imaging systems. It covers technology and hardware including detectors, detector materials, un-cooled focal plane arrays, high performance systems, camera characterization, electronics for on-chip image processing, optics, and cost-reduction designs. It then discusses the physiological basis of the thermal signature and its interpretation in a medical setting. The book also covers novel and emerging techniques, the complexities and importance of protocols for effective and reproducible results, storage and retrieval of thermal images, and ethical obligations. Of interest to both the medical and biomedical engineering communities, the book explores many opportunities for developing and conducting multidisciplinary research in many areas of medical infrared imaging. These range from clinical quantification to intelligent image processing for enhancement of the interpretation of images, and for further development of user-friendly high-resolution thermal cameras. These would enable the wide use of infrared imaging as a viable, noninvasive, low-cost, first-line detection modality.

Optical imaging methods help researchers interrogate complex biological and medical processes with the use of optical light, usually in the visible region (400-700 nm). We explore visualizing the state of liver diseases in living mice using non-invasive near infrared (NIR, 700-900 nm) and shortwave infrared (SWIR, 900-1700 nm) autofluorescence imaging. NIR/ SWIR imaging makes use of recently developed, high-sensitivity cameras, and relatively low en-ergy NIR excitation, which is less destructive to and penetrates deeper through biological tissue than conventional ultraviolet or visible light. Detecting longer NIR/ SWIR autoflu-orescence emission takes advantage of both the maximal transparency of biological tissue at these wavelengths, and could also enable greater specificity to disease associated signal, as there are very few autofluorescent materials from healthy tissue samples at these wave-lengths. We extend the imaging techniques with incorporation of background and shading correction methods from a suite of computer vision methods to determine autofluorescence signal levels in brain tissue, which consists of highly complex varying cellular types, helping us understand the applicability of our imaging techniques with advanced methods of im-age processing. In addition, we further investigate immunofluorescence methods with the incorporation of NIR/SWIR autofluorescence as a lipofuscin specific channel to digitally re-move autofluorescence from multi-fluorophore immuno-stained mouse liver samples. We also explore color deconvolution in histopathology imaging, and develop algorithms to support automated thresholding and segmentation for more accurate autofluorescence quantification. We show the development of NIR/SWIR experimental methods and computer vision processes to achieve a better understanding of extending NIR/SWIR imaging in pre-clinical and clinical settings for studying disease progression and regression.

This book is a guide for the constantly growing community of the users of medical thermal imaging. It describes where and how an infrared equipment can be used in a strictly standardised way and how one can ultimately comprehensively report the findings. Due to their insight into the complex mechanisms behind the distribution of surface temperature, future users of medical thermal imaging should be able to provide careful, and cautious, interpretations of infrared thermograms, thus avoiding the pitfalls of the past. The authors are well-known pioneers of the technique of infrared imaging in medicine who have combined strict standard-based evaluation of medical thermal images with their expertise in clinical medicine and related fields of health management.