Imaging with high spatial resolution and high specificity within intact tissues at depth has long been a critical research objective for implementation in biological studies. The development of imaging tools with the capability of deep imaging at cellular resolution would allow for more realistic and complicated biological hypotheses to be tested in their natural environment - intravitally. The most challenging aspects of such tool developments involve light scattering and aberration, which cause the light to distort along its propagation direction, limiting both its imaging depth and resolution. This thesis attempts to provide several solutions to overcomes these challenges. To overcome light scattering, imaging within the short-wave infrared region (SWIR, wavelength 1 - 2.5 micrometers) is explored in chapters 2-4. In chapter 2 and 3, reflectance confocal and fluorescence confocal microscopy are demonstrated providing 2-4 times deeper penetration than any previously reported work and preclude the possibility of using one-photon confocal microscopy for deep imaging, a method that has been rarely discussed. Furthermore, a study on the impact of staining inhomogeneity on the depth limit of fluorescence confocal microscopy also demonstrated the potential of confocal microscopy combined with SWIR and low staining inhomogeneity to achieve unprecedented imaging depth. After demonstrating the deep imaging capability of one-photon imaging at depth with a SWIR light source, a multimodal system combining three-photon, third-harmonic, and optical coherence microscopy (OCM) is demonstrated in chapter 4. This multimodal system was able to achieve simultaneous imaging depth comparable to imaging with multiple contrast mechanisms in terms of the fluorescence, the harmonic signal, and the backscattering. Furthermore, this multimodal system provided complementary information about the mouse in vivo and represented a powerful intravital biological imaging tool. To overcome light aberration, adaptive optical methods are demonstrated in chapters 5-7. In chapter 5, a sensorless, adaptive optics, and indirect wavefront sensing system is demonstrated to improve SWIR-excited three-photon imaging, achieving about 7x signal enhancement in the mouse hippocampus area. This method is based on using the nonlinear three-photon fluorescence signal as feedback and involves light exposure during the optimization process. To reduce light exposure, a more direct wavefront sensing method is explored using a SWIR OCM system to directly sense the complex field of a biological sample in chapter 6. The advantage of this system, including its potential high-speed wavefront sensing and offline wavefront estimation, and its limitations with respect to phase stability are discussed. Finally, in chapter 7, a direct wavefront sensing method based on a cheap silicon wavefront sensor is presented. This method provides a convenient approach for aberration measurement with any experiment that involves SWIR ultrafast laser. This thesis shows the great promise to achieve high-resolution deep tissue imaging at a larger depth by combining longer wavelength at short-wave infrared region and adaptive optics. It is anticipated that this thesis work will open doors to much more exciting biological research in the near future.

The use of light for probing and imaging biomedical media is promising for the development of safe, noninvasive, and inexpensive clinical imaging modalities with diagnostic ability. The advent of ultrafast lasers has enabled applications of nonlinear optical processes, which allow deeper imaging in biological tissues with higher spatial resolution. This book provides an overview of emerging novel optical imaging techniques, Gaussian beam optics, light scattering, nonlinear optics, and nonlinear optical tomography of tissues and cells. It consists of pioneering works that employ different linear and nonlinear optical imaging techniques for deep tissue imaging, including the new applications of single- and multiphoton excitation fluorescence, Raman scattering, resonance Raman spectroscopy, second harmonic generation, stimulated Raman scattering gain and loss, coherent anti-Stokes Raman spectroscopy, and near-infrared and mid-infrared supercontinuum spectroscopy. The book is a comprehensive reference of emerging deep tissue imaging techniques for researchers and students working in various disciplines.

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.

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.

In vivo fluorescence imaging is an emerging technique with potential for usage in non-invasive cancer screening, surveillance, real-time surgical guidance, and staging. Fluorescence imaging uses the interaction of non-ionizing optical radiation with endogenous fluorophores or fluorescent labels to provide real-time wide-field images of tissue structure and/or functional components. When imaging in vivo, excitation light must travel through overlying tissue to reach the fluorescent target and emitted fluorescence must then propagate back through the overlying tissue in order to be imaged onto a camera. Recently, fluorescent contrast agents have been developed with excitation and emission wavelengths in the near infrared (NIR) spectrum (~700 - 1,000 nm) in order to minimize attenuation and maximize the measured signal from tissue. While several clinical trials have shown the potential benefits of NIR contrast agents over visible fluorophores, there may still be room for improvement by moving to even longer wavelengths. As scattering is reduced as wavelength increases, some researchers are investigating fluorophores that emit in the short-wave infrared (SWIR) wavelength region (~1,000 - 2,300 nm). This study focuses on examining optical transmission and image contrast at NIR wavelengths and SWIR wavelengths to determine which wavelength region may be optimal for development of fluorescent contrast agents. Transmission and contrast measurements were performed on both tissue simulating phantoms and real biological tissues using 780 nm, 980 nm, and 1550 nm wavelengths. From the experiments conducted, it appears that fluorophore emissions should be chosen based on the goals of the specific application. For an application that requires simple detection of signal, near infrared wavelengths will be better as they can be detected with higher signal levels. For an application that focuses on imaging fluorophore-labeled tissues, short-wave infrared wavelengths will be the better option as they provided better image contrast.

Imaging of tissue blood flow (BF) distributions provides vital information for the diagnosis and therapeutic monitoring of various vascular diseases. The innovative near-infrared speckle contrast diffuse correlation tomography (scDCT) technique produces full 3D BF distributions. Many advanced features are provided over competing technologies including high sampling density, fast data acquisition, noninvasiveness, noncontact, affordability, portability, and translatability across varied subject sizes. The basic principle, instrumentation, and data analysis algorithms are presented in detail. The extensive applications are summarized such as imaging of cerebral BF (CBF) in mice, rat, and piglet animals with skull penetration into deep brain. Clinical human testing results are described by recovery of BF distributions on preterm infants (CBF) through incubator wall, and on sensitive burn tissues and mastectomy skin flaps without direct device-tissue interactions. Supporting activities outlined include integrated capability for acquiring surface curvature information, rapid 2D blood flow mapping, and optimizations via tissue-like phantoms and computer simulations. These applications and activities both highlight and guide the reader as to the expected abilities and limitations of scDCT for adapting into their own preclinical/clinical research, use in constrained environments (i.e., neonatal intensive care unit bedside), and use on vulnerable subjects and measurement sites.

Despite a number of books on biophotonics imaging for medical diagnostics and therapy, the field still lacks a comprehensive imaging book that describes state-of-the-art biophotonics imaging approaches intensively developed in recent years. Addressing this shortfall, Advanced Biophotonics: Tissue Optical Sectioning presents contemporary methods and