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Non-melanoma skin cancer is the most prevalent malignancy in the world, with over a million annual positive diagnoses in the United States. If left untreated, these cancers cause morbidity and in rare cases, can become life threatening. The key to identifying and characterizing these tumors in the earliest stages, where they are most treatable lie in margin delineation in order to prevent recurrence. The visual obscurity of tumor morphology and physiology can make early detection a difficult task for dermatologists, particularly in the initial stages of cancer development. Tumor resection is a common course of action once they are discovered; however, there is a high recurrence rate due to incomplete removal of the malignant tissue. This dissertation presents an imaging system that can capture the spectral signatures correlating with morphological and physiological changes that accompany skin dysplasia. With this system, we may improve tumor margin delineation, reducing the number of incomplete tumor biopsies and false negative screenings. As an initial step of this process, we begin with a non-contact point sampling diffuse reflectance probe that mitigates the adverse effects of traditional contact probing. Validation of this probe is performed using tissue simulating phantoms spanning a biologically relevant range of optical and physiological properties to ensure that the non-contact format will not hinder performance relative to the contact probe. Cross polarization and auto-focus mechanisms were included in the design to reduce specular reflections and movement artifacts from in vivo measurements. This non-contact design was further developed into a platform for investigating the role of sampling geometry on diffuse reflectance measurements with the addition of a DMD spatial filter. Finally, we developed a hyperspectral DRSi system for the acquisition of wide-field maps of optical and physiological properties that is currently being tested on patients undergoing skin cancer screenings. The spectral output of this system has been validated for scattering and absorption across biologically relevant ranges using tissue simulating phantoms. The DRSi system was optimized for portability, ergonomics and resolution. / text

In this thesis I will introduce a novel nonlinear optical microscopy method to address some of the shortcomings in the current nonlinear optical microscopy literature and offer a supplement to traditional fluorescent microscopy for label free optical biomedical imaging. In order to demonstrate this method I describe a method for the generation of a numerical sample of collagen fibrils, produce a set of numerical diffraction measurements. I introduce a novel Ptychography model for the simultaneous reconstruction of the components of the nonlinear optical susceptibility tensor and demonstrate the results of this model using numerically generated measurements from a numerical collagen sample. I additionally use the recovered information from Ptychography to retrieve new information about the structure of a sample.

Cancer cells require access to blood vessels for oxygen and nutrients to enable growth and metastasis, making the tumour vasculature an attractive potential target for cancer therapies. Recent evidence suggests that the tumour vasculature plays a significant role in tumour response to high dose radiation therapy; however this effect is not well characterized due to limitations in quantitative imaging of the microvasculature. Speckle variance optical coherence tomography is an emerging imaging modality capable of 3D, non-invasive imaging of in vivo microvasculature. This thesis outlines the work done to test the hypothesis that svOCT imaging can be used to quantitatively monitor the vascular effects of high dose radiotherapy in a preclinical model. This was achieved through the development of a quantification pipeline for longitudinal 3-D svOCT images of microvascular radioresponse.

Cancer cells require access to blood vessels for oxygen and nutrients to enable growth and metastasis, making the tumour vasculature an attractive potential target for cancer therapies. Recent evidence suggests that the tumour vasculature plays a significant role in tumour response to high dose radiation therapy; however this effect is not well characterized due to limitations in quantitative imaging of the microvasculature. Speckle variance optical coherence tomography is an emerging imaging modality capable of 3D, non-invasive imaging of in vivo microvasculature. This thesis outlines the work done to test the hypothesis that svOCT imaging can be used to quantitatively monitor the vascular effects of high dose radiotherapy in a preclinical model. This was achieved through the development of a quantification pipeline for longitudinal 3-D svOCT images of microvascular radioresponse.

The exploration of nanoparticles for applications in medicine has grown dramatically in recent years. Due to their size, nanoparticles provide an ideal platform for combining multiple functionalities and interfacing directly with the biological realm. Additionally, nanoparticles can have physical properties that don't naturally exist in biology. Metal nanoparticles in particular have unique optical and magnetic properties which have driven nanomaterials research. The optical properties of gold nanoparticles and the magnetic properties of iron nanoparticles make them suitable for use as contrast agents in diagnostics and for radiation enhancement in therapeutic applications. The strong optical absorption and scattering and the nature of the conduction electrons of gold particles makes them ideal contrast agents for two-photon microscopy, photoacoustic imaging, and photothermal therapy. The superparamagnetic nature of iron oxide nanoparticles is clearly visible in magnetic resonance imaging, rendering them suitable as whole-body imaging contrast agents. All nanoparticle types can serve as delivery vehicles for drugs consisting of small molecules, peptides, or nucleic acids. This multiplicity of characteristics renders nanoparticles suitable for use in combining diagnosis and therapy, such as using particles to first detect the spatial extent of a cancer, and then to enhance near-infrared radiation in the tissue optical window to induce localized heating of diseased tissue. This combined approach requires both a mechanism of enhanced imaging contrast and a localized therapeutic mechanism, and the studies presented in this dissertation present work both on these aspects. By coating iron oxide nanoparticle cores with gold shells, it is possible to obtain a nanoparticle with both magnetic and optical properties. While individual gold nanoparticles do not absorb light in the infrared, receptor-mediated aggregation and the plasmon coupling effect lead to enhanced optical absorption only in diseased tissue. In addition to exploring these advanced applications, this work presents a fundamental investigation into the stability of gold nanoparticles in biological media. A previously unknown mechanism of gold nanoparticle destabilization and opsonization is presented and supported, along with a technique for reducing this opsonization and greatly enhancing the stability of gold particles in biological applications. This work will provide guidance to future designs of nanoparticle systems. / text

Microelectronic vision prostheses aim to restore visual percepts through electrical stimulation of the surviving visual pathways in the blind. Electrical stimulation has been shown to produce spots of light in the visual field. A neurostimulator that forms the basis of a vision prosthesis was designed using a high voltage CMOS process to allow it to be able to stimulate when faced with high electrode-tissue impedances. It was implemented with novel features that allow it to be scalable, and to focus charge injection, and can stimulate multiple sites simultaneously using a current source and sink at each site. To reduce electrical cross-talk between multiple stimulation sites, six-return electrodes surround each stimulating electrode, electrically guarding them from each other. The six-return electrode configuration was shown to reduce electrical cross-talk in saline bath tests compared to single-return electrode configurations. The neurostimulator was used to evoke responses from cats through electrical stimulation via intravitreal ball electrodes, corneal electrodes, and planar electrode arrays in the suprachoroidal space. Responses were measured on the visual cortex through optical imaging of intrinsic signals, and through surface electrodes. Using the planar electrode array in the suprachoroidal space, responses were elicited to biphasic, bipolar and monopolar stimuli, with each stimulating electrode coupled with either six-return electrodes, two-return electrodes, or a single-return electrode. The average charge threshold to elicit a response for biphasic, bipolar stimulation with six-return electrodes was 76.47 ?? 8.76 nC (standard error of the mean). For biphasic, bipolar stimulation, the magnitude and area of cortical response with the six-return electrode configurations was on average 2.18 ?? 0.19 times smaller than single-return electrode configurations, and 1.89 ?? 0.19 times smaller than two-return electrode configurations (P < 0.0001). It was also found that for biphasic stimulation, a greater magnitude and area of response was elicited for monopolar stimulation compared to bipolar stimulation. This dissertation details the design and testing of a novel, scalable neurostimulator to focus charge injection. It also shows that suprachoroidal, bipolar stimulation can elicit visual responses, and that the area of cortical activation was more focused when using bipolar, biphasic stimulation, and six-return electrodes.

A new molecular imaging modality has been developed to detect and locate positive axillary and sentinel lymph nodes non-invasively in breast cancer patients undergoing lymphoscintigraphy. The modality is based on fluorescent photon detection to locate the presence of indocyanine green (ICG) in the lymph subsequent to peritumoral injection of ICG into the breast. The imaging system consists of a gain-modulated intensified charge-coupled device (ICCD) camera, which captures low-intensity, near-infrared, and frequency-modulated photons. A four-fold ‘optical lymphography’ study was conducted to (1) examine fluorescence depth penetration and ICCD system accuracy at clinically relevant depths, (2) compare image quality of the ICCD system vs. conventional gamma imaging, (3) measure ICG pharmacokinetics in vivo, and (4) develop a clinical protocol while examining pre-clinical factors such as the outcome of combining ICG with sulfur colloids used in lymphoscintigraphy. The frequency-domain ICCD system was found to precisely detect modulation amplitude, IAC, and phase, θ, at depths up to 9 cm and with IAC accuracy less than 20% and θ less than 2º using an 80-mW laser incident on phantoms having ranging tissue optical properties. Significant differences in the mean depth of penetration owing to 0.62-ns lifetime and 100-MHz frequency increases were detected. An in vivo optical vs. nuclear image quality comparison demonstrated statistically similar (α=0.05) target-to-background ratios for optical (1.4+/-0.3) and nuclear (1.5+/-0.2). Alternatively, resulting image signal-to-noise ratios (SNR) from the ICCD system were greater than that achieved with a conventional gamma camera (pvalue<<0.01). Analysis of SNR versus contrast showed greater sensitivity of optical over nuclear imaging for subcutaneous tumors. In vivo and rapid detection of ICG in the blood-stream of nude mice was accomplished with a home-built avalanche photodiode dynamic fluorescence measurement system. Intensity data upon i.v. injection were regressed with a pharmacokinetic model describing the partitioning of ICG from the blood to the surrounding tissues. ICG blood-clearance was detected approximately 15 min after injection. Lastly, a human subject protocol was written, practiced, and federally approved for the application of optical lymphography. Furthermore, ICG was unaffected when mixed with sulfur colloids thus supporting the feasibility for combining fluorescence imaging with lymphoscintigraphy in breast cancer patients.

A new molecular imaging modality has been developed to detect and locate positive axillary and sentinel lymph nodes non-invasively in breast cancer patients undergoing lymphoscintigraphy. The modality is based on fluorescent photon detection to locate the presence of indocyanine green (ICG) in the lymph subsequent to peritumoral injection of ICG into the breast. The imaging system consists of a gain-modulated intensified charge-coupled device (ICCD) camera, which captures low-intensity, near-infrared, and frequency-modulated photons. A four-fold ‘optical lymphography’ study was conducted to (1) examine fluorescence depth penetration and ICCD system accuracy at clinically relevant depths, (2) compare image quality of the ICCD system vs. conventional gamma imaging, (3) measure ICG pharmacokinetics in vivo, and (4) develop a clinical protocol while examining pre-clinical factors such as the outcome of combining ICG with sulfur colloids used in lymphoscintigraphy. The frequency-domain ICCD system was found to precisely detect modulation amplitude, IAC, and phase, θ, at depths up to 9 cm and with IAC accuracy less than 20% and θ less than 2º using an 80-mW laser incident on phantoms having ranging tissue optical properties. Significant differences in the mean depth of penetration owing to 0.62-ns lifetime and 100-MHz frequency increases were detected. An in vivo optical vs. nuclear image quality comparison demonstrated statistically similar (α=0.05) target-to-background ratios for optical (1.4+/-0.3) and nuclear (1.5+/-0.2). Alternatively, resulting image signal-to-noise ratios (SNR) from the ICCD system were greater than that achieved with a conventional gamma camera (pvalue<<0.01). Analysis of SNR versus contrast showed greater sensitivity of optical over nuclear imaging for subcutaneous tumors. In vivo and rapid detection of ICG in the blood-stream of nude mice was accomplished with a home-built avalanche photodiode dynamic fluorescence measurement system. Intensity data upon i.v. injection were regressed with a pharmacokinetic model describing the partitioning of ICG from the blood to the surrounding tissues. ICG blood-clearance was detected approximately 15 min after injection. Lastly, a human subject protocol was written, practiced, and federally approved for the application of optical lymphography. Furthermore, ICG was unaffected when mixed with sulfur colloids thus supporting the feasibility for combining fluorescence imaging with lymphoscintigraphy in breast cancer patients.

Blood flow and its payload of molecular oxygen are two parameters of most physiological interest. Systemic tissue health is routinely gauged through measurements of vitals and oxygen saturation to estimate the state of these physiological parameters. We design, develop, and deploy optical imaging systems for examining perfusion and oxygenation at the local tissue level and apply these techniques for elucidating the normal and pathological processes associated with neurovascular disease. Specifically, we develop and validate the ability to use Multi-Exposure Speckle Imaging (MESI) to estimate microvascular flow dynamics in rodents over acute and chronic periods. Next, we pose significant optimizations to improve the efficacy of the widefield imaging technique for adoption by bench-side and clinical perfusion studies. We also introduce re-interpretations of the underlying physics to advance the theory that quantifies motion from the imaged speckle patterns. Finally, the technique is deployed for chronic monitoring of cortical flow dynamics before after focal ischemia of the motor cortex as part of a behavioral study in rodents. At the microscale, we develop and validate Two Photon Phosphorescence Lifetime Microscopy (2PLM) to examine dissolved oxygen concentration in microvasculature in three dimensions. We examine the technique’s ability for functional mapping of the rodent cortical microvascular network by quantifying the partial pressure of oxygen (pO₂) before and after occlusion of critical arterioles. Automation of acquisitions and processing for robust oxygen mapping within the micro-vascular network are developed and evaluated. The in vivo results are presented in light of those from studies utilizing more invasive mapping electrodes to provide independent corroboration of the observed neurovascular oxygen distributions. The technique is deployed for examining high resolution functional and structural remodeling after focal cerebral ischemia. / text