Browsing by Subject "Optical imaging"

The American Cancer Society reported an estimated 300,000 new cases of breast cancer and 44,000 new breast-cancer related deaths in 2022 in the United States alone. With each new successfully treated primary tumor, there is a subsequent risk of disease recurrence. Recurrence poses a risk to 10% of patients within the first 5 years post treatment and a lifetime risk of 30% across all patients. While new tools are being developed to better understand and mitigate the risk of recurrence, triple negative breast cancers, which exhibit no targetable surface markers, offer little in the way of recurrence prediction or treatment. It is understood that tumor heterogeneity is a driving force in tumor recurrence. Temporal heterogeneity is associated with therapeutic treatment, where the administration either selects for resistance subpopulations of tumor cells that are able to recur or a de novo resistant phenotype arises that leads to recurrence. Additionally, it has been well documented that tumors vary spatially across a primary tumor. This heterogeneity takes the form of genetic, epigenetic, and phenotypic heterogeneity. One such phenotype of interest is metabolic heterogeneity. Metabolism is classified as a ‘Hallmark of Cancer’ and has been studied as a driver of tumor progression for almost a century since Otto Warburg first described the phenomenon of tumors exhibiting high rates of aerobic glycolysis. Optical imaging is well poised to study metabolic heterogeneity due to its ability to image cellular level features, to multiplex multiple endpoints, and the ability to image longitudinally. Endogenous fluorescence contrast of coenzymes NADH and FAD have been used to report on the redox state of in vivo tissue and distinguish cancerous from benign lesions. The Center for Global Women’s Health Technologies (GWHT) has employed the use of exogenous fluorescent contrast agents to provide substrate-specific metabolic information. Three fluorescent agents have been validated including: 2-[N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG), a glucose derivative that is able to report on glycolysis; Tetramethylrhodamine ethyl ester (TMRE), a cation that is selectively attracted to the charge gradient generated by the mitochondria during ATP synthesis, making it a reporter of OXPHOS; and Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Hexadecanoic Acid (Bodipy FL C16), a long chain saturated fatty acid is taken up by the cell and undergoes beta oxidation similar to native fatty acids. More recently, GWHT has begun combing these fluorescence agents for in vivo use to provide a wholistic understanding of cancer metabolism. The work here sets out to develop a novel optical imaging platform that is capable of imaging multiplexed metabolic endpoints, for quantitative intra-image analysis of metabolic gradients. This technology is built on the use of exogenous fluorescence contrast agents to report on substrate or pathway specific axes of metabolism. By simultaneously introducing multiple contrast agents, it is possible to capture a more wholistic snapshot of tissue metabolism. To encourage the adoption of this technology, a novel low-cost instrument will also be developed. Leveraging a consumer grade CMOS camera and variable focus lens, it is possible to image over multiple length scales, capturing both bulk tumor features and also single cell features. The flexibility offered by this simple innovation will allow for metabolic imaging to be applied over a variety sample type. Three specific aims were proposed to realize this goal by developing methods of multi-parametric exogenous contrast and low-cost instrumentation for multi-scale imaging of tumor metabolic heterogeneity in preclinical models. Aim 1 validated and demonstrated a method for the simultaneous injection and measurement of Bodipy FL C16 and TMRE to report on lipid uptake and mitochondrial activity, two potentially interrelated axes of metabolism. To validate this method, three sets of experiments were performed to establish that the two probes do not exhibit chemical, optical, or biological crosstalk. Chemical compatibility was established using liquid chromatography. Briefly, high molar concentration solutions of each individual probe (Bodipy FL C16, TMRE, and 2-NBDG) were created alongside a solution of all three probes at the same concentration. Chromatograms were collected immediately upon mixing, after 1 hour and after 24 hours. The area under the curve for each probe at each time point displayed an area under the curve (AUC) within 2% of the AUC of the single probe solutions, suggesting no chemical reactions. Optical crosstalk was assessed using optical spectroscopy and tissue mimicking phantoms. Optical phantoms were created with tissue mimicking optical properties and various concentration of Bodipy FL C16, TMRE, polystyrene microspheres (tissue scattering mimic), and hemoglobin (tissue absorption mimic). Leveraging an inverse Monte Carlo algorithm, we demonstrated that accurate values for each fluorescent probe could be measured regardless of the concentration of the other optical probe or level of optical scattering or absorption, indicating optical compatibility. To address biological crosstalk, two sets of 4T1 tumor bearing mice were subject to optical spectroscopy with either 1) Bodipy FL C16 alone, 2) TMRE alone, 3) a dual injection of Bodipy FL C16 and TMRE. Fluorescence spectra were measured 2-, 4-, 6-, 8-, 10-, 20-, 30-, 40-, 50-, and 60-minutes post-injection to establish uptake kinetics. It was found that the uptake kinetics of the dual probe group were not statistically different from the single probe group, indicating biological compatibility. With no observable crosstalk between Bodipy FL C16 and TMRE, the two probes method was applied to characterize murine mammary gland and two tumor of differing metastatic potential (4T1 and 67NR). In addition, to Bodipy FL C16 and TMRE, oxygen saturation and total hemoglobin were extracted from estimates of optical absorption, and these 4 endpoints were used to attempt to cluster groups of tumor and normal tissue. Difficulty clustering tumor groups of varying metastatic potential suggest a need for imaging technology. In Aim 2 a low-cost fluorescence microscope was developed capable of performing quantitative fluorescence imaging over a variety of samples. The goal of this work was to design a system that could be adapted to image a number of different sample types include core-needle tissue biopsies, preclinical window chambers, and in vitro organoids. To accomplish this a low-cost CMOS detector was used with a variable magnification lens allowing for imaging at multiple length scales. Uniform illumination was a necessary criterion for quantitative imaging. To generate uniform illumination that could be scaled across multiple length scales, an LED coupled 1:4 fanout optical fiber was employed alongside a computational model to determine the positioning of each fiber. To automate the design of illumination, a computational model was employed where each optical fiber was modeled as a Lambertian emitter in a spherical coordinate system. To determine the ideal placement of each fiber such that the individual illumination contributions of all fibers summed to a uniform distribution, a global optimizer was employed. A genetic pattern search allowed for the selection of coordinates to produce uniform illumination that could be feasibly employed at the benchtop. This integrated system is referred to as the CapCell microscope. Using this computational approach, two uniform illumination profiles were designed, one with a high aspect ratio (length ≫ width) and one with a low aspect ratio (length = width). To demonstrate the utility of optimized illumination, core needle biopsies from 4T1 tumors were stained with a tumor-specific fluorescent contrast agent, HS-27 and imaged with either optimized or unoptimized gaussian illumination. The repeatability of intra-image features was compared for the two illumination scenarios, and it was found that uniform illumination repeatedly revealed the same fluorescent features across the sample. These features were further confirmed with standard histology. Window chamber imaging demonstrated the importance of designing application specific illumination. 4T1 mammary tumors were grown orthotopically before a window chamber was surgically implanted. Animals were injected with either Bodipy FL C16, 2-NBDG, or HS-27 and imaged with both the high AR and low AR illumination platforms. As expected, the low AR, designed for window chambers, had a higher power density at the sample site and thus increased contrast compared to the low AR images. With a method and a system in place, the goal of Aim 3 was to apply the optical imaging platform to observe spatiotemporal metabolic heterogeneity. To achieve this, the CapCell microscope was upgraded to enhance contrast and improve resolution for the visualization of capillaries and single cells. This was demonstrated using 4T1 window chamber models stained with acridine orange, a nucleus specific stain, and green light reflectance to highlight hemoglobin absorption in microvessels. Given the interplay between metabolism and vasculature it was desirable to employ a vessel segmentation approach to describe vascular features within an image. A Gabor filter and Djikstra segmentation approach was employed on metabolic images to enable metabolic and vascular comparisons across an image field of view. To test the improved CapCell system, 4T1 tumors were treated with combretastatin A-1, a vascular disrupting agent. Across the course of treatment, the CapCell was able to observe bulk changes in metabolism and vascular density. Additionally, by employing high resolution imaging, it was possible to observe relationships between each metabolic probe and vessel tortuosity. This analysis allowed for the identification of metabolically unique regions within each group of animals, demonstrating the ability of this technology to parse metabolically distinct regions of tumor. In total, the work outlined here describes the development of a novel optical imaging platform capable of quantifying intratumor metabolic heterogeneity of multiple metabolic endpoints over multiple length scales. The system expands on previous work developing methods for simultaneous measurement of exogenous fluorescent contrast agents to report on lipid uptake and mitochondrial activity. The system also introduced a novel computational approach to design uniform illumination for a low-cost microscope capable of imaging across multiple sample types. Together these technologies were used to observe metabolic heterogeneity in preclinical window chamber models following chemical perturbation. The technology introduced here, is primed for future exploration. First, it would be desirable to integrate all three exogenous contrast agents for simultaneous imaging of three axes of metabolism in vivo. Once accomplished, the sample technology could be applied to study metabolic and vascular changes associated with residual disease and tumors that are entering recurrence.

The development of non-invasive retinal imaging systems has revolutionized the care and treatment of patients in ophthalmology clinics. Using high-resolution modalities such as scanning laser ophthalmoscopy (SLO) and optical coherence tomography (OCT), physicians and vision scientists are able detect previously unseen features on the subject retina which can 1) provide information for diagnosis, 2) identify disease biomarkers, 3) inform treatment or clinical trial regimens, and 4) improve understanding of underlying disease processes. Traditional SLO and OCT devices are designed as tabletop systems which are unable to accommodate vulnerable populations including intrasurgical patients and young children. Thus, the miniaturization of these systems into compact, handheld form factors is of great interest in both biomedical optics/imaging and medical research fields as they are essential to the proper care of patients. Previous studies have shown that handheld systems are instrumental in assessing overall health of young children and disease progressions in subjects of all ages. However, handheld systems are limited in optical performance as hardware selection is restricted to components of small size and low weight. Additionally, aberrations induced by both the system optics and the human eye degrade the resolution of the images. This work focuses on the integration of adaptive optics (AO) technology into handheld form factors to correct for aberrations and provide in vivo visualization of single cells such as cone photoreceptors and retinal pigment epithelium cells. We present two devices which demonstrate the first ever dual-modality AO-SLO and AO-OCT handheld imaging devices that push the limits of comprehensive, cellular-resolution retinal imaging. Finally, we investigate the use of 3x3 fused fiber couplers as a simple, compact coherent receiver design. Our novel balanced-detection topology achieves shot-noise limited performance in the presence of excess noise and shows improved SNR as compared to previous implementations. We detail its ability to enable instantaneous quadrature projection for applications in LiDAR, phase imaging, and optical communications.

This dissertation explores computational optical imaging methods to circumvent the physical limitations of classical sensing. An ideal imaging system would maximize resolution in time, spectral bandwidth, three-dimensional object space, and polarization. Practically, increasing any one parameter will correspondingly decrease the others.

Spectrometers strive to measure the power spectral density of the object scene. Traditional pushbroom spectral imagers acquire high resolution spectral and spatial resolution at the expense of acquisition time. Multiplexed spectral imagers acquire spectral and spatial information at each instant of time. Using a coded aperture and dispersive element, the coded aperture snapshot spectral imagers (CASSI) here described leverage correlations between voxels in the spatial-spectral data cube to compressively sample the power spectral density with minimal loss in spatial-spectral resolution while maintaining high temporal resolution.

Photography is limited by similar physical constraints. Low f/# systems are required for high spatial resolution to circumvent diffraction limits and allow for more photon transfer to the film plain, but require larger optical volumes and more optical elements. Wide field systems similarly suffer from increasing complexity and optical volume. Incorporating a multi-scale optical system, the f/#, resolving power, optical volume and wide field of view become much less coupled. This system uses a single objective lens that images onto a curved spherical focal plane which is relayed by small micro-optics to discrete focal planes. Using this design methodology allows for gigapixel designs at low f/# that are only a few pounds and smaller than a one-foot hemisphere.

Computational imaging systems add the necessary step of forward modeling and calibration. Since the mapping from object space to image space is no longer directly readable, post-processing is required to display the required data. The CASSI system uses an undersampled measurement matrix that requires inversion while the multi-scale camera requires image stitching and compositing methods for billions of pixels in the image. Calibration methods and a testbed are demonstrated that were developed specifically for these computational imaging systems.

Optical coherence tomography (OCT) is a non-invasive optical imaging modality which can provide high-resolution, cross-sectional images of retina and cornea. It has become a standard of care in ophthalmology for the diagnosis and monitoring of ocular diseases. However, current OCT systems face several major challenges, among which include: (1) difficult alignment and fixation in pediatric retinal imaging (2) limited cellular-level contrast for ophthalmic disease diagnosis and (3) expensive hardware and intensive computation requirements for real-time high-speed 3D imaging.

This dissertation describes the development of several novel optical design and signal processing approaches in OCT and optical coherence imaging technologies to address these limitations. We first describe a long working distance swept-source OCT system to facilitate retinal imaging in young children (chapter 2). The system incorporates two custom lenses and a novel compact 2f retinal scanning configuration to achieve a working distance of 350mm with a 16o OCT field of view. The system achieves high quality retinal imaging of children as young as 21 months old without sedation in the clinic. We then present a spectroscopic OCT technology that utilizes time-frequency analysis to obtain quantitative diagnostic information of cellular responses in the anterior chamber of the eye, which can indicate many ocular diseases such as hyphema and anterior uveitis. We demonstrate that this technology can differentiate and quantify the composition of anterior chamber blood cells such as red blood cells and subtypes of WBCs, including granulocytes, lymphocytes and monocytes (chapter 3 and 4). Finally, we describe a coherence-based 3D imaging technique that uses a grating for fast beam steering, a swept-source laser with long coherence length, and time-frequency analysis for depth retrieval (chapter 5). We demonstrate that the system can achieve high-speed 3D imaging with sub-millimeter axial resolution and tens of centimeters axial imaging ranging.

According to the World Health Organization, there were over 2 million new breast cancer cases in 2018. This number is projected to steadily increase year after year. American Cancer Society projections for 2020 list the breast as the leading cancer site for new cancer cases in females, estimating breast cancer to represent 30% of all new cases and 15% of cancer-related deaths.

A leading cause of breast cancer deaths is due to tumor recurrence following therapy. These tumors can recur years, sometimes decades, after treatment from reservoirs of residual cells that persist in a dormant state. Conversely, the absence of residual invasive disease following adjuvant therapy constitutes pathological complete response (pCR) and is positively associated with long-term relapse-free survival. This risk for recurrence is higher for women with human epidermal growth factor receptor 2 (Her2+) breast cancer or triple-negative breast cancer (TNBC). Approximately 50-70% of Her2+ patients and 40-55% of TNBC patients who undergo standard therapy achieve pCR; however, in the remaining patients, only a partial response occurs, leaving residual disease and an increased risk of relapse.

To mitigate the cancer burden, years of research have focused on several common biological capabilities of cancer, deemed the Hallmarks of Cancer, including sustained proliferation, genome mutations, replicative immortality, resistance to cell death, and a deregulated metabolism. Several recent studies have further reported that this last hallmark, metabolism, may be vital to understanding the underlying behavior of dormant and recurrent tumors. Once understood, these changes in metabolic pathways, referred to as metabolic reprogramming, can be leveraged as vulnerabilities and allow for the development of strategies to eliminate residual disease or prevent residual tumor cells’ subsequent reactivation into full recurrence.

For nearly 100 years, increased aerobic glycolysis has been considered a feature of rapidly proliferating primary tumors. This occurrence, where cells continue to use the metabolic pathway where glucose is converted to lactic acid to release its stored energy and produce adenosine triphosphate (ATP) despite the presence of oxygen, has been termed the Warburg Effect. Because of this, physicians frequently use nuclear medicine directly imaging glucose uptake, fluorodeoxyglucose (FDG) Positron Emission Tomography (PET) imaging, for the diagnosis and staging of cancer. In addition to glycolysis, mitochondrial metabolism through oxidative phosphorylation has grown in recognition as an additional energy source for cancer cells. In mitochondrial metabolism, the tricarboxylic acid (TCA) cycle generates energy carriers to be used in the electron transport chain. Here, the mitochondrial membrane potential provides a gradient to produce large amounts of ATP. Additionally, the TCA cycle can rely on sources of carbon besides glucose alone. A steadily growing consensus points to other energetic sources, such as glutamine, amino acids, and lipids, that are key to survival, especially following environmental stress, treatment, or before migration and metastasis.

Though metabolic reprogramming underpins aspects of tumor dormancy and recurrence, currently, there are no techniques available to provide a systems-level approach to investigate the major axes of metabolism. Several techniques that offer insights into cellular metabolism exist, such as the Seahorse assay, metabolomics, and FDG-PET imaging. They, however, are limited to in vitro model systems, single-time point analyses of in vivo model systems, or single-endpoint analysis of in vivo model systems, respectively. Further, neither the Seahorse assay nor metabolomics can capture information about both the tumor and its native microenvironment. Therefore, there is an unmet need for a method to study metabolism at a spatial resolution that can elucidate the metabolic modulation of residual cell populations longitudinally and across in vitro and in vivo models.

Optical imaging is well-suited to address this gap in technologies owing to its ability to measure multiple metabolic endpoints non-destructively and repeatedly. The Center for Global Women’s Health Technologies has developed protocols for the use of two optical probes 2-[N-(7-nitrobenz-2-oxa-1, 3-diaxol-4-yl) amino]-2-deoxyglucose (2-NBDG) and tetramethylrhodamine, ethyl ester (TMRE), to image glucose uptake and mitochondrial membrane potential, respectively, in preclinical cancer models. These endpoints are superior to imaging of the endogenous fluorescence of NADH and FAD (referred to as the redox ratio) by providing a direct measure of a substrate (glucose uptake) and metabolic output (mitochondrial metabolism). This optical, metabolic imaging approach fills a critical gap that exists between in vitro studies on single cells (Seahorse Extracellular Flux Assay) and whole-body imaging (FDG-PET imaging) and is complementary to metabolomics and immunohistochemistry (IHC) with endpoints measuring the major axes of metabolism.

The work described here details an innovative platform to image changes in the metabolism of primary tumors, residual disease, and recurrent tumors using a Her2+ genetically engineered mouse model. This model exhibits key features of dormancy and mimics sustained use of targeted therapy to facilitate understanding of tumor biology and function, assess recurrence risk, and design therapies to mitigate residual disease and recurrence altogether. Imaging at a cellular level resolution will not only document acute metabolic changes following Her2 downregulation but also allow for metabolic imaging of dormant cell populations that are typically too small to study in human patients, typically referred to as no evidence of disease (NED) in humans. This platform will push metabolic studies of tumor dormancy further.

Three specific aims were proposed towards this ultimate goal to develop a multiparametric platform to characterize the metabolic reprogramming of preclinical cancer models.

Aim 1 establishes the functional flexibility of the fluorescent glucose analog 2-NBDG to measure glycolytic demand and the fluorescent cation TMRE to measure mitochondrial membrane potential to report on the metabolic changes that occur throughout tumor progression, dormancy, and recurrence. Using a genetically engineered mouse-derived three-dimensional in vitro mammosphere model allowed for metabolic endpoints to be captured across key time points. Doxycycline (dox) addition and withdrawal modulates expression of Her2, which is overexpressed in primary and re-activated mammospheres, and downregulated in regressing and dormant mammospheres. The mammospheres were characterized using immunofluorescence to confirm phenotype. Ki67 expression was high in primary and re-activated mammospheres, confirming a proliferative phenotype typical of both primary and recurrent disease presented in the clinic. On the other hand, short-term dox withdrawal resulted in increased cleaved caspase 3 (CC3) expression, confirming apoptosis due to Her2 downregulation. Finally, both Ki67 and CC3 expression were negative in dormant mammospheres, demonstrating a viable, but non-proliferative, steady-state phenotype.

Metabolic imaging revealed unique metabolic phenotypes across the tumor development stages that were consistent with the gold standard assays. While primary mammospheres, overexpressing Her2, maintained increased glucose uptake (“Warburg effect”), after Her2 downregulation, regressing and residual disease mammospheres appeared to switch to oxidative phosphorylation. Interestingly, in mammospheres where Her2 overexpression was turned back on to model recurrence, glucose uptake was lowest, indicating a potential change in substrate preference following the reactivation of Her2, re-eliciting growth. These findings highlight the importance of imaging metabolic adaptations to gain insight into residual and recurrent disease’s fundamental behaviors.

This work paved the way for similar studies in vivo using a mammary window chamber with the ultimate goal of informing the potential impact of metabolically-targeted therapies on tumor dormancy and recurrence.

In Aim 2, 2-NBDG and TMRE imaging was applied to in vivo mammary tumors as they transitioned from primary tumors, through regression and dormancy, to regrowth as recurrent tumors. Two tumor models varying in periods of dormancy (termed slow recurring and fast recurring tumors) were selected to characterize the importance of either axis of metabolism in the context of recurrent disease. When comparing the glucose demand and mitochondrial membrane potential levels between slow and fast recurring tumors, both sets of primary tumors behaved similarly to the primary mammosphere cultures: increased 2-NBDG indicating highly glycolytic tumors with low TMRE indicating little mitochondrial activity. Following acute Her2 downregulation, there was an increase of mitochondrial activity that remained relatively constant through regression, dormancy, and recurrence for both tumor types. However, glucose uptake varied between the two tumor types following Her2 downregulation. The mice bearing slow-recurring tumors showed a resurgence of glucose uptake during recurrence; conversely, the mice bearing fast-recurring tumors maintained decreased glucose levels continually following Her2 downregulation. Because the fast-recurring tumors did not have a meaningful change in glucose uptake during recurrence, it was hypothesized that the fast-recurring tumors might have reprogrammed to use fatty acids as a fuel source. Indeed, inhibiting fatty acid oxidation in these tumors resulted in increased glucose uptake during regression. Additionally, following this acute change in metabolism due to the inhibition of fatty acid oxidation, the tumor’s dormancy period prior to recurrence was prolonged, pointing to lipids as a crucial fuel source for residual disease and recurrence in aggressive breast cancer.

Aim 2 showed the importance of lipid metabolism in residual disease and recurrence. Additionally, other groups have also shown increased reliance on fatty acid oxidation in breast cancer residual disease following oncogene downregulation. Thus, Aim 3 established a method of visualizing long-chain fatty acid uptake in breast cancer murine models. Until now, the ability to monitor such uptake has been limited to in vitro and ex vivo approaches. Here, an imaging strategy that combines a fluorescently labeled palmitate molecule, Bodipy FL c16, and intravital, optical imaging was developed to measure exogenous fatty acid uptake. Because the palmitate’s 16th carbon is fluorescently labeled, immediate degradation of the Bodipy dye during fatty acid oxidation (β-oxidation) is prevented, allowing for fatty acid to be visualized through fluorescence imaging.

This technique was validated in two breast cancer models: a MYC-overexpressing transgenic triple-negative breast cancer (TNBC) model, previously reported to dramatically upregulate fatty acid oxidation intermediates, and the murine model of the 4T1 family, a group of sibling tumor lines with a reported wide range of metabolic phenotypes.

Using a genetically engineered mouse-derived xenograft allowed for fatty acid uptake levels to be captured during MYC-overexpression and following oncogene downregulation. Similar to the previously described genetically engineered model, this model used doxycycline addition and withdrawal to modulate MYC expression.

Through in vivo Bodipy FL c16 imaging, fatty acid uptake was found to be increased in MYC-high tumors. This model showcased two critically needed features for clinically relevant study of fatty acid uptake: 1) longitudinal metabolite tracking in a single animal shown through intra-animal decreases in fatty acid uptake following MYC-downregulation; and 2) providing a link between oncogene expression, which can be modulated therapeutically, and metabolic endpoints. This decreased uptake is indicative of a less aggressive state and correlates with a visible reduction in tumor volume. Additionally, this method found an increased fatty acid uptake in tumors with high metastatic potential, as well as the ability of the system to monitor inhibition efficacy, potentially allowing for therapeutic pharmaceutical testing of drug efficacy.

This fast and dynamic approach to image fatty acid uptake in vivo is a tool relevant to study tumor metabolic reprogramming or the effectiveness of drugs targeting lipid metabolism.

Targeting a tumor’s metabolic dependencies is a clinically actionable therapeutic approach, but identifying subtypes of tumors that are likely to respond remains difficult. The work presented here indicates that an optical platform to image 2-NBDG, TMRE, and Bodipy FL c16 longitudinally is well suited to characterize breast cancer residual disease and recurrence’s critical metabolic features and to pinpoint metabolic vulnerabilities for potential treatments. While the primary goal was to develop an imaging strategy for the unprecedented assessment of residual and recurrent disease at high resolution in in vitro and in vivo models, this innovation also fits within the broader framework of existing metabolic assessment techniques and provides a systematic way to connect in vitro studies to whole-body imaging within the context of preclinical pharmacology research.

Future work will focus on establishing a combined imaging strategy for simultaneous imaging of all three endpoints, transitioning imaging to a hand-held microscope for wide-spread adoption and rapid metabolic phenotyping of clinical samples, and integrating optical spectroscopy with this imaging platform to track the long-term effects therapy has on an individual tumor’s metabolism. The third will enable the ability to retrospectively look for changes in primary and regressing phenotypes that might foreshadow dormant behavior or the risk of early recurrence.

The Majority of imaging systems require optical lenses to increase the light throughput as well as to form an isomorphic mapping. Advances in optical lenses improve observing power. However, as imaging resolution reaches about the magnitude of $10^8$ or higher, such as gigapixel cameras, the conventional monolithic lens architecture and processing routine is no longer sustainable due to the non-linearly increased optical size, weight, complexity and therefore the overall cost. The information efficiency measured by pixels per unit-cost drops drastically as the aperture size and field of view (FoV) march toward extreme values. On the one hand, reducing the up-scaled wavefront error to a fraction of wavelength requires more surfaces and more complex figures. On the other hand, the scheme of sampling 3-dimensional scenes with a single 2-dimensional aperture does not scale well, when the sampling space is extended. Correction for shift-varying sampling and non-uniform luminance aggravated by wide-field angles can easily lead to an explosion of the lens complexity.

Parallel cameras utilize multiple apertures and discrete focal planes to reduce camera complexity via the principle of divide and conquer. The high information efficiency of lenses with small aperture and narrow FoV is preserved. Also, modular design gives flexibility in configuration and reconfiguration, provides easy adaptation and inexpensive maintenance.

Multiscale lens design utilizes optical elements in various size scales. Large aperture optics collects light coherently, and small aperture optics enable efficient light processing. Monocentric multiscale (MMS) lenses exemplify this idea by adopting a multi-layered spherical lens as the front objective and an array of microcameras at the rear for segmenting and relaying the wide-field image onto disjoint focal planes. First generation as-constructed MMS lenses adopted Keplerian style, which features a real intermediate image surface. In this dissertation, we investigate another design style termed "Galilean", which eliminates the intermediate image surface, therefore leading to significantly reduced lens size and weight.

The FoV shape of a parallel camera is determined by the formation of the camera arrays. Arranging array cameras in myriad formations allows FoV to be captured in different shapes. This flexibility in FoV format arrangement facilitates customized camera applications and new visual experiences.

Parallel cameras can consist of dozens or even hundreds of imaging channels. Each channel requires an independent focusing mechanism for all in focus capture. The tight budget on packing space and expense desires small and inexpensive focusing mechanism. This dissertation addresses this problem with the voice coil motor (VCM) based focusing mechanism found on mobile platforms. We propose miniaturized optics in long focal length designs, thus reduces the traveling range of the focusing group, and enables universal focus.

Along the same line of building cost-efficient and small size lens systems, we explore ways of making thin lenses with low telephoto ratios. We illustrate a catadioptric design achieving a telephoto ratio of 0.35. The combination of high index material and meta-surfaces could push this value down to 0.18, as shown by one of our design examples.

Cancer is a significant threat to human health with more than eight million deaths each year in the world. Therefore, numerous technologies have been implemented or under development to effectively treat cancer.

One novel therapeutic platform is implemented using nanoparticle-mediated photothermal therapy. Gold NanoStars (GNS), are a unique form of gold nanoparticles (GNPs) that have unique therapeutic potential because of their star-shaped geometry. Enhanced light absorption and higher photon-to-heat conversion efficiency are introduced by GNS’s plasmonic properties. In the application of hyperthermia, this photothermal process can be exploited to specifically heat tumors and, more importantly, to amplify the antitumor immune response following the highly immunogenic thermal death of cancer cells. Meanwhile, when combined with immune checkpoint inhibition immunotherapy (IT), this SYnergistic iMmuno PHOtothermal NanotherapY (SYMPHONY) has been shown to reverse tumor-mediated immunosuppression, thereby leading to the treatment of not only primary tumors but also cancer metastasis. This phenomenon is called the “abscopal effect”. However, the immune response has not been clearly quantified yet. Our hypothesis was that different treatment modalities (PTT only, GNS-PTT, IT, SYMPHONY) will trigger different levels of immune response including the decrease of immunosuppressive cells and the influx of cytotoxic cells, which could be observed by imaging immune cell reporters.

Accordingly, two specific aims were set for this study: 1. to develop a pre-clinical murine model to quantify different levels of immune response mimicking the tumor metastatic environment; 2. to quantify immune response following SYMPHONY using imaging analysis techniques.

To achieve the specific aims, window chamber models combined with in vivo fluorescence imaging techniques provide an ideal platform to mimic cancer metastasis in the chamber and longitudinally monitor immune response through the prevalence of fluorescent reporters specifically localized to immune cells of interest. We utilized a dual tumor mouse model, consisting of a primary tumor grown in the flank of the mouse which received the SYMPHONY therapy, as well as a secondary tumor located in the window chamber through which we could image and observe the abscopal response to therapy. In this study, we demonstrate the optical imaging procedure following synergistic immune photothermal therapy (SYMPHONY) of bladder cancer using the immune-GFP-labeled murine window chamber model, for the purpose of quantifying the immune response at this mimicked-tumor metastasis site. Four groups were established: the SYMPHONY group, the photothermal therapy group, the immune therapy group, and the Gold NanoStars (GNS) control group.

Higher immune responses were observed in the tumor regions compared to the non-tumor regions. The in vivo fluorescence imaging along with the window chamber technique demonstrates the feasibility and convenience of following such a longitudinal study like SYMPHONY. However, although temporal changes in reporter intensity were observed, with a limited number of samples, we cannot thus far identify significant differences among the treatment groups. Approaches for further characterizing this model are discussed.

Hypoxic tumor microenvironments have a clear correlation with a lack of radiosensitivity and diminished therapy response. This relationship can be described through the use of fluorescent and phosphorescent nanoparticles optically imaged in a mouse model. Through the use of this ratiometric oxygen sensing, the hypoxic state of the cancerous tumor can be compared. Normally, the microscope imaging system requires the mouse to be imaged under anesthesia and data recorded for a short amount of time. This has led to challenges in clearly defining the oxygen saturation levels in hemoglobin because the anesthesia can affect the tumor vascular dynamics. Moreover, it is desirable to track blood flow and oxygenation changes over a longer period of time in order to characterize the dynamics of cycling hypoxia and therapeutic response. Therefore, a mobile imaging apparatus has been designed and built to directly attach to the dorsal skinfold window chamber installed on nude murine models. Current progress includes quantifiable ratiometric oxygenation in boron nanoparticle solutions imaged under UV light with the mobile unit. The concept has also been successful in in vivo studies for anesthetized mice. The mobile unit is capable of resolving vasculature and is sensitive enough to record nanoparticle emissions originating from tissue in a mouse window chamber model. This system will use dynamic microscopy to image the tumor’s hypoxic environment on un-anesthetized mice and yield insight into tumor biology and therapeutic response.

Although hypoxia in tumors has long been identified as a key contributor to poor patient outcome, clinical solutions to lessen its deleterious effects have not been forthcoming. One reason is that there have been few means to directly quantify hypoxia kinetics with adequate spatial and temporal resolution. Dual-emissive boron nanoparticles show promise in this regard. Fluorescence imaging of these dual-emissive boron nanoparticles allows quantification of oxygen tension in a tumor microenvironment; this is accomplished by calculating the ratio of oxygen-independent fluorescence signal to oxygen-dependent phosphorescence signal.

In this work, we demonstrate the ability of these nanoparticles to, in conjunction with hyperspectral imaging of hemoglobin saturation, quantitatively characterize the oxygenation state of irradiated murine tumors in vivo. Mice were implanted with E0771 tumors in dorsal window chambers, and tumors were irradiated with 12Gy. Fluorescence images of dual-emissive nanoparticles injected into the tumors, as well as hyperspectral images of hemoglobin saturation, were obtained 1 day before irradiation and 2 days after irradiation. 2-way ANOVA statistical analysis revealed that while oxygen supply to the tumors – indicated by hemoglobin saturation – did not change significantly after irradiation, fluorescence-to-phosphorescence ratios – which indicate oxygen tension in the tumor – increased significantly post-irradiation. Through its success in demonstrating radiation-induced reoxygenation in E0771 tumors, the nanoparticles show promise for further applications in characterizing oxygenation states of various tumors post-treatment. This finding also further underscores the importance of using direct techniques in quantitative studies of tumor oxygen tension.

This work presents a second technological objective: to determine if folic acid-conjugated nanoparticles can successfully target folate receptor-expressing cell lines. This is a key step towards more practical delivery of the nanoparticles to deep tumor tissue via intravenous administration, as well as to enable intracellular quantification of oxygen tension. MDA-MB-231 cells were incubated overnight with varying concentrations of folic acid-conjugated nanoparticles. Fluorescence images were obtained to determine uptake of nanoparticles. The results have been encouraging; nanoparticle internalization was observed in almost all cells at all tested concentrations, and higher concentrations of nanoparticles appeared to increase the amounts of nanoparticles internalized per cell.

This work has validated that dual-emissive boron nanoparticles can directly quantify tumor oxygen tension post-irradiation, and that folic acid-conjugated boron nanoparticles can successfully target folate receptor-overexpressing cells. Hence, the immediate next step is to introduce folate receptor-targeting abilities to dual-emissive nanoparticles for improved performance in tumor oxygen-sensing applications.