Browsing by Author "Wax, Adam P"

Cancer is now the leading cause of death in developed countries. Despite advances in strategies aimed at the prevention and treatment of the disease, early detection of precancerous growths remains the most effective method of reducing associated morbidity and mortality. Pathological examination of physical tissues that are collected via systematic biopsy is the current "gold standard" in this pursuit. Despite widespread acceptance of this methodology and high confidence in its performance, it is not without limitations. Recently, much attention has been given to the development of optical biopsy techniques that can be used clinically and are able to overcome these limitations. This dissertation describes one such optical biopsy technique, angle-resolved low coherence interferometry (a/LCI), its adaptation to a clinical technology, and its evaluation in clinical studies.

The dissertation presents the theory that underlies the operation of the a/LCI technique, the design and validation of the clinical instrument, and its evaluation by means of two clinical trials. First, an account of the manner in which the depth-resolved angular scattering profiles that are collected by a/LCI can be used to determine nuclear characteristics of the investigated tissues is given. The design of the clinical system that is able to collect these scattering profiles through an optical fiber probe that can be passed through the accessory channel of an endoscope for in vivo use is presented. To demonstrate the ability of this system to accurately determine the size of cell nuclei, a set of validation experiments are described.

In order to evaluate the clinical utility of this a/LCI system, two clinical trials intended to assess the ability of a/LCI to detect the presence of early, pre-cancerous dysplasias in human tissues are presented. The first of these, an in vivo study of Barrett's esophagus (BE) patients undergoing routine surveillance for the early signs of esophageal adenocarcinoma, is described. This study represents the first use of the a/LCI technique in vivo, and confirms its ability to provide clinically useful information regarding the disease state of the tissue that it examines, with performance that compares favorably to other optical biopsy techniques. Next, an ex vivo study of resected intestinal tissue is presented. The results of this study demonstrate the ability of a/LCI to provide information that can be used to detect dysplasia in the lower gastrointestinal tract with high accuracy. This study will enable future development of the technology to allow conduction of in vivo trials of intestinal tissue. The results of these two clinical studies demonstrate the clinical utility a/LCI, illustrating its potential as an optical biopsy technique that has great potential to provide diagnostically relevant information during surveillance procedures. This is particularly relevant in the case of BE, where its successful use has been demonstrated in vivo.

Optical imaging techniques generally offer shallow penetration depths due to high scattering in biological tissue. We have recently developed frequency domain multispectral multiple scattering low coherence interferometry (ms2/LCI) for deep tissue imaging. The ms2/LCI system offers unique spatial and angular rejection of out-of-focus photons by utilizing an off-axis interferometric setup. Multiply forward scattered light is preferentially detected for imaging at extended depths. Using tissue-mimicking phantoms that match the full scattering phase function of human dermal tissue, we demonstrate that the ms2/LCI system can provide a signal/noise ratio (SNR) improvement of 15.4 dB over conventional OCT at an imaging depth of 1 mm.

In vivo imaging is challenging for the ms2/LCI system due to its slow acquisition speed. To enable fast image acquisition, we have developed dual-axis optical coherence tomography (DA-OCT), which offers a 100-fold speed increase compared to the ms2/LCI system. Two DA-OCT systems were designed and constructed, operating at a center wavelength of 800 nm and 1300 nm respectively. The 1300 nm DA-OCT system offers up to 2 mm depth penetration in skin imaging, which is unprecedented for an OCT system. This significant improvement in penetration depth opens the door for various exciting applications in fields where conventional OCT imaging was limited by poor penetration depths.

Deep features revealed by DA-OCT can be confounded by speckle noise. Speckle is an intrinsic noise of interferometric signals which reduces contrast and degrades the quality of images. A novel frequency compounding speckle reduction technique using the Dual Window (DW) method was recently presented. Using the DW method, speckle noise is reduced without the need to acquire multiple frames. A ~25% improvement in the contrast-to-noise ratio (CNR) was achieved using the DW speckle reduction method with only minimal loss in resolution. The DW speckle reduction method can work on any existing OCT instrument without further system modification or extra components. This makes it applicable both in real-time imaging systems and during post-processing.

Finally, functional information was extracted from the raw interferometric data for diagnostic purposes. Depth-resolved spectra were calculated by a time-frequency analysis, which carry valuable localized tissue information. The spectroscopic information was first used to objectively evaluate burn injuries in an in vivo mouse model. Significant spectral differences were observed and correlated with the depth of the injury as determined by histopathology. Later, spectroscopic DA-OCT was used for the assessment of flap viability in an in vivo Macfarlane rat flap model, which exhibited a gradient in tissue perfusion along the length of the flap. These results suggest that the DA-OCT system can be used for objective evaluation of skin injuries at extended depths.

Cancer is a disease that affects millions of people each year. While methods for the prevention and treatment of the disease continue to advance, the early detection of precancerous development remains a key factor in reducing mortality and morbidity among patients. The current gold standard for cancer detection is the systematic biopsy. While this method has been used for decades, it is not without limitations. Fortunately, optical detection of cancer techniques are particularly well suited to overcome these limitations. This dissertation chronicles the development of one such technique called Fourier domain low coherence interferometry (fLCI).

The presented work first describes a detailed analysis of temporal and spatial coherence. The study shows that temporal coherence information in time frequency distributions contains valuable structural information about experimental samples. Additionally, the study of spatial coherence demonstrates the necessity of spatial resolution in white light interferometry systems. The coherence analysis also leads to the development of a new data processing technique that generates depth resolved spectroscopic information with simultaneously high depth and spectral resolution.

The development of two new fLCI optical systems is also presented. These systems are used to complete a series of controlled experiments validating the theoretical basis and functionality of the fLCI system and processing methods. First, the imaging capabilities of the fLCI system are validated through scattering standard experiments and animal tissue imaging. Next, the new processing method is validated by a series of absorption phantom experiments. Additionally, the nuclear sizing capabilities of the fLCI technique are validated by a study measuring the nuclear morphology of in vitro cell monolayers.

The validation experiments set the stage for two animal studies: an initial, pilot study and a complete animal trial. The results of these animal studies show that fLCI can distinguish between normal and dyplastic epithelial tissue with high sensitivity and specificity. The results of the work presented in this dissertation show that fLCI has great potential to develop into an effective method for early cancer detection.

Minimally invasive surgical procedures using arthroscopic, catheterized, or laparoscopic devices are an increasingly preferred alternative to more labor-intensive open surgeries and typically associated with less pain, shorter hospital stays and fewer complications [1, 2]. However, these surgical models rely heavily on a combination of preoperative imaging and white-light intraoperative imaging technologies for guidance, which often severely limit the surgeon's depth perception and visualization of critical surface and sub-surface anatomy during endoscopic surgery.Optical coherence tomography (OCT) is a noninvasive optical imaging technique that captures volumetric imaging data of tissues at nearly cellular resolution [3]. Endoscopic OCT presents significant advantages over traditional white-light intraoperative imaging technologies enabling real-time volumetric visualization and quantitative feedback during surgery [4]. However, although this technology has been well demonstrated in research settings, endoscopic OCT has yet to find widespread use in the operating suite. This is primarily due to a lack of effective instrumentation for obtaining adequate depth penetration in highly scattering tissue, surgical/ergonomic useability in a minimally invasive setting, and a limited proof-of-concept demonstrations in non-retinal tissues. To address these challenges, this dissertation presents a collection of scientific works to extend the range of OCT for surgery by v adapting low-cost OCT technology and a dual-axis architecture to solve unmet clinical needs. First, a method for extending the imaging plane of a low-cost OCT system using a commercially available narrow rigid borescope is reported. This approach enables the application of the system in otherwise hard-to-access regions necessary for endoscopic adoption. This design's clinical potential is demonstrated by quantifying articular cartilage thickness, a primary biomarker of joint health during osteoarthritis (OA), for real-time feedback during arthroscopic surgery at a substantial reduction in cost compared to current protocols. Second, OCT is applied to evaluate the small bowel tissues of two rhesus macaques undergoing intestinal transplantation of the ileum using a handheld surgical probe. The correspondence between traditional assessment from routine histological observation and structures visualized with OCT were compared to assess the diagnostic capabilities of OCT for revealing changes associated with intestinal transplant rejection. Third, the image penetration of OCT is extended at a clinically relevant scale by adopting Dual-Axis OCT (DA-OCT) technology for increased depth priority in highly-scattering media. OCT imaging past 2 mm is measured using 1.3 μm wavelengths and enhanced depth of field (DOF) using a dynamic focusing method. Deep imaging performance for DA-OCT and conventional OCT is extensively compared using contrast-to-noise (CNR) analysis, highlighting the importance of high-order scattering anisotropy in tissue to maintain DA-OCT's depth advantage. Finally, DA-OCT vi is translated from a proof-of-concept research device into a low-cost and portable handheld device suitable for adoption in the operating suite. A handheld deep-tissue imaging device was developed with a narrow distal profile required for oral surgery. Preliminary studies are reported to validate the system's ability to image dental tissue. By making OCT more accessible and expanding the range of tissues that may be evaluated in vivo, these advancements demonstrate the potential of OCT for broad EMIS adoption.

Alzheimer’s disease (AD) is a neurodegenerative disease currently affecting 5.8 million Americans and more than 50 million people worldwide. It is a progressive disease that destroys cognitive functions, leading to dementia. With increasing life-expectancy, important efforts have been made to clinically diagnose this age-related disease. However, definitive diagnosis of AD has been challenging, especially at an early stage, as there is a lack of quantifiable changes. Recently, many researchers have shown retinal changes as an extension of the brain pathology, leading to a window to study AD using fast and high-resolution retinal imaging tools. This dissertation will be focused on the development of low-cost imaging tools aimed to extract retinal biomarkers for AD. Specifically, the use of optical coherence tomography (OCT) and angle-resolved low-coherence interferometry (a/LCI) will be described, with steps leading to a combined optical system for retinal imaging in humans. OCT has already been established as the gold standard in ophthalmology due to its excellent axial resolution and high sensitivity. Similar to OCT, a/LCI is another interferometric technique that provides depth resolution. Previous work has supported the ability of a/LCI to retrieve depth-resolved light scattering measurements of nuclear morphology in dysplastic tissue. The use of OCT as image guidance for a/LCI can strengthen the technique, providing sample orientation as well as retinal layer segmentation to pinpoint a/LCI measurements. The dissertation starts with the development and clinical application of a low-cost OCT system. Despite the prevalence of OCT, its high-cost nature has limited its access to large eye centers and away from low-resource settings. Clinical feasibility of a complete low-cost OCT system will be evaluated, and its imaging performance compared to a commercial system. System design will be discussed, followed by a comprehensive image processing pipeline to characterize image quality for subsequent low-cost systems. The subsequent portions outline studies using a/LCI and the extraction of light scattering parameters in an AD mouse model. A benchtop co-registered system using a/LCI guided by OCT allowed measurements of depth-resolved light scattering measurements in an AD mouse retina model. Resulting parameters serve as unique quantification of AD tissue structure with potential to be translated to future human studies. A scanning mechanism for 2D a/LCI is also presented, which also allowed for the characterization of a/LCI sensitivity to anisotropic scattering that is often present in the complex retinal tissue. The last portion discusses the development of a second-generation low-cost OCT system which will be integrated in a combined imaging system for eventual AD studies in human patients. Several technical improvements are shown to facilitate clinical retinal imaging at the point-of-care. A characterization of this system in a small clinical study will illustrate the system’s capability to screen AD patients, and to serve as a morphological image guide for a clinical a/LCI system. Finally, a discussion of how the low-cost OCT system can be integrated to a multimodal imaging system for AD human retinal biomarker extraction will be provided.

The goal of this dissertation is to develop multimodal light scattering techniques using optical coherence tomography (OCT) to improve clinical diagnosis. OCT is a non-invasive optical imaging technique that utilizes low coherence interferometry to detect reflected and scattered light from a sample to produce depth resolved images. OCT is an emerging technology for a wide range of biomedical applications, with its largest impact in the field of ophthalmology to assess retinal morphology and abnormalities. Due to its excellent axial resolution, OCT has been often jointly used with a variety of other optical techniques in multimodal platforms for enhanced characterization of biological tissues.

The first section discusses the development of a multimodal optical system that combines OCT and angle-resolved low coherence interferometry (a/LCI). Similar to OCT, a/LCI utilizes low coherence interferometry for depth gating, but instead of imaging, it measures the angular dependence of scattered light as a function of depth to retrieve depth-resolved nuclear morphology measurements. However, since a/LCI is not an imaging modality, it can produce ambiguous results when the measurements are not properly oriented to the tissue structure. Utilization of OCT can resolve this problem, by providing real time image guidance for a/LCI and ensuring proper sample orientation. Moreover, OCT enables the co-registration of light scattering measurements to specific histological layers, which significantly improves the effectiveness of nuclear morphology determination. Thus, a multimodal system that combines OCT and a/LCI can provide a unique analysis of tissue structure that cannot be assessed using a standalone modality. Using the combined modality, this research develops quantitative biomarkers from ex vivo tissue samples to discriminate disease states.

The second portion of the work describes the development of a low-cost, portable OCT system that could significantly increase ease of access, particularly targeted for low resource settings. Although OCT has been adopted as the gold standard for retinal imaging in ophthalmology, the high cost of the clinical system has restricted access to mostly large eye centers and laboratories. Cost reduction and portability have been of interest for numerous optical technologies. Providing a comprehensive low-cost OCT system will open the doors for a wide variety of potential opportunities of OCT guided diagnosis. This section discusses the design and the implementation of a low-cost system, as well as a demonstration of the imaging capabilities that could meet the required performance for retinal imaging in clinical and laboratory studies.

The last section discusses the performance of imaging fiber bundles for light delivery and collection in endoscopic a/LCI. The use of imaging bundle for coherent imaging application has been limited since coherent imaging relies on single mode illumination, which requires expensive scanning optics, to reject higher mode interference. This section investigates the application of more affordable fiber bundles to replace such costly systems. A number of commercial and custom fiber bundles that could be used for light delivery and collection for the endoscopic probe have been carefully characterized. This characterization will not only help with developing a novel probe design for a/LCI, but also provide valuable insights into the potential application of coherent bundles for general coherent imaging including OCT.

Esophageal cancer is a very aggressive form of cancer, and in the past decade, the incidence rate of esophageal cancer is rising faster than any other malignancy in the U.S. Luckily, most precancers are preventable given timely surveillance and proper treatments. Despite the recent success of current screening methodologies, these techniques are still costly and limited. As an alternative, angle-resolved low-coherence interferometry (a/LCI) is an optical technique which enables depth-resolved measurements of nuclear morphology, a biomarker for precancer. In this dissertation, several advances in a/LCI technology were presented. First, computational analysis of a previous clinical a/LCI dataset was used to provide design guidance for future a/LCI designs. The impact of reductions in angular range and angular sampling frequency on the diagnostic performance of a/LCI was analyzed and discussed. Next, in order to improve the clinical utility of a/LCI, a novel processing algorithm based on deep learning was presented for identifying dysplasia from depth-resolved angular scattering scans collected by a/LCI with high accuracy and speed. Future development of this algorithm may open to possibilities for real-time clinical analysis of a/LCI data, and improve the clinical utility of the instrument during in vivo clinical trials for real-time screening of the tissue. In addition, instrumentational advances in a/LCI were also demonstrated. Development of the opto-mechanical instrumentation using a single multimode fiber was presented to overcome the limiting factor of using fiber bundles for a/LCI imaging, as these fiber bundles are fragile, expensive, and exhibits low optical throughput. The technique was validated using microsphere phantoms, and showed excellent agreement with the actual size. This technique was also insensible to the displacement of the fiber, and showed great potential for future endoscopic applications for medical diagnostics. Finally, a combined a/LCI and OCT imaging platform was developed and adapted for esophageal imaging, and a clinical study was performed to determine the effectiveness of using this combined instrumentation for screening dysplasia in patients with Barrett’s esophagus, a biomarker for dysplasia. Optical biopsies were taken from 50 distinct tissue biopsy sites and compared to histopathological analysis of co-registered tissue biopsies. Analysis of the a/LCI scans demonstrated perfect sensitivity (100%) for detection of esophageal dysplasia, and the increase in specificity (from 84% to 93%) compared with a previous clinical study demonstrated the ability of OCT in targeting potential diseased biopsies, suggesting that optical biopsy characterization using a/LCI nuclear morphology measurements with real-time OCT imaging guidance would aid the clinician in identifying dysplastic tissue sites in vivo, leading to improved screening protocols, and ultimately, better patient outcomes.

Cervical cancer is one of the most common gynecologic malignancies with significant morbidity and mortality globally. However, most pre-cancers are easily treatable such that early detection of cervical abnormalities is critical in improving patient prognosis. Despite the success of current cervical cancer screening methodologies, these techniques are still limited in accuracy, leading to undetected cervical lesions or unnecessary biopsies.

This dissertation will focus on the development of two optical modalities for early detection of cervical dysplasia: angle-resolved low coherence interferometry (a/LCI) and multiplexed low coherence interferometry (mLCI). Originally, a/LCI was developed as a clinical technique for detecting esophageal dysplasia by detecting nuclear enlargement in the basal layer of the epithelium. To improve the clinical utility of a/LCI, a novel processing algorithm was developed using a continuous wavelet transform (CWT) based analysis of the a/LCI data which demonstrated significant improvement in processing speed compared to previous analysis techniques. Future development of this algorithm may open the possibility for real-time clinical analysis of a/LCI data, improving the clinical utility of the instrument.

In addition, the a/LCI instrument was adapted for cervical imaging, and a clinical feasibility study was performed to determine the effectiveness of using a/LCI nuclear morphology measurements for detecting cervical dysplasia. a/LCI optical biopsies were taken from 63 distinct tissue biopsy sites and compared to histopathological analysis of co-registered tissue biopsies. Analysis of the a/LCI nuclear morphology data found a significant increase in the nuclear diameter in the basal layer of dysplastic tissue sites and demonstrated high sensitivity and specificity (both >0.80) for detection of cervical dysplasia and high-grade squamous intraepithelial lesions.

Secondly, mLCI was adapted for collecting A-scans over the cervical epithelium. An mLCI cervical probe was designed and a clinical study was conducted to image 50 patients with the new device. Linear discriminant analysis was performed on the mLCI data to automatically classify the cervical A-scans as either endocervical or ectocervical tissue towards the goal of automatic delineation cervical transformation zone. This device can be combined with a/LCI to direct optical biopsy scans to areas on the cervix which are most likely to harbor tissue dysplasia.

Finally, the two technologies were incorporated into a single multimodal imaging system. First, a benchtop scanning a/LCI system was developed by incorporating an image rotator and 2D scanner into the system to enable radial scanning on the sample. This system was integrated into a handheld probe for cervical imaging. Volumetric imaging using sparse depth scans and the scanning a/LCI technology was validated with a polystyrene microsphere phantom, and a pilot study was conducted to demonstrate the feasibility of using this instrument for comprehensive screening of cervical tissue for precancerous cells.

Cellular mechanobiology has been of great to scientists, as changes in stiffness has been linked to various biological phenomena. Specifically, variation of cellular viscoelasticity occurs in cancerous cells, likely due to abnormal behavior of the cytoskeleton. Current standard methods for probing cellular stiffness are slow, laborious, and utilize complex and sometimes indirect detection mechanisms that stymy the progress of research in the field. As such, we developed a tool using quantitative phase imaging (QPI) to directly measure mechanical displacement in living cells in response to static loading. In this dissertation, instrumentation and methodologies are developed to probe mechanical differences in living cells, in a typical culture environment, without complex external devices or exogenous contrast agents.

An off-axis quantitative phase microscope was constructed and used to image cells subjected to shear flow within a flow cell. Cellular center-of-mass deviations were fitted to simple one-dimensional, viscoelastic, mechanical models to model cellular deformation and extract a shear stiffness parameter. Cells were tested at multiple flow rates to confirm linear viscoelasticity. Shear stiffness parameters between normal and pharmacologically disrupted cell lines were compared to demonstrate the assay’s ability to segregate different mechanical populations. The assay was then applied to a carcinogenesis model, mimicking transformation of normal bronchial epithelium into carcinogenic phenotypes via intracellular (arsenic) and extracellular (soft agar) changes. Changes in mean stiffness and the relative standard deviation of stiffness were found amongst all groups, indicating mechanical changes occur during oncogenesis.

In addition to the shear flow assay, a method for measuring cellular microstructure via the framework of disorder strength was created using QPI images. Disorder strength was measured by assessing phase variance over small windows across QPI images. Sensitivity to disordered media was verified by phase variance measurements of polystyrene bead solutions. Disorder strength was measured for multiple cell lines, and was found to closely resemble previous measurements. Additionally, disorder strength was found to be strongly correlated to shear stiffness, indicating that inferences of mechanical integrity could be acquired from QPI images alone. To verify these findings and to assert QPI’s utility for mechanical measurements, a QPI method for measuring shear modulus was devised and directly compared to atomic force microscopy (AFM) measurements of Young’s modulus. Shear modulus and disorder strength, via QPI, were measured across six groups of cells and compared to AFM-based Young’s modulus measurements. Comparison of shear modulus and Young’s modulus were found to strongly agree with theory, confirming the integrity of QPI based mechanical measurements. Shear modulus and Young’s modulus were found to be negatively correlated to disorder strength, reinforcing previous findings of a relationship between microstructure and cellular mechanical status. Finally, a combined QPI and fluorescence set up was constructed to add molecular measurements to the morphological imaging capability of QPI. Specifically, FRET based apoptosis sensors were used to monitor morphological parameters of HeLa cells during apoptosis. Cells were found to have optical volume and disorder strength modulation in response to caspase-3 mediated apoptosis. Additionally, cells transfected with FRET-based vinculin tension sensors (VinTS) were analyzed while subjecting cells to static loading. QPI was used to ensure mechanical loading occurred via measured center-of-mass displacements, while VinTS reported the relative tensional changes via changes in FRET index before and after flow subjugation. Indeed, tension was found to increase due to shear flow in response to mechanical displacement, after contributions from focal shifts and photobleaching were accounted for.

These results demonstrated the applicability of QPI as a tool for analyzing cellular mechanical parameters. Additionally, QPI elucidated changes in mechanical status during oncogenic transformation and affirmed the connection between microstructure and mechanical integrity. These measures were confirmed to be valid when compared to gold standard methodologies for cellular mechanical interrogation. Finally, the system was outfitted with molecular imaging capabilities, enabling co-registered measurements of cellular morphological changes with molecular specific changes during apoptosis and static shear loading. In summary, QPI can be an indispensable tool for evaluating mechanical characteristics of cells.

Without exogenous contrast agents, imaging semitransparent samples such as biological cells can be difficult with light microscopy that measures modulation in the intensity of transmitted light. Quantitative phase microscopy (QPM) that measures the phase delays imparted by the objects is often used to examine the spatial and temporal dynamics of individual cells without chemical modification.

In order to demonstrate its effectiveness as a diagnostic tool, quantitative phase microscope is used to image red blood cells (RBCs) specifically those infected with malaria parasites, Plasmodium falciparum. RBCs are a favorite target for QPM studies, in particular because they have little internal structure and thus can be represented by a homogenous refractive index. The ability to profile RBCs is an important capability of QPM since these cells are affected by different disease stages, and thus they are essential in human health diagnostics. Classifiers built using QPM are able to distinguish and classify uninfected red blood cells from those infected with P. falciparum using morphological features of the cells with high accuracy. The study shows that QPM can be a very useful diagnostic modality and that it can be more clinically relevant by developing into a high throughput holographic imaging system.

In addition to the morphological measurement of erythrocytes, biophysical properties of RBCs under mechanical stress are measured by incorporating microfluidic chips into the QPM platform. Highly precise microfluidic chips are manufactured with specific dimensions that will stress the RBCs at designated positions as they flow through them. Using a high refractive index medium, QPM is used to measure the optical volume changes associated with efflux of water through the membrane of the cells under mechanical stress. The OV changes in response to mechanical force are compared for different storage periods to evaluate the changes in response to deformation throughout the ageing cells.

Finally, a novel approach for high throughput screening is developed based on holographic imaging of the cells flowing through microfluidic chips. To enable high throughput imaging, we have implemented a new quantitative phase imaging modality, holographic cytometry. Holographic cytometry maintains the high sensitivity of QPM while imaging a large number of cells flowing through the microfluidic devices. As demonstrated in previous studies, morphological parameters are extracted from these images to assess their changes over the storage time and classify them according to the time in the blood units.

Microscopic objects such as biological cells produce only minor modulation in the intensity of transmitted light, leading many researchers to add exogenous contrast agents for image enhancement. However, cells and other semitransparent objects that have not been chemically modified impart phase delays to the transmitted electromagnetic fields, which can be measured using interferometric microscopy methods. In this dissertation, instrumentation and methods are developed to investigate the spatiotemporal dynamics and spectral signatures of individual cells and semitransparent polymer film samples.

An off-axis quantitative phase microscope is applied to (1) quantitatively image the two-dimensional refractive index distributions of microbicide films undergoing hydration and compare effects of thickness and composition on dissolution dynamics, and (2) investigate the morphological and volumetric changes of individual RBCs undergoing mechanical flow stresses in in vitro models of capillaries. The quantitative phase microscope is further modified to capture high-resolution hyperspectral holographic phase and amplitude images. This novel hyperspectral imaging system is applied to compare the sensitivity of phase-based and amplitude-based spectral quantification of optically-absorbing molecules, and then used to measure spectroscopic changes in RBCs that take place during infection by P. falciparum parasites.

Measurements of an object's optical volume, which is defined as a novel metric for characterizing objects whose refractive index and thickness profiles are not known a priori. The composition and thickness of microbicide films are both found to impact spatiotemporal dissolution kinetics. A comparison of fluorophore concentration determination by amplitude and phase spectra indicates that both methods of quantification have comparable sensitivity, and that the two may be combined to improve the precision of quantity determination. Both optical volume and hemoglobin mass measurements are seen to decrease in cells infected by P. falciparum, although the two metrics are only loosely correlated. Finally, RBCs flowing through in vitro capillary models exhibit large changes in optical volume when deforming in response to mechanical stresses, which is attributed to a combination of cytosolic volume changes as well as conformational changes in the intracellular protein configuration.

These results demonstrate the applicability of QPM as a tool for evaluating (1) microbicide film performance, (2) spectroscopic changes in infected individual RBCs, and (3) novel biophysical changes observed in RBCs under mechanical stresses.

The redistribution of incident light into scattered fields ultimately limits the ability to image into biological media. However, these scattered fields also contain information about the structure and distribution of protein complexes, organelles, cells and whole tissues that can be used to assess the health of tissue or to enhance imaging contrast by excluding confounding signals. The interpretation of scattered fields depends on a detailed understanding of the scattering process as well as sophisticated measurement systems. In this work, the development of new instruments based on low coherence interferometry (LCI) is presented in order to perform precise, depth-resolved measurements of scattered fields. Combined with LCI, the application of new light scattering models based on both analytic and numerical methods is presented in order to interpret scattered field measurements in terms of scatterer geometry and tissue health.

The first portion of this work discusses the application of a new light scattering model to the measurement recorded with an existing technique, Angle Resolved Low Coherence Interferometry (a/LCI). In the a/LCI technique, biological samples are interrogated with collimated light and the energy per scattering angle at each depth in the volume is recorded interferometrically. A light scattering model is then used to invert the scattering measurements and measure the geometry of cell nuclei. A new light scattering model is presented that can recover information about the size, refractive index, and for the first time, shape of cell nuclei. This model is validated and then applied to the study of cell biology in a series of experiments measuring cell swelling, cell deformation, and finally detecting the onset of apoptosis.

The second portion of this work introduces an improved version of a/LCI based on two dimension angle resolved measurement (2D a/LCI) and Fourier domain low coherence interferometry (FD-LCI). Several systems are presenting for high speed and polarization-resolved measurements of scattered fields. An improved light scattering model based on fully polarization and solid angle resolved measurements is presented, and then efficiently implemented using distributed computing techniques. The combined system is validated with phantoms and is shown to be able to uniquely determine the size and shape of scattering particles using a single measurement.

The third portion of this work develops the use of angle-resolved interferometry for imaging through highly scattering media by exploiting the tendency of scatterers to forward scatter light. A new interferometers is developed that can image through very large numbers of scattering events with acceptable resolution. A computational model capable of reproducing experimental measurements is developed and used to understand the performance of the technique.

The final portion of the work develops a method for processing 2D angle resolved measurements using optical autocorrelation. In this method, measurements over a range of angles are fused into a single depth scan that incorporates the component of scattered light only from certain spatial scales. The utility of the technique is demonstrated using a gene knockout model of retinal degeneration in mice. Optical autocorrelation is shown to be a potentially useful biomarker of tissue disease.

The behavior of light after interacting with a biological medium reveals a wealth of information that may be used to distinguish between normal and disease states. This may be achieved by simply imaging the morphology of tissues or individual cells, and/or by more sophisticated methods that quantify specific surrogate biomarkers of disease. To this end, the work presented in this dissertation demonstrates novel tools derived from low coherence interferometry (LCI) that quantitatively measure wavelength-dependent scattering and absorption properties of biological samples, with high spectral resolution and micrometer spatial resolution, to provide insight into disease states.

The presented work first describes a dual window (DW) method, which decomposes a signal sampled in a single domain (in this case the frequency domain) to a distribution that simultaneously contains information from both the original domain and the conjugate domain (here, the temporal or spatial domain). As the name suggests, the DW method utilizes two independently adjustable windows, each with different spatial and spectral properties to overcome limitations found in other processing methods that seek to obtain the same information. A theoretical treatment is provided, and the method is validated through simulations and experiments. With this tool, the spatially dependent spectral behavior of light after interacting with a biological medium may be analyzed to extract parameters of interest, such as the scattering and absorption properties.

The DW method is employed to investigate scattering properties of samples using Fourier domain LCI (fLCI). In this method, induced temporal coherence effects provide insight into structural changes in dominant scatterers, such as cell nuclei within tissue, which can reveal the early stages of cancerous development. fLCI is demonstrated in complex, three-dimensional samples using a scattering phantom and an ex-vivo animal model. The results from the latter study show that fLCI is able to detect changes in the morphology of tissues undergoing precancerous development.

The DW method is also employed to enable a novel form of optical coherence tomography (OCT), an imaging modality that uses coherence gating to obtain micrometer-scale, cross-sectional information of tissues. The novel method, named molecular imaging true color spectroscopic OCT (METRiCS OCT), analyses the depth dependent absorption of light to ascertain quantitative information of chromophore concentration, such as hemoglobin. The molecular information is also processed to yield a true color representation of the sample, a unique capability of this approach. A number of experiments, including hemoglobin absorbing phantoms and in-vivo imaging of a chick embryo model and dorsal skinfold window chamber model, demonstrate the power of the method.

The final method presented in this dissertation, consists of a spectroscopic approach that interrogates the dispersive biochemical properties of samples to independently probe the scattering and absorption coefficients. To demonstrate this method, named non-linear phase dispersion spectroscopy (NLDS), a careful analysis of LCI signals is presented. The method is verified using measurements from samples that scatter and absorb light. Lastly, NLDS is combined with phase microscopy to achieve molecular imaging with sub-micron spatial resolution. Imaging of red blood cells (RBCs) shows that the method enables highly sensitive measurements that can quantify hemoglobin content from single RBCs.

Noble metal nanoparticles exhibit unique optical properties that are beneficial to a variety of applications, including molecular imaging. The large scattering cross sections of nanoparticles provide high contrast necessary for biomarkers. Unlike alternative contrast agents, nanoparticles provide refractive index sensitivity revealing information regarding the local cellular environment. Altering the shape and composition of the nanoparticle shifts the peak resonant wavelength of scattered light, allowing for implementation of multiple spectrally distinct tags. In this project, nanoparticles that scatter in different spectral windows are functionalized with various antibodies recognizing extra-cellular receptors integral to cancer progression. A hyperspectral imaging system is developed, allowing for visualization and spectral characterization of cells labeled with these conjugates. Various molecular imaging and microspectroscopy applications of plasmonic nanoparticles are then investigated. First, anti-EGFR gold nanospheres are shown to quantitatively measure receptor expression with similar performance to fluorescence assays. Second, anti-EGFR gold nanorods and novel anti-IGF-1R silver nanospheres are implemented to indicate local cellular refractive indices. Third, because biosensing capabilities of nanoparticle tags may be limited by plasmonic coupling, polarization mapping is investigated as a method to discern these effects. Fourth, plasmonic coupling is tested to monitor HER-2 dimerization. Experiments reveal the interparticle conformation of proximal HER-2 bound labels, required for plasmonic coupling-enhanced dielectric sensing. Fifth, all three functionalized plasmonic tags are implemented simultaneously to indicate clinically relevant cell immunophenotype information and changes in the cellular dielectric environment. Finally, flow cytometry experiments are conducted utilizing the anti-EGFR nanorod tag to demonstrate profiling of receptor expression distribution and potential increased multiplexing capability.

Even though women make up over half the population in the United States, medical advancements in areas of women's health have typically lagged behind the rest of the medical field. Specifically, two major threats to women's reproductive health include human immunodeficiency virus (HIV), and cervical cancer with accompanying human papillomavirus (HPV) infection. This dissertation presents the development and application of two novel optical imaging technologies aimed at improving these aspects of women's reproductive health.

The presented work details the instrumentation development of a probe-based, dual-modality optical imaging instrument, which uses simultaneous imaging of fluorimetry and multiplexed low coherence interferometry (mLCI) to measure in vivo microbicide gel thickness distributions. The study explores the optical performance of the device and provides proof of concept measurements on a calibration socket, tissue phantom, and in vivo human data. Once the instrument is fully characterized, it is applied in a clinical trial in which in vivo human vaginal gel thickness distributions. The gel distribution data obtained by the modalities are compared in order to assess the ability of mLCI making accurate in vivo measurements. Differences between the fluorimetry and mLCI modalities are then exploited in order to show a methodology for calculating the extent of microbicide gel dilution with the dual-modality instrument data.

Limitations in cervical cancer screening are then addressed as angle-resolved low coherence interferometry (a/LCI) is used in an ex vivo pilot study to assess the feasibility of a/LCI in identifying dysplasia in cervical tissues. The study found that the average nuclear diameter found by a/LCI in the basal layer of ectocervical epithelium showed a statistically significant increase in size in dysplastic tissue. These results indicate that a/LCI is capable of identifying cervical dysplasia in ectocervical epithelium. The results of the work presented in this dissertation show that dual-modality optical imaging with fluorimetry and mLCI, and the a/LCI technique show promise in advancing technologies that are used in the field of women's reproductive health.

Cancer, despite its status as the second leading cause of death worldwide, is often preventable given proper surveillance and timely intervention. In the esophagus, metaplastic changes linked to reflux disease lead to alterations in cellular DNA, abnormal growth, and eventually, metastatic cancer. Fortunately, this process takes place over a period of several years, during which treatment and eradication of the precancerous lesions is possible if discovered at a sufficiently early time.

Current protocols for surveillance of the esophagus are costly and limited. As an alternative, angle-resolved low-coherence interferometry (a/LCI) is an optical technique which enables depth-resolved measurements of nuclear morphology, a biomarker of precancer. a/LCI allows for real-time identification of precancerous lesions, which may be treated using radiofrequency ablation or related techniques.

In this dissertation, several advances in a/LCI technology are presented. Measurements of the nuclear refractive index, an important parameter for a/LCI inverse light scattering analysis, are offered to settle an important debate in the literature regarding the relative density of the cell nucleus. Instrumentational advances in a/LCI are demonstrated to address the need for implementing scanning capability, which is important for screening of larger tissues such as the cervix. The properties of commercially available fiber optic imaging bundles are investigated for their capacity to support coherence-based imaging, and when these are found to be lacking, an a/LCI device based on single-mode optical fibers is designed and validated using an array of pathlength-matched individual fibers, which exhibits significant advantages over previous image bundles. Computational analysis of a previous clinical a/LCI dataset is used to provide design guidance for this methodology. Finally, this new a/LCI device is combined with a rotational endoscopic optical coherence tomography (OCT) probe to create a multimodal imaging system for comprehensive evaluation of the esophageal epithelium. The complete system includes a paddle form factor which allows it to be affixed to the exterior of a commercial endoscope for clinical compatibility, similar to related endoscopic devices. These advances demonstrate the continued applicability of a/LCI for evaluating epithelial health, and present new and attractive options for surveillance and early intervention against cancer.

Cancer encompasses many diseases affecting all parts of the body, is characterized by rapid, abnormal cell growth, and is one of the most common causes of death worldwide. Certain cancers, including cervical and colorectal cancer, exhibit very slow growth and histological evidence on or close to the tissue surface of precancerous development. With appropriate methods, the cancer can be detected before it has the chance to actually become malignant, and safely removed, preventing additional risk from more advanced disease. Developments in optical diagnostic techniques have shown promise in early detection of colorectal and cervical cancer, potentially able to improve upon existing preventative screening techniques in terms of accuracy and detection time.

This dissertation is focused on optical methods based on low-coherence interferometry towards early detection of cervical and colorectal cancer. Angle-resolved low coherence interferometry (a/LCI) was used to build a system with scanning capabilities that was used for in vivo assessment of cervical dysplasia in a clinical study involving 20 patients. The results and prospective comparison to a previous a/LCI study had high accuracy (85%) as well as consistent numerical bright lines for normal vs. dysplastic tissue, showing that it is a promising technique towards diagnosing cervical dysplasia.

Spectroscopic optical coherence tomography (SOCT) is another technique based on low-coherence interferometry that was investigated towards early detection of colorectal cancer (CRC). The large amount of genetic contributions to adenomatous polyps result in a large diversity of polyp morphologies, each with their own unique malignant potential. The depth-resolution capabilities as well as rich spectral information available from SOCT allows for computation of optical biomarkers such as scattering attenuation coefficient (????) or scattering power (SP) to be determined by imaging tissue, leading to quantitative parameters that can define different morphologies of adenoma. Mouse models representing different severities of CRC were imaged with SOCT and separated with high accuracy. Deep learning methods were applied to both mouse and subsequent human data with high accuracy, with benign human tissues separable from tubular and tubulovillous adenomas with 93.1% accuracy.