The purpose of this project is to develop a retinal imaging system suitable for routine examination or screening of mouse models that acquires fundus and Optical Coherence Tomography (OCT) images. The imaging system is composed of a digital camera with an objective for biomicroscopic examination of the fundus, an OCT interferometer, an OCT beam delivery system designed for the mouse eye, and a mouse positioning stage. The image acquisition is controlled with software that displays the fundus and OCT images in real-time, and allows the user to control the position of the OCT beam spot on the fundus image display. The system was used to image healthy mice and a mouse model of glaucoma. Fundus images and OCT scans were successfully acquired in both eyes of all mice with eyes that had clear optics. The study demonstrates the feasibility of acquiring simultaneous fundus and OCT images of the mouse retina, by a single operator, in a manner suitable for rapid evaluation of mouse models of retinal disease.

Measuring the Mueller matrix with optical coherence tomography (OCT) makes it possible to acquire the complete polarization properties of scattering media with three-dimensional spatial resolution. We first proved that the measured degree-of-polarization (DOP) of the backscattered light by OCT remains unity-a conclusion that validated the use of Jones calculus in OCT. A multi-channel Mueller-matrix OCT system was then built to measure the Jones-matrix, which can be transformed into a Mueller matrix, images of scattering biological tissues accurately with single depth scan. We showed that when diattenuation is negligible, the round-trip Jones matrix represents a linear retarder, which is the foundation of conventional PS-OCT, and can be calculated with a single incident polarization state although the one-way Jones matrix generally represents an elliptical retarder; otherwise, two incident polarization states are needed. We discovered the transpose symmetry in the roundtrip Jones matrix, which is critical for eliminating the arbitrary phase difference between the two measured Jones vectors corresponding to the two incident polarization states to yield the correct Jones matrix. We investigated the various contrast mechanisms provided by Mueller-matrix OCT. Our OCT system for the first time offers simultaneously comprehensive polarization contrast mechanisms including the amplitude of birefringence, the orientation of birefringence, and the diattenuation in addition to the polarization-independent intensity contrast, all of which can be extracted from the measured Jones or the equivalent Mueller matrix. The experimental results obtained from rat skin samples, show that Mueller OCT provides complementary structural and functional information on biological samples and reveal that polarization contrast is more sensitive to thermal degeneration of biological tissues than amplitude-based contrast. Finally, an optical-fiber-based multi-channel Mueller-matrix OCT was built and a new rigorous algorithm was developed to retrieve the calibrated polarization properties of a sample. For the first time to our knowledge, fiber-based polarization-sensitive OCT was dynamically calibrated to eliminate the polarization distortion caused by the single-mode optical fiber in the sample arm, thereby overcoming a key technical impediment to the application of optical fibers in this technology.

This project initiated with the aim for improving the ultrahigh resolution optical coherence tomography (UHR-OCT) system performance by considering the limitations to the axial OCT resolution for in vivo imaging of human and animal retina. To this end, a computational model was developed to simulate the effect of wavelength-dependant water absorption on the detected spectral shape of the broad-bandwidth light source used in UHR-OCT at 1060nm wavelength region, which effectively determines the axial OCT resolution in the retina. For experimental verification of the computational model, a custom built light source with a re-shaped spectrum (Superlum Inc.) was interfaced to the state-of-the-art UHR-OCT system. About 30% improvement of the axial OCT resolution in the rat retina and ~12% improvement of the axial OCT resolution in the human retina was achieved compared to the case of the almost Gaussian shaped spectrum of the standard, commercially available SLD. Although water absorption in the 1060nm spectral region strongly affects the sample beam, selecting a suitable light source with specific spectral shape can compensate for the undesired water absorption effect and thus result in significantly improved axial resolution in in vivo OCT retinal images. To demonstrate the advantages of the state-of-the-art OCT technology for non invasive retinal imaging, an established animal model of outer retina degeneration (sodium iodate (NaIO3)-induced retina degeneration) was employed for longitudinal monitoring of the degeneration and investigation of possible early and dynamic signs of damage undetected by other imaging modalities. The long-term (up to 3 months) and short-term (up to 12 hours) effect of sodium iodate toxicity on the layered structure of retina was monitored longitudinally and in vivo for the first time using OCT. An initial acute swelling of the retina, followed by progressive disruption and degeneration of outer retina was observed as a result of sodium iodate-induced damage. Changes in the thickness and optical reflectivity of individual retinal layers were extracted from the OCT images to quantify the changes occurring at different stages of the disease model. Results from this project present the theoretical and practical limits to the highest axial OCT resolution achievable for retina imaging in the 1060nm spectral range both in small animals and humans, and provided a framework for future development of novel light sources. Furthermore, UHR-OCT imaging was shown to be an effective and valuable modality for in vivo, non invasive investigation of retina degenerative disease.

Abstract Both in the closed and open eye state the superior limbus is covered by the upper lid. This region is of physiological interest and clinical importance because in chronic hypoxia, neovascularization of the cornea commonly occurs here. The limbal region in general is additionally of importance as the stem cells which are the source of the new corneal cells are located in the epithelium of the limbus and these are vital for normal functioning and are affected under certain adverse conditions. Purpose: In this experiment I examined corneal morphology in the limbal area and in particular under the upper lid in order to primarily examine the variation in the corneal limbal epithelial and total thickness as well as epithelial and endothelial cell density. Methods: I measured 30 eyes OD/OS (chosen randomly) of thirty healthy subjects aged from 18 to 55 years in the first study and twelve participants in the second study, with refractive error ≤ ±4 D and astigmatism ≤ 2 D. The thickness and cell density of five positions: superior, inferior, temporal, nasal limbal and central cornea was determined with optical coherence tomography (OCT) and confocal microscopy. At least three scans of each position were taken in both studies with OCT. At least 40 of 100 adjacent sagittal scans of each image were measured using OCT software program. In the confocal study, image J software was used to determine cell densities. Results: The epithelial and corneal limbal thickness were significantly thicker than the epithelial and central corneal thickness (p<0.05). The limbal, inferior cornea is thinner than the three other positions and the temporal region of the cornea is the thickest both in epithelial and total cornea. Epithelial cell density was significantly lower in the superior cornea than the four other positions. There was no significant difference in the endothelial cell density. Conclusions: Using OCT with high resolution and cross-sectional imaging capability and confocal microscope with high magnification, I found that the limbal cornea is significantly thicker than the central cornea both in total and in epithelial thickness. In the limbus, one might expect the superior cornea (under the lid) to be thickest (because of the expected hypoxia) whereas I found the temporal cornea was thickest. The epithelial cell density was lower in the superior cornea but there was no significant difference in cell densities in the endothelium. Further morphological investigation is of interest.

Abstract Both in the closed and open eye state the superior limbus is covered by the upper lid. This region is of physiological interest and clinical importance because in chronic hypoxia, neovascularization of the cornea commonly occurs here. The limbal region in general is additionally of importance as the stem cells which are the source of the new corneal cells are located in the epithelium of the limbus and these are vital for normal functioning and are affected under certain adverse conditions. Purpose: In this experiment I examined corneal morphology in the limbal area and in particular under the upper lid in order to primarily examine the variation in the corneal limbal epithelial and total thickness as well as epithelial and endothelial cell density. Methods: I measured 30 eyes OD/OS (chosen randomly) of thirty healthy subjects aged from 18 to 55 years in the first study and twelve participants in the second study, with refractive error ≤ ±4 D and astigmatism ≤ 2 D. The thickness and cell density of five positions: superior, inferior, temporal, nasal limbal and central cornea was determined with optical coherence tomography (OCT) and confocal microscopy. At least three scans of each position were taken in both studies with OCT. At least 40 of 100 adjacent sagittal scans of each image were measured using OCT software program. In the confocal study, image J software was used to determine cell densities. Results: The epithelial and corneal limbal thickness were significantly thicker than the epithelial and central corneal thickness (p<0.05). The limbal, inferior cornea is thinner than the three other positions and the temporal region of the cornea is the thickest both in epithelial and total cornea. Epithelial cell density was significantly lower in the superior cornea than the four other positions. There was no significant difference in the endothelial cell density. Conclusions: Using OCT with high resolution and cross-sectional imaging capability and confocal microscope with high magnification, I found that the limbal cornea is significantly thicker than the central cornea both in total and in epithelial thickness. In the limbus, one might expect the superior cornea (under the lid) to be thickest (because of the expected hypoxia) whereas I found the temporal cornea was thickest. The epithelial cell density was lower in the superior cornea but there was no significant difference in cell densities in the endothelium. Further morphological investigation is of interest.

Recently, with the escalating demands for optical communications, the need for bandwidth in optical communication network has increased. The technology breakthrough indry fiber fabrication opens the possibility for fiber bandwidth form 1.3 to 1.6 £gm. Cr4+:YAG double-clad crystal fiber (DCF) grown by the co-drawing laser-heated pedestal growth method has a strong spontaneous emission spectum form 1.3 to 1.6 £gm. Such fiber is therefore, eminently suitable for optical coherence tomography (OCT), broadband optical amplifier, amplifier spontaneous emission (ASE) light source, and tunable solid-state laser applications. In this thesis, multilayer dielectric thin films were directly deposited by E-gun coating onto the end faces of the Cr4+:YAG DCF. To improve the thin-film quality, to increase transmittance of laser output, and to design for the high power laser. For broadband optical amplifier in dual-pump and double-pass scheme, a 3.0-dB gross gain, a 3.0-dB insertion loss, and a 0-dB net gain at 1.4-£gm signal wavelength have been successfully developed with HR coating onto one of the Cr4+:YAG DCF end faces. In addition, we have successfully developed the Cr4+:YAG DCF laser by direct HR coatings onto fiber end faces and increase transmittance of laser output. A record-low threshold of 31.2 mW with a slope efficiency of 7.5% was achieved at room temperature.

Molecular imaging using fluorescence spectroscopy-based techniques is generally inefficient due to the low quantum yield of most naturally occurring biomolecules. Current fluorescence imaging techniques tag these biomolecules chemically or through genetic manipulation, increasing the complexity of the system. A technique capable of imaging these biomolecules without modifying the chromophore and/or its environment could provide vital biometric parameters and unique insights into various biological processes at a molecular level. Pump probe spectroscopy has been used extensively to study the molecular properties of poorly fluorescing biomolecules, because it utilizes the known absorption spectrum of these chromophores. Optical Coherence Tomography (OCT) is an optical imaging modality that harnesses the power of low coherence interferometry to measure the 3-D spatially resolved reflectivity of a tissue sample. We plan to develop a new molecular imaging modality that combines these techniques to provide 3-D, highresolution molecular images of various important biomolecules. The system uses a Fourier domain OCT setup with a modified sample arm that combines the "pump" and "probe" beams. The pump beam drives the molecules from the ground state to excited state and the probe interrogates the population change due to the pump and is detected interferometrically. The pump and the probe beam wavelengths are optimized to maximize absorption at the pump wavelength and maximize the penetration depth at the probe wavelength. The pump-probe delay can be varied to measure the rate at which the excited state repopulates the ground state, i.e., the ground state recovery time. The ground state recovery time varies for different chromophores and can potentially be used to identify different biomolecules. The system was designed and optimized to increase the SNR of the PPOCT signals. It was tested by imaging hemoglobin and melanin samples and yielded encouraging results. Potential applications of imaging hemoglobin using this technique include the mapping of tissue microvasculature and measuring blood-oxygen saturation levels. These applications could be used to identify hypoxic areas in tissue. Melanin imaging can provide means of demarcation of melanoma in various organs such as skin, eye and intestines.

Glaucoma is the second leading cause of blindness worldwide after cataract. Retinal nerve fiber layer thickness (RNFLT), phase retardation, and birefringence measured by Polarization Sensitive Optical Coherence Tomography (PS-OCT) have been used for glaucoma diagnosis. We first investigated two different image registration algorithms, a mutual information (MI) based algorithm and a log-polar transform cross-correlation (LPCC) based algorithm, on both human and non-human primate models. We evaluated the effects of image registration on longitudinal analysis of RNFLT in non-human primates using PS-OCT. Then, we investigated thickness, phase retardation, birefringence, and reflectance of the retinal nerve fiber layer as measured by PS-OCT in normal and glaucomatous non-human primates in a longitudinal study. We defined a new Reflectance Index (RI) and demonstrated that it might be an earlier indicator of glaucoma onset than RNFLT, phase retardation, or birefringence. Finally, we validated this finding on cross-sectional clinical study on human eyes measured by PS-OCT and RTVue OCT. For the data measured by PS-OCT, we showed that for distinguishing between glaucomatous and healthy eyes, as well as for distinguishing between glaucoma suspect and healthy eyes, our new normalized RNFL reflectance index (NRRI) performs significantly better than phase retardation and birefringence. The performances of NRRI and RNFL thickness in both conditions were statistically indistinguishable in this study, which is likely due to the limited sample size. For the data measured by RTVue OCT, the performances of NRRI and RNFL thickness were statistically indistinguishable for distinguishing between glaucomatous and healthy eyes. NRRI performs significantly better than RNFL thickness for distinguishing between glaucoma suspect and healthy eyes. NRRI also performs significantly better than temporal, superior, nasal, inferior and temporal (TSNIT) average and nerve fiber indicator (NFI) from GDx VCC for distinguishing between glaucoma suspect and healthy eyes. NRRI is a promising parameter for distinguishing glaucoma suspect and healthy eyes and may indicate disease in the pre-perimetric stage. / text

Abstract Cardiovascular disease is one of the leading causes of death in Canada. Atherosclerosis is considered the primary cause for cardiovascular disease. Optical coherence tomography (OCT) provides a means to minimally invasive imaging and assessment of textural features of atherosclerotic plaque. However, detecting atherosclerotic plaque by visual inspection from Optical Coherence Tomography (OCT) images is usually difficult. Therefore we developed unsupervised segmentation algorithms to automatically detect atherosclerosis plaque from OCT images. We used three different clustering methods to identify atherosclerotic plaque automatically from OCT images. Our method involves data preprocessing of raw OCT images, feature selection and texture feature extraction using the Spatial Gray Level Dependence Matrix method (SGLDM), and the application of three different clustering techniques: K-means, Fuzzy C-means and Gustafson-Kessel algorithms to segment the plaque regions from OCT images and to map the cluster regions (background, vascular tissue, OCT degraded signal region and Atherosclerosis plaque) from the feature-space back to the original preprocessed OCT image. We validated our results by comparing our segmented OCT images with actual photographic images of vascular tissue with plaque. / October 2015

This study investigates three high resolution optical imaging modalities to better detect and diagnose skin cancer. The ideal high resolution optical imaging system can visualize pre-malignant tissue growth non-invasively with resolution comparable to histology. I examined 3 modalities which approached this goal. The first method examined was high magnification microscopy of thin stained tissue sections, together with a statistical analysis of nuclear chromatin patterns termed Karyometry. This method has subcellular resolution, but it necessitates taking a biopsy at the desired tissue site and imaging the tissue ex-vivo. My part of this study was to develop an automated nuclear segmentation algorithm to segment cell nuclei in skin histology images for karyometric analysis. The results of this algorithm were compared to hand segmented cell nuclei in the same images, and it was concluded that the automated segmentations can be used for karyometric analysis.The second optical imaging modality I investigated was Optical Coherence Tomography (OCT). OCT is analogous to ultrasound, in which sound waves are delivered into the body and the echo time and reflected signal magnitude are measured. Due to the fast speed of light and detector temporal integration times, low coherence interferometry is needed to gate the backscattered light. OCT acquires cross sectional images, and has an axial resolution of 1-15 µm (depending on the source bandwidth) and a lateral resolution of 10-20 µm (depending on the sample arm optics). While it is not capable of achieving subcellular resolution, it is a non-invasive imaging modality. OCT was used in this study to evaluate skin along a continuum from normal to sun damaged to precancer. I developed algorithms to detect statistically significant differences between images of sun protected and sun damaged skin, as well as between undiseased and precancerous skin.An Optical Coherence Microscopy (OCM) endoscope was developed in the third portion of this study. OCM is a high resolution en-face imaging modality. It is a hybrid system that combines the principles of confocal microscopy with coherence gating to provide an increased imaging depth. It can also be described as an OCT system with a high NA objective. Similar to OCT, the axial resolution is determined by the source center wavelength and bandwidth. The NA of the sample arm optics determines the lateral resolution, usually on the order of 1-5 µm. My effort on this system was to develop a handheld endoscope. To my knowledge, an OCM endoscope has not been developed prior to this work. An image of skin was taken as a proof of concept. This rigid handheld OCM endoscope will be useful for applications ranging from minimally invasive surgical imaging to non-invasively assessing dysplasia and sun damage in skin.