Retinal diseases such as age-related macular degeneration (AMD) are the major cause of blindness in the developed world. Early diagnosis of these diseases is difficult as symptoms appear only at advanced stages. Nevertheless, several studies suggest that impairment of dark adaptation (the ability of the retina to adapt to low lighting) is a cue to AMD. Dark adaptation is the result of the regeneration of light sensitive pigments after having reacted to light (bleaching). This PhD aims at developing a tool for objective measurements of the quantity of photopigment and the kinetics of dark adaptation. This work comprises a thorough review of the absorbing and reflecting properties of the different ocular structures, giving rise to a new model of retinal (or fundus) reflectance. This model provides a detailed description of the different pathways of light through the photoreceptor layer and was able to explain measurements and findings of the literature, in particular the effect of the photoreceptor matrix interstices. An extensive study of the influence of the different model parameters on the total fundus reflectance led to the proposal of a new objective and comparable measure of quantity of photopigment (QoP). This measure is obtained by fitting a constrained version of the new model to a double density difference (DDD) measurement (i.e. the logarithmic difference between reflectances of a retinal area in bleached and dark adapted states). This approach was validated by correctly fitting several DDD measurements from the literature. Future experimental studies are needed to confirm the relevance of the new QoP measure and specify its application in clinical diagnosis. Several fundus reflectometry instruments have been able to measure the DDD in human eyes however because of practical and technological limitations none of these instruments were suitable for clinical use. Here, these limitations are discussed and two new imaging fundus reflectometers are presented. Developed respectively by 4D Optics Ltd. and the Vision Research group at the University of Manchester, these two systems, based on modified fundus cameras, are ongoing development work towards clinically suitable imaging fundus reflectometry. Example data obtained with these two instruments exhibits aberrant points and low signal to noise ratio (SNR). The main issues encountered were camera noise and stability, uneven retinal illumination, and subject’s eye movements and changes of alignment. It is believed that these issues can be overcome with current technologies. One important impediment to the use of the dark adaptation experiment in clinical practice is the time it takes for photopigments to completely regenerate (up to 40 min in normal eyes). A theoretical data analysis strategy using the new model of fundus reflectance and the Marhoo, Lamb and Pugh model of photopigment regeneration kinetics is proposed to rapidly diagnose an abnormal regeneration, hence reducing considerably the duration of the experiment. This idea has not been tested on experimental data but may become relevant once better quality measurements of DDD are obtained.

Optical coherence tomography systems are used to image the retina in 3D to allow ophthalmologists diagnose ocular disease. These systems yield large data sets that are often labor-intensive to analyze and require significant expertise in order to draw conclusions, especially when used over time to monitor disease progression. Spectral Domain Optical Coherence Tomography (SD-OCT) instantly acquires depth profiles at a single location with a broadband source. These systems require mechanical scanning to generate two- or three-dimensional images. Instead of mechanically scanning, a beamlet array was used to permit multiple depth measurements on the retina with a single snapshot using a 3x 3 beamlet array. This multi-channel system was designed, assembled, and tested using a 1 x 2 beamlet lens array instead of a 3 x 3 beamlet array as a proof of concept prototype. The source was a superluminescent diode centered at 840nm with a 45nm bandwidth. Theoretical axial resolution was 6.92um and depth of focus was 3.45mm. Glass samples of varying thickness ranging from 0.18mm to 1.14mm were measured with the system to validate that correct depth profiles can be acquired for each channel. The results demonstrated the prototype system performed as expected, and is ready to be modified for in vivo applicability.

Research into automatic retina image analysis has become increasingly important, not just in ophthalmology but also in other clinical specialities such as cardiology and neurology. In the retina, blood vessels can be directly visualised non-invasively in-vivo, and hence it serves as a "window" to cardiovascular and neurovascular complications. Biomarker research, i.e. investigating associations between the morphology of the retinal vasculature (as a means of revealing microvascular health or disease) and particular conditions affecting the body or brain could play an important role in detecting disease early enough to impact on patient treatment and care. A fundamental requirement of biomarker research is access to large datasets to achieve sufficient power and significance when ascertaining associations between retinal measures and clinical characterisation of disease. Crucially, the vascular changes that appear can affect arteries and veins differently. An essential part of automatic systems for retinal morphology quantification and biomarker extraction is, therefore, a computational method for classifying vessels into arteries and veins. Artery-vein classification enables the efficient extraction of biomarkers such as the Arteriolar to Venular Ratio, which is a well-established predictor of stroke and other cardiovascular events. While structural parameters of the retinal vasculature such as vessels calibre, branching angle, and tortuosity may individually convey some information regarding specific aspects of the health of the retinal vascular network, they do not convey a global summary of the branching pattern and its state or condition. The retinal vascular tree can be considered a fractal structure as it has a branching pattern that exhibits the property of self-similarity. Fractal analysis, therefore, provides an additional means for the quantitative study of changes to the retinal vascular network and may be of use in detecting abnormalities related to retinopathy and systemic diseases. In this thesis, new developments to fully automated retinal vessel classification and fractal analysis were explored in order to extract potential biomarkers. These novel processes were tested and validated on several datasets of retinal images acquired with fundus cameras. The major contributions of this thesis include: 1) developing a fully automated retinal blood vessel classification technique, 2) developing a fractal analysis technique that quantifies regional as well as global branching complexity, 3) validating the methods using multiple datasets, and 4) applying the proposed methods in multiple retinal vasculature analysis studies.

The focus of this thesis is the improvement of acquisition and processing of Mueller matrix polarimetry using a confocal scanning laser ophthalmoscope (CSLO) and the application of Mueller matrix polarimetry to image the retina. Stepper motors were incorporated into a CSLO to semi-automate Mueller matrix polarimetry and were used in retinal image acquisition. Success rates of Fourier transform based edge detection filters, designed to improve the registration of retinal images, were compared. The acquired polarimetry images were used to reassess 2 image quality enhancement techniques, Mueller matrix reconstruction (MMR) and Stokes vector reconstruction (SVR), focusing on the role of auto-contrasting or normalization within the techniques and the degree to which auto-contrasting or normalization is responsible for image quality improvement of the resulting images. Mueller matrix polarimetry was also applied to find the retardance image of a malaria infected retinal blood vessel imaged in a confocal scanning laser microscope (CSLM) to visualize hemozoin within the vessel. Image quality enhancement techniques were also applied and image quality improvement was quantified for this blood vessel. The semi-automation of Mueller matrix polarimetry yielded a significant reduction in experimental acquisition time (80%) and a non-significant reduction in registration time (44%). A larger sample size would give higher power and this result might become significant. The reduction in registration time was most likely due to less movement of the eye, particularly in terms of decreased rotation seen between registered images. Fourier transform edge detection methods increased the success rate of registration from 73.9% to 92.3%. Assessment of the 2 MMR images (max entropy and max signal-to-noise ratio (SNR)) showed that comparison to the best CSLO images (not auto-contrasted) yielded significant average image quality improvements of 158% and 4% when quantified with entropy and SNR, respectively. When compared to best auto-contrasted CSLO images, significant image quality improvements were 11% and 5% for entropy and SNR, respectively. Images constructed from auto-contrasted input images were of significantly higher quality than images reconstructed from original images. Of the 2 other images assessed (modified degree of polarization (DOPM) and the first element of the Stokes vector (S0)), DOPM and S0 yielded significant average image quality improvements quantified by entropy except for the DOPM image of the RNFL. SNR was not improved significantly when either SVR image was compared to the best CSLO images. Compared to the best auto-contrasted CSLO images, neither DOPM nor S0 improved average image quality significantly. This result might change with a larger number of participants. When MMR were applied to images of malaria infected retinal slides, image quality was improved by 19.7% and 15.3% in terms of entropy and SNR, respectively, when compared to the best CSLO image. The DOPM image yielded image quality improvements of 8.6% and -24.3% and the S0 image gave improvements of 9.5% and 9.4% in entropy and SNR, respectively. Although percent increase in image quality was reduced when images were compared to initial auto-contrasted CSLO images, the final image quality was improved when auto-contrasting occurred prior to polarimetry calculations for max SNR and max entropy images. Quantitative values of retardance could not be found due to physical constraints in the CSLM that did not allow for characterization of its polarization properties and vibrational noise. Mueller matrix polarimetry used to find the retardance image of a malaria infected retina sample did yield visualization of hemozoin within the vessel but only qualitatively. In conclusion, improvements in the acquisition and registration of CSLO images were successful in leading to considerably shorter experimentation and processing times. In terms of polarimetric image quality improvement techniques, when compared to the best CSLO image. A large proportion of the improvement was in fact due to partially or completely stretching the pixel values across the dynamic range of the images within the algorithm of each technique. However, in general the image quality was still improved by the Mueller matrix reconstruction techniques using both entropy and SNR to generate the CSLO retinal images and the CSLM imaged malaria infected sample. In the malaria sample, retinal blood vessel visualization was also qualitatively improved. The images yielded from Mueller matrix polarimetry applied to a malaria infected retinal sample localized hemozoin within the blood vessel, but a quantitative image of the phase retardance could not be achieved.

Adaptive optics (AO) systems make use of active optical elements, namely wavefront correctors, to improve the resolution of imaging systems by compensating for complex optical aberrations. Recently, magnetic fluid deformable mirrors (MFDM) were proposed as a novel type of wavefront correctors that offer cost and performance advantages over existing wavefront correctors. These mirrors are developed by coating the free surface of a magnetic fluid with a thin reflective film of nano-particles. The reflective surface of the mirrors can be deformed using a locally applied magnetic field and thus serves as a wavefront corrector. MFDMs have been found particularly suitable for ophthalmic imaging systems where they can be used to compensate for the complex aberrations in the eye that blur the images of the internal parts of the eye. However, their practical implementation in clinical devices is hampered by the lack of effective methods to control the shape of their deformable surface. The research work reported in this thesis presents solutions to the surface shape control problem in a MFDM that will make it possible for such devices to become integral components of retinal imaging AO systems. The first major contribution of this research is the development of an accurate analytical model of the dynamics of the mirror surface shape. The model is developed by analytically solving the coupled system of fluid-magnetic equations that govern the dynamics of the surface shape. The model is presented in state-space form and can be readily used in the development of surface shape control algorithms. The second major contribution of the research work is a novel, innovative design of the MFDM. The design change was prompted by the findings of the analytical work undertaken to develop the model mentioned above and is aimed at linearizing the response of the mirror surface. The proposed design also allows for mirror surface deflections that are many times higher than those provided by the conventional MFDM designs. A third contribution of this thesis involves the development of control algorithms that allowed the first ever use of a MFDM in a closed-loop adaptive optics system. A decentralized proportional-integral (PI) control algorithm developed based on the DC model of the wavefront corrector is presented to deal mostly with static or slowly time-varying aberrations. To improve the stability robustness of the closed-loop AO system, a decentralized robust proportional-integral-derivative (PID) controller is developed using the linear-matrix-inequalities (LMI) approach. To compensate for more complex dynamic aberrations, an Hinf controller is designed using the mixed-sensitivity Hinf design method. The proposed model, design and control algorithms are experimentally tested and validated.

Adaptive optics (AO) systems make use of active optical elements, namely wavefront correctors, to improve the resolution of imaging systems by compensating for complex optical aberrations. Recently, magnetic fluid deformable mirrors (MFDM) were proposed as a novel type of wavefront correctors that offer cost and performance advantages over existing wavefront correctors. These mirrors are developed by coating the free surface of a magnetic fluid with a thin reflective film of nano-particles. The reflective surface of the mirrors can be deformed using a locally applied magnetic field and thus serves as a wavefront corrector. MFDMs have been found particularly suitable for ophthalmic imaging systems where they can be used to compensate for the complex aberrations in the eye that blur the images of the internal parts of the eye. However, their practical implementation in clinical devices is hampered by the lack of effective methods to control the shape of their deformable surface. The research work reported in this thesis presents solutions to the surface shape control problem in a MFDM that will make it possible for such devices to become integral components of retinal imaging AO systems. The first major contribution of this research is the development of an accurate analytical model of the dynamics of the mirror surface shape. The model is developed by analytically solving the coupled system of fluid-magnetic equations that govern the dynamics of the surface shape. The model is presented in state-space form and can be readily used in the development of surface shape control algorithms. The second major contribution of the research work is a novel, innovative design of the MFDM. The design change was prompted by the findings of the analytical work undertaken to develop the model mentioned above and is aimed at linearizing the response of the mirror surface. The proposed design also allows for mirror surface deflections that are many times higher than those provided by the conventional MFDM designs. A third contribution of this thesis involves the development of control algorithms that allowed the first ever use of a MFDM in a closed-loop adaptive optics system. A decentralized proportional-integral (PI) control algorithm developed based on the DC model of the wavefront corrector is presented to deal mostly with static or slowly time-varying aberrations. To improve the stability robustness of the closed-loop AO system, a decentralized robust proportional-integral-derivative (PID) controller is developed using the linear-matrix-inequalities (LMI) approach. To compensate for more complex dynamic aberrations, an Hinf controller is designed using the mixed-sensitivity Hinf design method. The proposed model, design and control algorithms are experimentally tested and validated.

The ROx is a retinal oximeter under development with the purpose of non-invasively and accurately measuring oxygen saturation (SO₂) in vivo. It is novel in that it utilizes the blue-green oximetry technique with on-axis illumination. ROx calibration tests were performed by inducing hypoxia in live anesthetized swine and comparing ROx measurements to SO₂ values measured by a CO-Oximeter. Calibration was not achieved to the precision required for clinical use, but limiting factors were identified and improved. The ROx was used in a set of sepsis experiments on live pigs with the intention of tracking retinal SO₂ during the development of sepsis. Though conclusions are qualitative due to insufficient calibration of the device, retinal venous SO₂ is shown to trend generally with central venous SO₂ as sepsis develops. The novel sepsis model developed in these experiments is also described. The method of cecal ligation and perforation with additional soiling of the abdomen consistently produced controllable severe sepsis/septic shock in a matter of hours. In addition, the ROx was used to collect retinal images from a healthy human volunteer. These experiments served as a bench test for several of the additions/modifications made to the ROx. This set of experiments specifically served to illuminate problems with various light paths and image acquisition. The analysis procedure for the ROx is under development, particularly automating the process for consistency, accuracy, and time efficiency. The current stage of automation is explained, including data acquisition processes and the automated vessel fit routine. Suggestions for the next generation of device minimization are also described.

The focus of this thesis is the improvement of acquisition and processing of Mueller matrix polarimetry using a confocal scanning laser ophthalmoscope (CSLO) and the application of Mueller matrix polarimetry to image the retina. Stepper motors were incorporated into a CSLO to semi-automate Mueller matrix polarimetry and were used in retinal image acquisition. Success rates of Fourier transform based edge detection filters, designed to improve the registration of retinal images, were compared. The acquired polarimetry images were used to reassess 2 image quality enhancement techniques, Mueller matrix reconstruction (MMR) and Stokes vector reconstruction (SVR), focusing on the role of auto-contrasting or normalization within the techniques and the degree to which auto-contrasting or normalization is responsible for image quality improvement of the resulting images. Mueller matrix polarimetry was also applied to find the retardance image of a malaria infected retinal blood vessel imaged in a confocal scanning laser microscope (CSLM) to visualize hemozoin within the vessel. Image quality enhancement techniques were also applied and image quality improvement was quantified for this blood vessel. The semi-automation of Mueller matrix polarimetry yielded a significant reduction in experimental acquisition time (80%) and a non-significant reduction in registration time (44%). A larger sample size would give higher power and this result might become significant. The reduction in registration time was most likely due to less movement of the eye, particularly in terms of decreased rotation seen between registered images. Fourier transform edge detection methods increased the success rate of registration from 73.9% to 92.3%. Assessment of the 2 MMR images (max entropy and max signal-to-noise ratio (SNR)) showed that comparison to the best CSLO images (not auto-contrasted) yielded significant average image quality improvements of 158% and 4% when quantified with entropy and SNR, respectively. When compared to best auto-contrasted CSLO images, significant image quality improvements were 11% and 5% for entropy and SNR, respectively. Images constructed from auto-contrasted input images were of significantly higher quality than images reconstructed from original images. Of the 2 other images assessed (modified degree of polarization (DOPM) and the first element of the Stokes vector (S0)), DOPM and S0 yielded significant average image quality improvements quantified by entropy except for the DOPM image of the RNFL. SNR was not improved significantly when either SVR image was compared to the best CSLO images. Compared to the best auto-contrasted CSLO images, neither DOPM nor S0 improved average image quality significantly. This result might change with a larger number of participants. When MMR were applied to images of malaria infected retinal slides, image quality was improved by 19.7% and 15.3% in terms of entropy and SNR, respectively, when compared to the best CSLO image. The DOPM image yielded image quality improvements of 8.6% and -24.3% and the S0 image gave improvements of 9.5% and 9.4% in entropy and SNR, respectively. Although percent increase in image quality was reduced when images were compared to initial auto-contrasted CSLO images, the final image quality was improved when auto-contrasting occurred prior to polarimetry calculations for max SNR and max entropy images. Quantitative values of retardance could not be found due to physical constraints in the CSLM that did not allow for characterization of its polarization properties and vibrational noise. Mueller matrix polarimetry used to find the retardance image of a malaria infected retina sample did yield visualization of hemozoin within the vessel but only qualitatively. In conclusion, improvements in the acquisition and registration of CSLO images were successful in leading to considerably shorter experimentation and processing times. In terms of polarimetric image quality improvement techniques, when compared to the best CSLO image. A large proportion of the improvement was in fact due to partially or completely stretching the pixel values across the dynamic range of the images within the algorithm of each technique. However, in general the image quality was still improved by the Mueller matrix reconstruction techniques using both entropy and SNR to generate the CSLO retinal images and the CSLM imaged malaria infected sample. In the malaria sample, retinal blood vessel visualization was also qualitatively improved. The images yielded from Mueller matrix polarimetry applied to a malaria infected retinal sample localized hemozoin within the blood vessel, but a quantitative image of the phase retardance could not be achieved.

INTRODUCTION: Schizophrenia (SZ) and bipolar disorder (BD) are neurodegenerative psychotic disorders hallmarked by reductions in gray and white matter volume. Limitations in neuroimaging have led to the use of OCT to study retinal layer biomarkers and their relation to brain pathology. This thesis includes a meta-analysis of current literature and an exploratory analysis of retinal layer thickness in relation to SZ and BD. METHODS: For the meta-analysis, twelve articles were identified using PubMed, Web of Science, and Cochrane database. Diagnostic groups were proband (SZ and BD combined), SZ only, BD only, and healthy control (HC) eyes. Analyses utilized fixed and random effects models, in addition to assuring that bias was adjusted for and that results were cross-validated. Statistical analyses were performed using the “meta” package in R, with results reported as standard mean differences (SMD). The exploratory analysis included a total of 38 subjects (24 probands and 14 HC). Retinal measures were co-varied for age, sex, race, body mass index (BMI), and best-corrected visual acuity (BCVA). Correlations between retinal and clinical and cortical measures were also performed. Clinical data included illness duration, symptom severity, antipsychotic dosage, and smoking status. Neuroimaging data included gray matter (GM) thickness, gray matter volume, and intracranial volume (ICV). Linear effects and mixed effects models were used to study mean eye and right/left eye measures, respectively. Statistical analysis was done in R. RESULTS: A total of 820 patient eyes (541 SZ and 279 BD) and 904 HC eyes were used for the meta-analysis. Compared to HC eyes, probands, SZ, and BD eyes showed significant thinning the peripapillary retinal nerve fiber layer (RNFL), with atrophy greatest in the nasal, temporal, and superior regions. In addition, all diagnostic groups demonstrated significant reductions in the combined ganglion cell layer and inner plexiform layer (GCL-IPL) compared to HC. No significant differences were found for choroidal and macular measures. No significant relationships were seen from meta-regression analysis for clinical measures. For the exploratory analysis, retinal measures from a total of 24 probands (18 SZ and 6 BD) and 14 HC was studied. Compared to HC, probands showed reductions in overall RNFL in mean eye measures, while increases in the inner and outer RNFL were seen in left eye measures. No significant group differences were seen in the GCL, IPL, and inner nuclear layer (INL). The outer plexiform layer (OPL) showed significant thickening in probands and SZ compared to HC for all eye measures. Probands showed trending reductions in the outer nuclear layer (ONL) in the left eye compared to HC. No significant correlations were found between retinal layers and illness duration, overall PANSS (Positive and Negative Syndrome Scale) score, PANSS negative symptom subscore, and smoking status. PANSS positive symptom subscore showed significant and trending negative correlations to the RNFL and GCL, respectively. Antipsychotic medication dosage displayed a trending negative relationship with the IPL. GM thickness showed a significant and trending negative correlation to the RNFL and ONL, respectively. Furthermore, a trending inverse relationship was observed between GM volume and the OPL. Finally, ICV demonstrated a trending and significant negative relationship with GCL and OPL thickness, respectively. CONCLUSION: The meta-analysis showed that atrophy in RNFL and GCL-IPL measures are widely associated with psychosis. Furthermore, it supports previous findings of gray and white matter reductions in SZ and BD. The exploratory analysis showed psychosis-associated reductions in the RNFL and ONL layers, consistent with previous literature. Contradictory findings, the thickening of the ONL, can be attributed to the conflicting findings, but might also be explained by neuro-inflammatory pathways related to psychotic disorders.

The purpose of this research was to investigate detect and monitor laser-tissue interactions at threshold and potentially sub-threshold levels of injury. High resolution imaging modalities can provide a deeper understanding of candidate biomarkers disease and injury at the molecular, cellular, and tissue-levels which can be used to identify and diagnose early stages disease and damage. In addition, multi-scale and multi-modal imaging have also been used to identify inherent biomarkers of retinal disease and injury. Monitoring tissue changes can be mapped back to biological changes at the cellular and sub-cellular level. Diseases often alter tissue on the ultra-structural level yet retinal clinical diagnosis often monitor changes in tissue at the organ level. If injury and disease is detected and diagnosed during an “early” stage of development, treatments and drug interventions may prevent further spread of the pathology. Non-invasive imaging is expected to be a valuable tool for in vivo medical research as well as for the diagnosis and management of disease. In addition to developing new imaging tools and techniques to image the retina, the identification of inherent biomarkers of disease and health using diagnostic methods are almost equally as important. Using the inherent optical properties of retinal tissue, we can non- invasively quantify differences in the absorption and reflection of light to gauge the risk for visual disability or worse yet irreversible vision loss as a result of retinal disease and chronic light exposure. The research presented with in this dissertation is three separate studies aimed at identifying light injury and potential biomarkers indicating the risk of light mediated development of disease. / text