This dissertation documents the development of a new illumination technique for use in the studies of retinal oximetry and fundus spectroscopy. Intravitreal illumination is a technique where the back of the eye is illuminated trans-sclerally using a scanning monochromator coupled into a fiber optic illuminator. Retinal oximetry is the processof measuring the oxygen saturation of blood contained in retinal vessels by quantitative measurement of the characteristic color shift seen as blood oxygen saturation changes from oxygenated blood (reddish) to deoxygenated blood (bluish). Retinal oximetry was first attempted in 1963 but due to a variety of problems with accuracy and difficulty of measurement, has not matured to the point of clinical acceptabilityor commercial viability.Accurate retinal oximetry relies in part on an adequate understanding of the spectral reflectance characteristics of the fundus. The use of intravitreal illumination allows new investigations into the spectral reflectance properties of the fundus. The results of much research in fundus reflectance and retinal oximetry is detailed in thisdocument, providing new insight into both of these related fields of study.Intravitreal illumination has been used to study retinal vessel oximetry and fundus reflectometry resulting in several important findings that are presented in this document. Studies on enucleated swine eyes have provided new insight into the bidirectional reflectance distribution function of the fundus. Research on live swine hasshown accurate measurement of retinal vessel oxygen saturation and provided the first in vivo spectral transmittance measurement of the sensory retina. A secondary discovery during this research suggests that vitrectomy alters the retinal vasculature,a finding that should spawn new research in its own right.

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.