Retinal hypoxia and inadequate oxygen delivery have been implicated as causal for the development of several eye diseases, including diabetic retinopathy, glaucoma, and retinopathy of prematurity. The imaging of oxygen tension in the retina, generated from a measure of the phosphorescence lifetimes of bolus-injected palladium-porphyrin probes, has been used successfully to study retinal oxygen dynamics in numerous animal models. However, the specific parameters for applying this technique in the mouse have not been thoroughly investigated. The goals of this project were to calibrate a newly-constructed phosphorescence lifetime imaging instrument and data analysis software against known oxygen concentrations, to determine specific parameters for probe excitation and image collection and analysis in the mouse eye, and to assess any damage caused to the eye by the technique using histological analysis. An in vitro system was developed for calibration of the probe and for estimation of power of excitation light and camera settings necessary to produce acceptable oxygen maps. In vivo experiments were then performed, and plots indicating camera settings necessary for producing varying qualities of oxygen maps were constructed. Trypsin digestion of retinal tissue was used in an attempt to assess any damage to experimental subjects, but this histological technique was deemed inadequate for analyzing the capillary structures of the mouse eye. Alternatively, damage was assessed using the instrument itself to calculate changes in oxygen tension during the experimental process. The results of this work will allow the phosphorescence lifetime imaging system to be used in the mouse to study how changes in retinal oxygen tension correlate with the progression of eye diseases where oxygen is implicated, including diabetic retinopathy.

Thesis (M.S.)--Worcester Polytechnic Institute. / Keywords: Diabetes; imaging; phosphorescence; retina. Includes bibliographical references (p. 69-70).

Retinal hypoxia is associated with many retinal diseases, such as diabetic retinopathy. Current retinal research suggests that retinal hypoxia appears prior to the onset of diabetic retinopathy. The preliminary association of retinal hypoxia to the early stages of diabetic retinopathy is stimulating the development of new technologies to measure the oxygen content of retinal tissue. Frequency domain phosphoresence lifetime imaging (PLI) is a promising technology that enables the mapping of the oxygen content across the entire retina in the form of two-dimensional images. The two-dimensional images generated from the PLI process are a spatial mapping of the retinal tissue's oxygen tension. Currently, the phosphorescent based oxygen tension PLI measurements contain contaminating auto-fluorescent signals in addition to the desired phosphorescent signals. These auto-fluorescent signals artificially inflate the oxygen tension readings due to the nature of fluorescent signals in phosphorescent imaging. Additionally, the maps generated through PLI appear to contain oxygen tension information from both the retinal vasculature and the choroidal vasculature. The choroidal vasculature is situated directly behind the retina and can have a different oxygen tension value than the retinal vasculature. This research enhanced the PLI system by mathematically eliminating the contaminating auto-fluorescent signals and investigated the methods aimed at separating the PO2s of the retinal and choroidal vasculature beds. In addition, the application of the enhanced PLI technology to the investigation of retinal oxygen changes in a rat model of type I diabetes yielded results that suggest a hyperoxic to hypoxic trend prior to the onset of diabetic retinopathy.