Optimization of a technique for phosphorescence lifetime imaging of oxygen tension in the mouse retina

by Kight, Amanda C.

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 newlyconstructed 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. iii