Two-photon adaptive optics fluorescence lifetime imaging ophthalmoscopy

Abstract

Thesis (Ph. D.)--University of Rochester. The Institute of Optics, 2018.There are many critical processes involved in keeping the retina functioning properly. Two of these, the visual cycle and the metabolism of the cell, are tied together by their conversion of important molecules from one form to another. In the visual cycle, 11-cis-retinal is regenerated so that it can combine with a rhodopsin molecule and initiate phototransduction. In cellular metabolism, the cell undergoes many steps to generate adenosine triphosphate, the energy unit of the cell. These mechanisms are critical in maintaining a functioning retina, however they have been difficult to directly interrogate in the living eye. A technique which can quantitatively measure these processes could allow researchers and clinicians to examine them in healthy subjects and how they change under conditions of disease. The goal of this work is to develop a technique which will allow us to investigate these measures of retinal function quantitatively and in a repeatable way. Advantageously, molecules which are converted during the visual cycle or cellular metabolism are accessible using adaptive optics aided two-photon fluorescence ophthalmoscopy. Furthermore, I develop a new technique, adaptive optics fluorescence lifetime ophthalmoscopy, which provides a robust and quantitative measure of a key property of retinal fluorescence. Initially, this method was deployed in a new two-photon adaptive optics ophthalmoscope designed for imaging mice. Exogenous fluorophores with known fluorescence lifetimes were used to validate the initial measurements, before using the new technique to establish baseline measurements for a sensor of cellular metabolism in the mouse eye. Following successful implementation in the mouse, the fluorescence lifetime method was translated to a system dedicated to imaging the macaque retina. By measuring the fluorescence lifetime of endogenous fluorescence originating in the photoreceptors, I found that rods and cones exhibit different fluorescence lifetimes. Further development of this technology may advance research in widespread areas including fluorophore identification in the retina, mechanisms of retinal metabolism, and as a clinical diagnostic