Retinal Oxygen Imaging in Mice by Phosphorescence Lifetime

Abstract

Abstract: : Purpose: Regional tissue hypoxia may be the cause or early contributing factor that leads to retinal and choroidal neovascularization. The purpose of this study is to develop an imaging system to generate a high–resolution oxygen distribution map of the mouse retina using phosphorescence lifetime. Methods: Oxygen–sensitive phosphor molecules when excited by a certain wavelength of light can be used to accurately measure oxygen pressure in vitro and in vivo based on oxygen’s specific ability to quench the molecule’s phosphorescent decay or lifetime. Three different phosphors with absorption bands for excitation at wavelengths from 420 nm to 635 nm and phosphorescence emission from 660 nm to 820 nm were used. Eyes were dilated using 1% tropicamide, 10% phenylephrine, or 1% atropine sulfate. Phosphor dissolved in saline was injected intravenously into anesthetized adult C57BL/6 mice. On axis illumination by a modulated LED light source was used to excite the phosphor and the phosphorescence was imaged at different phase delays relative to excitation using an intensified CCD camera through microscope optics. The phosphorescence lifetime was calculated and oxygen pressure was calculated for each pixel from the oxygen dependence of the phosphorescence lifetime. The wavelengths of excitation and emission were specific to the phosphor used. Results: The different phosphors and dilating solutions were compared based on the measured oxygen pressures in the retinal arteries, veins and capillary beds and the resolution of the oxygen pressure maps. We report the optimal conditions for oxygen pressure measurements in the mouse retina and how the different phosphors compared with respect to the values of the measured oxygen pressures and the extent to which the comparisons allow selective measurement of retinal and/or choroidal oxygen pressures. Conclusions: Phosphorescence lifetime imaging can provide high–resolution maps of the oxygen distribution in the retina of the mouse eye when optimal conditions are used. This minimally invasive real time oxygen imaging technology can be used to determine the role of oxygen pressure in retinal pathology and may lead to novel diagnostic clinical imaging systems.

Keywords: retinal neovascularization • imaging/image analysis: non–clinical • ischemia