Abstract
Purpose :
Retinal oximetry can provide early detection of retinal disease: typically, abnormal metabolic activity is inferred from abnormal vascular oxygenation. However, inter- and intra-subject variability reduces the efficacy of vascular oximetry as a prognostic indicator. We report a feasibility study into imaging oxygen metabolism in tissue directly, by measuring flavin adenine dinucleotide (FAD) fluorescence, an indicator of oxygen metabolism. Crucial challenges are low intensity of FAD and clutter from ocular autofluorescence (AF). We report a study of the feasibility of spectral and fluorescence lifetime imaging (FLIM) for quantitative retinal mapping of FAD.
Methods :
An LSM710 laser-scanning confocal microscope (Zeiss, Germany) was used to acquire images of FAD fluorescence in the mouse retina. Excitation (488nm) and emission (505-575nm) wavelengths were chosen to image FAD (laser power=100μW, acquisition time 25sec). We also report the design of combined spectro-lifetime fluorescence imaging, and its application to in vitro imaging of FAD in a model eye. A mathematical model was developed for predicting optical intensities and spectral-lifetime fluorescence in the human eye, assuming similar metabolic activity.
Results :
The pilot study conducted with mouse retina under normal and hypoxic conditions showed a decrease in FAD signal during hypoxia, indicating the feasibility of mapping metabolic activity. The fluorescence signal in a human eye, despite its smaller numerical aperture, is calculated to be comparable to that of the mouse eye. A non-confocal SLO, or a fundus camera, is calculated to offer a 20-fold increase in optical efficiency, but also an increase in ocular-autofluorescence clutter. We will present spectral-fluorescence-lifetime imaging of FAD in phantom eyes, which provide promise for enhanced discrimination of FAD signatures in the presence of these higher levels of clutter.
Conclusions :
FAD fluorescence is shown to be modulated by oxygen metabolism in the retina. Our experiments and modelling indicate that the use of a fundus camera with spectral/FLIM imaging of fluorescence provides a route for unmixing the FAD signal from ocular AF, with sufficient signal for robust detection.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.