Abstract
Purpose :
AF imaging in AOSLO has enabled in vivo study of RPE cells by capitalizing on the AF of lipofuscin, excited with visible light, and melanin, excited with near infrared (NIR) light. Lipofuscin is more efficient but visible light presents safety and patient comfort concerns. NIR-AF can be used to image RPE cells but has not been systematically interrogated across spectrum. Further, the spatial distribution of fluorophores within RPE cells and their spectral AF may shift in aging and disease. Here we aimed to evaluate the multi-spectral AF of the RPE cell mosaic in healthy eyes and establish techniques for studying the spatial and spectral changes to RPE cells in aging and disease.
Methods :
Five healthy participants (age 25-61) were imaged from the fovea to 12° with AOSLO using eight excitation and emission bands (see Fig. 1). Excitation powers varied in accordance with the ANSI limits and all exposures were ≤50% of the ANSI MPE. AF images were co-registered using simultaneously acquired confocal images and averaged; signal to noise ratio (SNR) and Michelson contrast were computed. All 8 bands were aligned and averaged to generate composite images for comparison.
Results :
Individual RPE cells were visible in all bands but some cells appeared consistent across bands (e.g. Fig. 1 yellow arrows) while others varied (e.g. Fig. 1 purple arrows). Subjectively, the intermediate excitation wavelengths (720-760nm) produced the best images of individual cells with longer and shorter bands appearing visibly noisier. SNR and Michelson contrast were also greatest for these intermediate bands and diminished at both ends (Fig 1).
Conclusions :
Though shorter wavelengths are more efficient than longer ones for exciting AF from the RPE, we found 720-760nm to be the most effective excitation wavelengths amongst those tested here. This likely reflects that any increased excitation efficiency at the shorter wavelengths tested (660-700) is unable to compensate for the lower light levels that are required for patient safety. The spatial variation of fluorescence across spectra may reflect differences in the abundance and concentration of fluorophores. Future work is needed to understand how the spatial distribution of fluorophores and spectrum of RPE AF changes with aging and in disease.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.