Abstract
Purpose :
Fluorescence adaptive optics scanning light ophthalmoscopy (AOSLO) images individual RPE cells by utilizing the intrinsic AF of RPE fluorophores such as lipofuscin, melanin and melanolipofuscin. However, the contribution of each fluorophore to the AF signal remains controversial. Furthermore, evidence suggests that the composition of RPE fluorophores and their spectral fluorescence may change with age. Here, we used multi-spectral AF imaging in AOSLO to detect and quantify variations in AF of individual RPE cells in living human eyes as a function of wavelength, age, and eccentricity.
Methods :
11 healthy participants (age: 20-73) were sequentially imaged from the fovea to 12° temporal using eight different excitation wavelengths (ex: 660-795nm; em: 708-850nm); power levels varied and were below 50% of the ANSI MPE. Cellpose, a generalist machine learning cell segmentation tool, was used to retrain a model and segment the RPE cells. Segmentation of the cell borders allowed for morphometric analysis (e.g., cell area, aspect ratio) and evaluation of the AF signal at the cellular level. After normalization of the raw AF signal, AF heterogeneity of each wavelength was examined by calculating deviations from the mean AF signal.
Results :
143 retinal locations were imaged and ~68,000 RPE cells were segmented (Fig 1a) for analysis. 16 locations (12%) were excluded from analysis due to poor image quality. 720nm light produced images with highest AF intensity (37%). Of the RPE cells that showed AF variance, 660 and 795nm light deviated most from the mean AF signal intensity (96%). Some RPE cells showed more AF variance than others (Fig 1b). The younger cohort (20-40 yrs.) showed more deviation from the mean (0.88 SD) than the older (0.82 SD).
Conclusions :
AF signal varied substantially across wavelengths. The smaller deviation across spectrum in the older cohort is suggestive of more colocalization of RPE AF signal with age. This finding is suggestive of a potential biomarker of RPE cell age, warranting further study in a larger cohort. Better understanding of how RPE multi-spectral AF changes with age may provide improved diagnostic tools to evaluate RPE health across the lifespan and in diseases affecting the RPE, such as age-related macular degeneration.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.