Abstract
Purpose :
Hyperspectral imaging can generate different spectral profiles that correlate to distinct fluorophore families present in human RPE. One of these profiles has been previously associated with the localization of melanosomes-melanolipofuscin (M/ML) granules in eyes with late stage AMD (Ben Ami ARVO 2016). The presence and localization of these spectra may represent important physiologic and pathologic steps in human RPE. This study sought to explore the spatial correlation of these spectra with M/ML granules in normal and pathological specimens.
Methods :
Hyperspectral AF images were captured at multiple locations from 19 RPE/Bruch’s-membrane flat-mounts of donor eyes with and without AMD, yielding 68 images. Imaging was performed at 40x magnification using autofluorescence at 3 excitation bands of 436, 480, and 505nm. Emissions were collected from 420-720nm and decomposed using NMF as previously described (Ben Ami ARVO 2016). Five spectra were generated from each image. For all spectra collected from all images, those with an S3 peak with an emission maximum at 620nm representing M/ML (Ben Ami PMID 27226929) were isolated and compared to brightfield images.
Results :
At 436 nm excitation, a broad AF signal S3 (Fig. 1: Spectra & S3) was extracted for 25 locations. The S3 abundance image demonstrated spatial correlation with areas containing M/ML granules on BF images in 16/25 locations (64%) (Fig. 1: BF). This group included 4/5 previously known sub RPE deposits in the data set (80%). Additionally, 8/11 (72.7%) of eyes with AMD and S3 spectra demonstrated overlap with M/ML.
Conclusions :
Hyperspectral imaging of RPE in normal and AMD eyes was able to isolate a spectrum that corresponds to M/ML granules in most samples. Further development could improve the specificity of this signal, enabling more detailed studies.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.