Hyperspectral Autofluorescence Characterization of Transition to Atrophy in Donor Eyes with Advanced Age-Related Macular Degeneration (AMD)

Abstract

Purpose : Obtain spectral properties of transition zones between atrophy and intact retinal pigment epithelium (RPE) in donor eyes with late AMD (geographic atrophy-GA and neovascularization-NV).

Methods : Hyperspectral AF images were acquired from 6 flatmounts of RPE/ Bruch’s membrane (BrM) (1 GA donor, 2 tissues; 3 NV donors (4 tissues)) at peak excitations λ_ex = 436 and λ_ex = 480 nm for emissions 420-700 nm, 10 nm intervals. A matrix factorization algorithm recovered fluorophore emission spectra and respective basis images.

Results : Basis spectra S1, S2 and S3 were recovered from lipofuscin/melanolipofuscin (LF/ML) in RPE (Fig 1), intact or not, and were similar in shape, peak emission wavelength, and tissue localization (Fig 2) to those reported for normal RPE/BrM (PMID 25574430). The spectrum S0 (seen in normal BrM) was weak. Spectrum SD, which is sensitive and specific for drusen and sub-RPE deposits (ARVO 2015: Program Number: 3956), localized to sub-RPE deposits (Fig 1), peaked near 500 nm at λ_ex = 436 nm, and was more visible in atrophic areas than in areas with surviving RPE (Fig 2). S2 was weak in two CNV cases (not shown) and strong in other tissues. The S3 spectrum of ML was less intense in GA relative to LF spectra S1 and S2 (Fig 1), consistent with decreased melanin, but not in CNV tissues.

Conclusions : The spectral characteristics of normal RPE were largely preserved in the transition zones between healthy cells and atrophy in advanced AMD. Normal RPE constituents may persist even in areas with severe AMD damage, consistent with reports of numerous RPE derived cells in atrophic areas (PMID 25813989).

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

 

Figure 1. Basis spectra. 81-year-old male donor. GA transition zone. Emission spectra from excitation at λ_ex= 436 nm (solid) and 480 nm (dashed) (green, blue, red) resemble spectra S1, S2 and S3 reported in normal RPE. The azure spectrum is SD a marker for drusen and sub-RPE deposits. S0 (BrM, gray) is weak.

 

Figure 2. Composite AF and spectral basis images for fluorophores of Fig 1. Upper left, composite RGB image of total AF emission. The basis images of the LF/ML fluorophores S1, S2 and S3 show comparable intensity and relatively normal distributions in both disrupted and intact RPE. The spectrum SD localizes to sub-RPE deposits in the atrophic area (lower R). S0 (BrM) localizes to exposed BrM.