gms | German Medical Science

Background: We have previously demonstrated that hyperspectral imaging is capable of extracting and localizing individual signals of abundant fluorophore families from normal RPE and Bruch’s-membrane (BrM) ex-vivo (Smith et al, Biomed Opt Express 2014). Herein we describe new spectral/spatlal fluorophore characteristics in donor eyes with AMD.

Methods: Hyperspectral AF images were captured from 11 locations containing drusen and 35 other locations with other pathologies in 9 RPE/BrM flat-mounts from donor eyes with AMD. Imaging was performed at 2 excitation bands, 436-460 and 480-510 nm, with emission captured between 420 and 720 nm in 10 nm intervals using a Nuance FX camera. Gaussian mixtures and mathematical factorization were applied to extract 4-5 abundant emission spectra from each tissue.

Results: Drusen: There is a consistent emission spectrum peaking at ~510 nm (exc 436 nm) that appears to be both sensitive and specific for drusen and sub-RPE deposits. One or more abundant RPE spectra (lipofuscin and/or melanolipofuscin) also co-localize with, or form haloes around drusen. Other Pathology: Compared to the fairly similar spatial localizations of the main RPE AF signatures in normal tissue, there is remarkably heterogeneous lesion-associated grouping of signals in, for example, extruded LF packets described by Ach et al (ARVO, 2014) and other structures (basal laminar deposits, atrophic zones, and abnormal, enlarged or emptying RPE cells).

Conclusions: There is a remarkable dissociation of RPE fluorophore families as eyes develop AMD, as well as a distinct new AF signal from drusen, that are well demonstrated by hyperspectral imaging. Molecular identification of these families of fluorophores with imaging mass spectroscopy is a critically needed next step towards understanding the biochemistry of the RPE in normal aging and AMD.