Drusen are one of the hallmark lesions of AMD. Our purpose is to characterize drusen by their spectral AF signatures, for a better molecular understanding of AMD.
Retinal pigment epithelium (RPE)/Bruch’s membrane (BrM)-flatmounts were prepared from 5 donors with AMD. Hyperspectral AF imaging was performed at 11 locations containing drusen at 2 excitation bands, 436-460 and 480-510 nm, with emissions captured between 420 and 700 nm in 10 nm intervals, with the Nuance FX camera. Abundant individual spectra with corresponding spatial localizations were recovered with custom software based on a nonnegative tensor factorization algorithm .
In a typical example the original AF image (RGB) is presented with 5 spatial abundances (Fig. 1) of 5 recovered individual spectra (Fig. 2). In particular, spectrum C4 (Fig. 2, cyan line) localizes precisely and specifically to the drusen and also a diffuse region (upper left Fig. 1, abundance C4) with a peak wavelength of 510 nm. A spectrum with this characteristic peak and shape was recovered from all drusen and variably from diffuse regions in 10 of the 11 locations, with one spectrum peaking at 490 nm. Further, the dominant fluorophore (spectrum C1 and abundance C1 herein) consistently shows moderate co-localization with drusen, indicating this fluorophore overlying as granules and/or within drusen. This fluorophore spectrum is remarkably blue-shifted about 50 nm from the corresponding dominant fluorophore spectrum recovered from previously reported normal donors .
With hyperspectral AF image analysis, we found a single spectral AF signature for drusen and diffuse sub-RPE deposits in AMD that appears to be highly sensitive for drusen. The possibility that this signal also localizes to basal linear deposit is being addressed. A second drusen-associated AF spectral signature shares commonalities with RPE lipofuscin. These hyperspectral AF characterizations of drusen may aid the molecular understanding of AMD.