Hyperspectral Autofluorescence Imaging of Drusen and Retinal Pigment Epithelium in Donor Eyes With Age-Related Macular Degeneration

Abstract

Purpose To elucidate the molecular pathogenesis of age-related macular degeneration (AMD) and interpretation of fundus autofluorescence (AF) imaging, we identified spectral AF characteristics of drusen and retinal pigment epithelium (RPE) in donor eyes with AMD. Methods Macular RPE/Bruch’s membrane (BrM) flat mounts were prepared from 5 donor eyes with AMD. In 12 locations (1–3/eye), hyperspectral AF images in 10-nm-wavelength steps were acquired at 2 excitation wavelengths (λex 436 nm, 480 nm). A non-negative tensor factorization algorithm was used to recover 5 abundant emission spectra and their corresponding spatial localizations. Results At λex 436 nm, we consistently localized a novel spectrum (SDr) with a peak emission near 510 nm in drusen and sub-RPE deposits. Abundant emission spectra seen previously (S0 in BrM and S1, S2, and S3 in RPE lipofuscin/melanolipofuscin (L/ML), respectively), also appeared in AMD eyes, with the same shapes and peak wavelengths as in normal tissue. L/ML spectra localizations in AMD eyes varied widely in their overlap with drusen, ranging from none to complete. Conclusions An emission spectrum peaking at ~510 nm (λex 436 nm) appears to be sensitive and specific for drusen and sub-RPE deposits. One or more abundant spectra from RPE organelles exhibit characteristic relationships with drusen.

Fundus Autofluorescence Lifetimes and Spectral Features of Soft Drusen and Hyperpigmentation in Age-Related Macular Degeneration

“…This confirms earlier studies that found shorter emission peak wavelengths for sub-RPE deposits, drusen, and Bruch's membrane compared to RPE in confocal microscopy of histologic sections of donor eyes. 19,20 Also, widefield epifluorescence microscopy of RPE/Bruch's membrane flat mounts showed shorter emission wavelengths for drusen than for the RPE. 20 Nonnegative tensor factorization 21 revealed a drusen-specific spectrum.…”

Repeatability and reliability of quantitative fundus autofluorescence imaging in patients with early and intermediate age‐related macular degeneration

“…The major fluorophores of the human macula are thus currently unknown and are subject to research (Tong et al. ). In vivo lipofuscin is excited by wavelengths of light ranging from 400 to 590 nm, with a peak between 490 and 510 nm.…”

“…Fundus autofluorescence (AF) of lipofuscin has long been thought to originate from bisretinoid fluorophores in the RPE cells, particularly A2E Yamamoto et al 2012), but more recently this most studied bisretinoid (and hence its derivatives) has been found in low quantities in the macula of humans (Ablonczy et al 2013). The major fluorophores of the human macula are thus currently unknown and are subject to research (Tong et al 2016). In vivo lipofuscin is excited by wavelengths of light ranging from 400 to 590 nm, with a peak between 490 and 510 nm.…”

Spectral analysis of fundus autofluorescence pattern as a tool to detect early stages of degeneration in the retina and retinal pigment epithelium

“…In addition, other components of cells, including flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide phosphate (NADPH), and the carotenoids zeaxanthin and lutein, have weak fluorescence in the same spectral area as LGs [7][8][9]. In addition, spectral autofluorescence characteristics of drusen have been observed in donor eyes with AMD [10].…”