Identify and quantify candidate AF signals from BrM in RPE/BrM with flat-mounts of human donor eyes using ex vivo hyperspectral AF imaging and mathematical modeling.
Flat-mounts from 11 human eyes lacking chorioretinal pathology (6 donors <50 yrs, 5 >80 yrs) were prepared by removing the retina and choroid and studied at 3 locations with distinct photoreceptor content in overlying retina: fovea, perifovea (2-4mm superior to fovea), and periphery (10-12mm superior to fovea). RPE was further removed from a region at each location to provide 33 samples of isolated BrM for spectral microscopy (Zeiss Axio Imager A2 microscope (Carl Zeiss, Jena, Germany) with Plan-Apochromat objective optics (excitation: 430 nm; emission: long-pass fluorescence filter). Hyperspectral AF images were captured at emissions between 420 to 720 nm in 10 nm intervals using the Nuance FX camera (Caliper Life Sciences, US) and saved as data hypercubes with two spatial and one wavelength dimension.
Gaussian mixture modeling and mathematical factorization of the hypercubes were applied to extract 4 dominant BrM candidate spectra from each sample. Comparison with lipofuscin spectra independently obtained from these locations showed two shorter wavelength peaks that were unique to BrM, one always present near 533nm and another near 488nm that was present at a statistically significantly higher rate in the older donor populations (Fischer exact test, p = 0.0272) (table1). There was also a trend for the 488nm wavelength to be present more peripherally (table 2). Two other peaks were found near 600nm and 690nm. The mean values (nm) of peak centers were: Fovea: 698 ± 22.4, 605 ± 15.7, 534 ± 4.0, 489 ± 2.9 Parafovea: 688 ± 28.2, 603 ± 19.4, 536 ± 7.2, 492 ± 4.4 Periphery: 689 ± 12.6,, 602 ± 11.4, 534 ± 3.7, 489 ± 3.3
Candidate individual emission spectra for BrM suggest a population of fluorophors. A well-defined source with emission at 488nm appears to increase with age. Peaks at 600-690nm resemble those independently determined for RPE lipofuscin at the same locations. Whether these represent bis-retinoids requires further elucidation in tissues subject to lipid extraction. Biochemical identification of these species will be important in understanding BrM physiology in health and disease and for interpreting clinical hyperspectral imaging.