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Jacob Rosenbloom, Yuehong Tong, Taariq Mohammed, Nayanika Challa, R Theodore Smith; Classification of Fluorophore Hyperspectral Signatures in Canine Best Disease. Invest. Ophthalmol. Vis. Sci. 2019;60(9):206.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations in the BEST1 gene cause bestrophinopathies in both humans and canines. These lesions share certain features in the retina across species. Hyperspectral autofluorescence (AF) imaging in humans ex vivo has identified particular molecular signatures in both normal eyes and those with age-related macular degeneration (AMD). Analysis of spectra and distribution of fluorophores in both canine Best (cBest) disease and normal canine donor eyes can elucidate understanding of retinal physiology of both species.
Thirty one images were prepared from fifteen tissue cross sections generated from three cBest specimens and one normal canine specimen. The images were captured using 40X field hyperspectral AF imaging (Figure 1d). Excitation wavelengths of 436, 480, and 505 nm were used with emissions collected from 420 nm to 720 nm, and the resulting multi-excitation hyperspectral data was unmixed using a non-negative matrix factorization into fluorophore emission spectra and spatial distributions.
Two of the RPE fluorophore spectral profiles recovered in cBest and normal canine (Figure 1a, spectra C1 and C2) match those previously described in normal human eyes as S1 and S2 (Ben Ami et al., PMID 27226929). A profile recovered almost exclusively in cBest specimens matches the S3 peak previously described in normal human eyes (Figure 2a, spectra C3). In all samples, the S1-like peak is represented by a broad peak with maximal height at 520 - 530 nm. S2-like peak is represented by a bimodal peak with maximal height at 580 nm. S3-like peak is defined by a trimodal peak with relative maxima at 630 and 650.
The presence of S3-like autofluorescent spectral peaks in the cBest specimens when compared to normal canine specimen suggests the role of melanolipofuscin accumulation in the progression of Best in canines. Due to the similarity of S2-like and S3-like peaks to S2 and S3 peaks found in human RPE (Ben Ami et al., PMID 27226929), this finding may provide key insight into the physiology of human retinal diseases.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
Figure 1: The image, spectra, and abundances of a normal canine specimen AF image decomposed with non-negative factorization.
Figure 2: cBest Spectra C1, C2, C3. Abundance C1: BrM, collagen and PR outer segments. Abundance C3: specifically RPE and subretinal granules. Abundances C1 and C3: color-coded blue and red and overlaid. Note mutually exclusive localizations.
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