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Yuehong Tong, Jennifer H. Acton, Jilin Zhou, Janet R. Sparrow, Theodore Smith; Hyperspectral Imaging of RPE Fluorophores in vitro. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3087.
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Lipofuscin is deposited in the retinal pigment epithelium (RPE) in normal eyes, and is an important disease marker. We used hyperspectral autofluorescence (AF) imaging to characterize these fluorophores in vitro.
RPE was studied in two forms: a) non-diseased retinal tissue from an elderly male donor was fixed and sectioned for microscopic examination; and b) ARPE-19 cells that had accumulated A2E were plated and fixed. Hyperspectral AF images with blue light excitation were captured using the Nuance FX camera (Caliper Life Sciences, US) at wavelengths between 420 and 720nm in 10 nm intervals, then mathematically unmixed into background and RPE-specific spectra.
The tissue section hyperspectral composite image (Fig 1A) shows green background AF primarily due to collagen in the choroid and the RPE lipofuscin AF as an orange band. Unmixing of the hyperspectral signal gave the spectral component images (Fig 1B & C). Figure 1D shows the spectral signature of the separate components, in particular reproducing that of lipofuscin. For the cell culture model, hyperspectral unmixing of the raw image showed A2E localizing around the nuclei in RPE cells (Fig 2), with its characteristic spectrum.
We applied hyperspectral imaging to characterize the bisretinoid fluorophores of the RPE, in one case the complex mixture of lipofuscin and in the other, pure A2E. In each case, the spectra recovered agreed with published results and colocalized correctly. This is a promising technique to investigate complex fluorophore biomarkers, and one which can also be adapted to in vivo imaging for non-invasive research on the role of these compounds in retinal disease.
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