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P. J. Coffey, N. Patel, P. Lundh von Leithner; Spectral Profiling of Pigments of the Retinal Epithelium in Excised Human Donor Tissue Using Confocal Microscopy. Invest. Ophthalmol. Vis. Sci. 2009;50(13):6216.
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Derive optimal excitation and emission bands for multimodal ultrastructural investigation of retinal pigment epithelium (RPE), with particular emphasis on correlation of in vivo and histological data.
Lipofuscin fluorescence emission spectra and melanin absorption spectral functions were derived from flatmounted sections of intact retinae (n=4) from frozen human post-mortem eyes (>60 years of age) with no known ocular disease. Tissue samples were spectrally profiled and results were compared with in vivo high magnification autofluorescence images of Macaque RPE.
RPE cell and cell matrix morphology became increasingly visible as excitation and emission bands were shifted towards longer wavelengths (figure). However, autofluorescence emission spectra were not found to change across the surface of the RPE cell. Fixation of tissues for histological investigation shifted and broadened RPE related fluorescence emission spectra. Fixation of donor tissue with 4% paraformaldehyde resulted in a shift of the peak AF emission by -50nm and emission its profile was broadened. Measuring the RPE AF when the inner retina is removed from the RPE/Choroidal tissue samples results in a shift towards longer wavelengths.
This study shows that multimodal structural imaging of the RPE, using endogenous fluorescence emission is best done using longer wavelength excitation at 633nm with corresponding shift in AF emission bandpass filtering, due to reduced melanin absorption, and tissue scatter. 633nm wavelength excitation also allows for higher levels of irradiation without risk for ocular damage.
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