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J. Qu, D. Chen, G. Xu, Y. Sun, H. Niu; Autofluorescence Diagnostic Spectro–Microscopy for Light–Induced Damage of Retinal Pigment Epithelium . Invest. Ophthalmol. Vis. Sci. 2006;47(13):3301.
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© ARVO (1962-2015); The Authors (2016-present)
Gradual vision impairment caused by light–induced damage is invisible, which often leads to irreversible vision loss before clinical diagnosis and treatment. The purpose of this work is to differentiate the damaged retinal pigment epithelium (RPE) cells with high spatial and spectral resolutions using a novel autofluorescence diagnostic spectro–microscopy and discuss the potential applications of this method in clinics.
Porcine eyes were enucleated 4 hours after death and preserved at 4°C before experiments. 1cm–diameter RPE–choroid–sclera complex near the optic nerve was cut off by trepanation. Light–induced damage was produced by an argon ion laser (=488nm) focused on the RPE layer, and damage degree was controlled by the exposure time. Then, the autofluorescence spectrum of the damaged RPE cells was measured by a single–photon excitation confocal microscopy system with the same laser.
Light–induced morphological and spectral change of RPE cells was dependent on the exposure time. Before exposure to the laser light, cells were distributed orderly and there was a fluorescence emission peak near 604–624nm. With a very brief exposure, light–induced damage was slight, and the diameter of cells increased a little. Although the morphological change was imperceptible, the emission spectrum change was distinct. The original emission peak diminished, whereas a new peak longer than 750nm appeared. Spectrally resolved imaging from 610 to 690nm and 720 to 840nm provides a unique contrast mechanism between damaged and normal cells. When the exposure time was increased, light–induced damage became more severe and significant pigment accumulation was observed. The original emission peak almost disappeared, but the intensity of the new emission peak increased significantly. Damage degree can be evaluated from the intensity of the emission peak. The new emission peak indicated that a new fluorophore was produced during the exposure process.
Primary results suggested that the autofluorescence spectro–microscopy achieved detection of the light–damaged cells, and the intensity of the emission peak provided a criterion for evaluating the light–induced damage degree of RPE.
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