Characterization of laser-induced fluorescence in the canine retinal pigment epithelium

The adult retinal pigment epithelium (RPE) exhibits fluorescence caused by various cytoplasmic flurophors and by lipofuscin. Microscopic investigations of fluorescence generally utilize continuum excitation sources (xenon, mercury) and varying filter combinations. Consequently, discrepant emission characteristics are often encountered in the literature. In the present study, an epi-fluorescence microscope linked to a laser source with defined excitation characteristics was used to examine fluorescence in normal, adult canine RPE. Excitation at 337 nm produced maximal fluorescence at 450 nm (blue), while excitation at 439 nm induced 550 nm (yellow) fluorescence. Continuous exposure of cells to 439 nm light caused an initial rapid reduction of intensity, with a subsequent slower decrease of the signal. After solvent extraction, fluorescence at 600 and 650 nm showed no major alterations relative to the values recorded at 550 nm. Our findings demonstrate that varying excitation wavelengths produce discrepant fluorescence data from RPE cells and indicate caution in direct comparisons between experiments where different excitation sources and filter combinations are used.