In Situ Morphologic and Spectral Characterization of Retinal Pigment Epithelium Organelles in Mice Using Multicolor Confocal Fluorescence Imaging.

Purpose: To investigate the major organelles of the retinal pigment epithelium (RPE) in wild-type (WT, control) mice and their changes in pigmented Abca4 knockout (Abca4-/-) mice with in situ morphologic, spatial, and spectral characterization of live ex vivo flat-mounted RPE using multicolor confocal fluorescence microscopy (MCFM). Methods: In situ imaging of RPE flat-mounts of agouti Abca4-/- (129S4), agouti WT (129S1/SvlmJ) controls, and B6 albino mice (C57BL/6J-Tyrc-Brd) was performed with a Nikon A1 confocal microscope. High-resolution confocal image z-stacks of the RPE cell mosaic were acquired with four different excitation wavelengths (405 nm, 488 nm, 561 nm, and 640 nm). The autofluorescence images of RPE, including voxel-by-voxel emission spectra, were acquired and processed with Nikon NIS-AR Elements software. Results: The 3-dimensional multicolor confocal images provided a detailed visualization of the RPE cell mosaic, including its melanosomes and lipofuscin granules, and their varying characteristics in the different mice strains. The autofluorescence spectra, spatial distribution, and morphologic features of melanosomes and lipofuscin granules were measured. Increased numbers of lipofuscin and reduced numbers of melanosomes were observed in the RPE of Abca4-/- mice relative to controls. Conclusions: A detailed assessment of the RPE autofluorescent granules and their changes ex vivo was possible with MCFM. For all excitation wavelengths, autofluorescence from the RPE cells was predominantly contributed by lipofuscin granules, while melanosomes were found to be essentially nonfluorescent. The red shift of the emission peak confirmed the presence of multiple chromophores within lipofuscin granules. The elevated autofluorescence levels in Abca4-/- mice correlated well with the increased number of lipofuscin granules.