Multicolor confocal fluorescence microscopy provides the enhanced visualization and fluorescent emission properties of intrinsic fluorophores in retinal pigment epithelium cells

Abstract

Purpose : To investigate the intrinsic autofluorescent (AF) granules in the retinal epithelium (RPE) cells of mouse retina and their AF emission properties using multicolor confocal fluorescent microscopy (MCFM). Compare the in vivo recorded spectra with ex vivo measured spectra with MCFM for the mouse model of Stargardt’s disease (Abca4^-/- [129S4]) and agouti WT control.

Methods : In situ imaging of RPE flat-mounts from agouti Abca4^-/- (129S4) and agouti WT (129S1/SvlmJ) controls, 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 (405nm, 488nm, 561nm, and 640nm). In vivo AF spectra for same four excitation wavelengths are acquired with a spectrometer-integrated SLO system from both mice strains. The autofluorescence images of RPE, including voxel-by-voxel emission spectra, were acquired, and processed with Nikon NIS-AR Elements software.

Results : MCFM provided an enhanced visualization of the RPE cell mosaic, including melanosomes and lipofuscin granules, and their variations in Abca4^-/- and agouti WT control mice. MCFM allowed the extraction of AF emission spectrum from the individual granules. Acquired in vivo spectra are compared with the ex vivo measured spectra for both mice strains.

Conclusions : MCFM can delineate individual RPE autofluorescent granules and provide their characteristics AF emission spectra. Lipofuscin granules and melanosomes are the two major pigmented granules. Lipofuscin granules are identified as the major source of AF from RPE whereas, melanosomes completely lack fluorescence in visible excitation ranges. The in vivo acquired spectra are in good agreement with the ex vivo spectra. This comparison also revealed the source of FAF and their changes in disease model.

This is a 2021 ARVO Annual Meeting abstract.