Confocal laser microscopy and Fluorescence lifetime Imaging microscopy of Retinal pigment epithelium of mice

DOMINGUEZ LUCÍA; TORRES RODRIGO; ETCHART JUAN; ADUR, JAVIER

Encuentro; ARVO 2022 -The Association for Research in Vision and Ophthalmology; 2022

An ex-vivo experiment was designed for developing and employing an imaging technique performed with a scanning laser confocal microscope for measuring fluorescent lifetime of endogenous fluorophores of the retinal mice.METHODS:The enucleated eyes of BALBC/c mice are used. They are placed on a slide and observed with a confocal laser scanning microscope, LSM 880 – Carl Zeiss, by a transscleral approach.This microscope is composed of continuous wave lasers from 405 nm to 613 nm which are used to analyze the intensity and location of the fluorescence signal for each wavelength, and a fluorescence lifetime imaging microscopy (FLIM) system, which is fitted by micropulse lasers of 405 and 440 nm, synchronized with a photomultiplier, and a time correlated single photon counting (TCSPC) device. That system works by exciting fluorophores and measuring their fluorescence lifetime.RESULTS:We obtained images of a monolayer of autofluorescent hexagonal cells with large nuclei compatible with the retinal pigment epithelium (RPE) using a 488 nm wavelength laser, as is shown in Fig. 1. The mean cell diameter is 5.3 mm and the nucleus diameter is 2.7 mm.The fluorescence lifetimes measured were 0.8 ns and 2.2 ns, with an excitation wavelength of 405 nm and an emission wavelength range of 465-515 nm, compatible with melanin granules andFlavin adenine dinucleotide(FAD) free, as is observed in Fig. 2.CONCLUSIONS:During the development of this setup for capturing images of the retina ex-vivo it was possible to penetrate sclera and chroroid with the scanning laser until reaching the RPE in the enucleated eyes. Images were taken and the fluorescence lifetime of the endogenous fluorophores was measured.The next step is to perform in-vivo experiments with anesthetized animals and mouse holders.