ABSTRACT We present an innovative transscleral imaging technique to obtain high‐quality, non‐destructive images of the retinal pigment epithelium (RPE) of enucleated eyes. This approach combines confocal microscopy with fluorescence lifetime imaging to assess both morphological and fluorescence lifetime characteristics of the RPE in murine models. Unlike traditional transpupillary methods, our transscleral approach significantly reduces autofluorescence interference from the cornea and lens, providing clearer images and accurate fluorescence lifetime measurements. Using a continuous‐wave 405 nm laser, we achieved high‐resolution imaging of the RPE without the need for complex pulsed laser setups. Morphological characterization demonstrated that the transscleral approach effectively visualizes hexagonal, mono‐ and binucleated RPE cells with clear delineation. Fluorescence lifetime analysis using pulsed 405 nm laser revealed distinct autofluorescent signatures corresponding to putative endogenous fluorophores. Notably, fixed tissue samples showed altered fluorescence lifetimes compared to fresh samples, highlighting the influence of preservation on biochemical assessment. Quantitative fluorescence lifetime data revealed differences between autofluorescent granules and the surrounding cytoplasm, indicating the potential to obtain information about changes in the cellular chemical environment. Our results suggest that transscleral FLIM imaging is a viable tool for non‐destructive RPE analysis, offering potential applications in studying retinal diseases.
Dominguez et al. (2026) studied this question.