Two-photon autofluorescence lifetime assay of rabbit photoreceptors and retinal pigment epithelium during light-dark visual cycles in rabbit retina.
Trung Duc NguyenYuan-I ChenAnh-Thu NguyenSiem YonasManasa P SripatiYu-An KuoSoonwoo HongMitchell LitvinovYujie HeHsin-Chih YehH Grady RylanderPublished in: Biomedical optics express (2024)
Two-photon excited fluorescence (TPEF) is a powerful technique that enables the examination of intrinsic retinal fluorophores involved in cellular metabolism and the visual cycle. Although previous intensity-based TPEF studies in non-human primates have successfully imaged several classes of retinal cells and elucidated aspects of both rod and cone photoreceptor function, fluorescence lifetime imaging (FLIM) of the retinal cells under light-dark visual cycle has yet to be fully exploited. Here we demonstrate a FLIM assay of photoreceptors and retinal pigment epithelium (RPE) that reveals key insights into retinal physiology and adaptation. We found that photoreceptor fluorescence lifetimes increase and decrease in sync with light and dark exposure, respectively. This is likely due to changes in all-trans-retinol and all-trans-retinal levels in the outer segments, mediated by phototransduction and visual cycle activity. During light exposure, RPE fluorescence lifetime was observed to increase steadily over time, as a result of all-trans-retinol accumulation during the visual cycle and decreasing metabolism caused by the lack of normal perfusion of the sample. Our system can measure the fluorescence lifetime of intrinsic retinal fluorophores on a cellular scale, revealing differences in lifetime between retinal cell classes under different conditions of light and dark exposure.
Keyphrases
- diabetic retinopathy
- optical coherence tomography
- optic nerve
- single molecule
- energy transfer
- high throughput
- high resolution
- endothelial cells
- stem cells
- mesenchymal stem cells
- bone marrow
- cell proliferation
- living cells
- oxidative stress
- photodynamic therapy
- signaling pathway
- case control
- induced pluripotent stem cells
- fluorescent probe