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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.
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Probing the Requirements of a Bruch's Membrane for Retinal Pigment Epithelial Cells
Age related macular degeneration (AMD) is the leading cause of blindness in developed countries. AMD occurs due to dysfunction of the retinal pigment epithelial (RPE) cell basement membrane, the Bruch’s membrane. Previous work in the lab demonstrated that retinal pigment epithelial cells preferred stiff substrates to soft ones, and that RGD-conjugated polyethylene glycol (PEG) hydrogels alone were not sufficient to support long term RPE cell health. There is evidence that epithelial and neural cells prefer laminin-derived peptides over fibronectin-derived peptides. Therefore, we examined the fate of RPE cells when seeded on PEG hydrogels conjugated with synthetic laminin peptides.
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- Award ID(s):
- 1752079
- PAR ID:
- 10139164
- Date Published:
- Journal Name:
- Biomedical Engineering Society
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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