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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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Measuring the spatial distribution of multiply scattered light using a de-scanned image sensor for examining retinal structure contrast
An optical platform is presented for examining intrinsic contrast detection strategies when imaging retinal structure usingex vivotissue. A custom microscope was developed that scans intact tissue and collects scattered light distribution at every image pixel, allowing digital masks to be applied after image collection. With this novel approach at measuring the spatial distribution of multiply scattered light, known and novel methods of detecting intrinsic cellular contrast can be explored, compared, and optimized for retinal structures of interest.
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- Award ID(s):
- 1845801
- PAR ID:
- 10208684
- Publisher / Repository:
- Optical Society of America
- Date Published:
- Journal Name:
- Optics Express
- Volume:
- 29
- Issue:
- 2
- ISSN:
- 1094-4087; OPEXFF
- Format(s):
- Medium: X Size: Article No. 552
- Size(s):
- Article No. 552
- Sponsoring Org:
- National Science Foundation
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