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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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Assessing recovery of Drosophila melanogaster photoreceptors with different wavelengths of red and infrared light
ABSTRACT It has previously been shown that near-infrared light can positively affect the physiology of damaged tissue. This is likely mediated by the modulation of metabolic activity via cytochrome c oxidase (COX), the rate of ATP production and the generation of reactive oxygen species. It has been suggested that this process is influenced by the wavelength of near-infrared light, with different wavelengths having different efficacy. The impact of these effects on retinal health is not yet well understood. To answer this question, we first induced photoreceptor damage in the eyes of white mutant D. melanogaster through prolonged exposure to bright light. We then investigated the recovery of retinal health following exposure to different wavelengths of near-infrared light (670, 750, 810, 850 and 950 nm) over the course of 10 days. Retinal health was assessed through electroretinograms and fluorescence imaging of live photoreceptors. We found that all treatments except for 950 nm light facilitated the recovery of the electroretinogram response in previously light-damaged flies – though efficacy varied across treatments. All near-infrared light-exposed groups showed at least some improvement in retinal organization and auto-fluorescence compared with an untreated recovery control. We also showed that our results do not stem from a fly-specific artifact relating to opsin photoconversion. Finally, we made use of a bioassay to show enhanced ATP levels in light treatments. This study represents a much-needed direct comparison of the effect of light of a multitude of different wavelengths and contributes to an emerging body of literature that highlights the promise of phototherapy.
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- Award ID(s):
- 1856241
- PAR ID:
- 10610145
- Publisher / Repository:
- Company of Biologists
- Date Published:
- Journal Name:
- Journal of Experimental Biology
- Volume:
- 228
- Issue:
- 6
- ISSN:
- 0022-0949
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript
- Journal Article:
- https://doi.org/10.1242/jeb.250043
