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Yoko Miura, Alessa Hutfilz, Britta Lewke, Carolina Coelius, Salvatore Grisanti, Ralf Brinkmann; Fluorescence Lifetime Imaging Ophthalmoscopy of ex-vivo retinal pigment epithelium after selective laser irradiation. Invest. Ophthalmol. Vis. Sci. 2018;59(9):5855. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Fluorescence lifetime (FLT) of intracellular molecules may indicate the metabolic status of cells and tissues. Fluorescence lifetime imaging ophthalmoscopy (FLIO) was introduced as the novel method that enables the measurement of the FLT of the human fundus. In this study, the FLT of the ex-vivo retinal pigment epithelium (RPE) after selective RPE laser irradiation was investigated.
RPE-sclera explants from freshly enucleated porcine eyes were used. The explants were maintained in a culture medium under 5% CO2 at 37°C. The explants were irradiated with a SRT (selective retina therapy) laser (Medical Laser Center Luebeck, 527 nm, pulse duration: 1.7 µs, repetition rate: 100 Hz, irradiation time: 300 ms, spot diameter: 200 µm). In one tissue, the RPE was irradiation with a few parallel columns of a pulse energy sequence of 80-150 uJ (10 µJ increment) with 800-1000 µm distances between columns. The FLT was measured with a FLIO prototype system (Heidelberg Engineering GmbH) at 24h and 72h after laser irradiation. Following the last FLIO measurement at 72h the cell viability and morphology were investigated with Calcein-AM staining.
At 24h as well as 72h after laser irradiation, mean FLT (tm) of the RPE around the laser spots (up to 2000 µm away) were significantly longer than the FLT of the RPE in the farther region, while the intensity of the autofluorescence showed no differences. In particular, the FLT of the RPE between laser spots was strongly extended in dependence with the irradiation energy with which the RPE nearby was irradiated (tm after 24h: outside area: 251.8± 15.5 ps, area between 100 µJ-spots: 384.8± 21.1 ps, area between 150 µJ-spots: 419.2± 25.6 ps. P<0.01). Although the difference became smaller after 72h, there were still significant differences in tm between non-irradiated area and the area around irradiation (tm after 72h: outside area: 368.6 ± 29.3 ps, area between 100 µJ-spots: 414.3± 30.5 ps, area between 150 µJ-spots: 460.2± 20.6 ps, p<0.05). Calcein-AM tests proved the RPE cell viability at 72h post irradiation and narrowing the RPE defect at the laser spots.
FLIO revealed the apparent extension of the FLT of the RPE even located far away (a few millimeter) from the laser spots after selective laser irradiation. It suggests that FLIO might indicate the change in metabolic activity of the RPE after laser treatment.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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