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Yoko Miura, Natalie Blimke, Katharina Kern, Daniel Weng, Joachim Pruessner, Gereon Huttmann, Ralf Brinkmann; Fluorescence lifetime measurements during laser-induced wound healing of retinal pigment epithelial cells. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4876.
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© ARVO (1962-2015); The Authors (2016-present)
The fluorescence lifetime (FLT) is an imaging parameter that may provide information regarding anatomical and metabolic status of living tissues. Purpose of this study is to investigate FLT of ex-vivo retinal pigment epithelium (RPE) after thermal laser irradiation using fluorescence lifetime imaging ophthalmoscopy (FLIO).
Porcine RPE-Bruch’s membrane-choroid explants were set in ring holders, placed in a custom-made artificial eye filled with phosphate buffered saline (PBS). Laser irradiation was performed using a continuous wave laser (λex = 532 nm) with a spot diameter of 200 μm, irradiation duration of 100 ms, and a power from 50 to 150 mW. After laser irradiation the tissues were maintained in the culture medium at 37°C in a 5% CO2 incubator. At 1, 24 and 48 h after irradiation FLT was measured at 473 nm excitation with a prototype Fluorescence Lifetime Imaging Ophthalmoscope (FLIO) build by Heidelberg Engineering GmbH. It comprises a scanning laser Ophthalmoscope with a fluorescence lifetime imaging system using a time-correlated single photon counting method. Following FLT measurements, cell morphology was investigated by staining cellular F-actin using FITC-phalloidin.
F-actin staining showed RPE destruction already 1 h after irradiation with the lowest power (50 mW). The damaged area increased power-dependently. One hour after irradiation, only subtle autofluorescence (AF) but apparent FLT alterations were visible at the spots irradiated with higher powers (>80-100 mW, varied dependent on pigmentation), where the mean FLT (τm) was significantly increased in the damaged region (from around 200 ps to over 2000 ps). Spots after lower power irradiations were not detectable both with AF and FLIO after 1 h. After 48h, F-actin staining showed that all laser wounds were under wound healing processes, where the defect after irradiations with 50-80 mW had been closed, whereas the wounds made with higher powers were still open. While AF alterations were rarely detectable in all spots, FLT clearly visualized all irradiated regions, even where the defect was already closed. Center of the spots showed the longest FLT, surrounded by a ring region with the intermediate length of FLT between the central region and the surroundings, whose width increased over time.
FLIO might non-invasively detect the RPE cells in the process of wound healing.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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