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Joachim Pruessner, Natalie Blimke, Lars Alt, Gereon Huttmann, Ralf Brinkmann, Yoko Miura; Fluorescence Lifetime Imaging Ophthalmoscopy of in-vitro retinal pigment epithelial cells under different glucose conditions. Invest. Ophthalmol. Vis. Sci. 2016;57(12):266.
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© ARVO (1962-2015); The Authors (2016-present)
Fluorescence lifetime imaging ophthalmoscopy (FLIO) is a new method for measuring fluorescence lifetimes (FLT) of endogenous fundus autofluorescence (AF). The metabolically active retinal pigment epithelium (RPE) may largely contribute to the fundus AF signal, and thus it is of great importance to elucidate its FLT characteristics under different pathological conditions. The aim of this study is to utilize the FLIO system to investigate in-vitro RPE cells cultivated under different glucose conditions.
Second passage confluent porcine RPE cells cultured on 12-mm Transwell® membrane inserts were used for the study. The cells were cultured under glucose concentrations of 1000 (low glucose: LG), 4500 (normal glucose: NG) or 9000 (high glucose: HG) mg/L for 6 days. FLT was measured on day 3 and day 6, using a prototype system of FLIO (Heidelberg Engineering), which is based on a laser scanning ophthalmoscope with excitation at 473 nm and two emission spectral channels (ch. 1: 498-560 nm, ch. 2: 560-700 nm). Measurements were performed by placing the membrane insert into a custom-made artificial eye model. RPE cell viability was evaluated using calcein-AM cell viability indicator.
There was no significant change in RPE cell viability among different glucose conditions. A biexponential fit was performed for FLT decay analysis, thus each FLT consists of the short (t1) and long (t2) components. Under LG and HG conditions, elongation of t1 and t2 was typically seen (e.g. in ch. 1 on day 3, t1 was 355 ps ± 6.6, 366 ps ± 12, and 368 ps ± 16, and t2 was 2101 ps ± 64, 2123 ps ± 129 and 2179 ps ± 100, under NG, LG and HG conditions, respectively). The apparent difference between LG and HG conditions was shown in tm (mean FLT) of both channels on both days, where tm under LG is significantly longer than under other conditions. Regarding component contribution ratio (a1/a2), LG and HG conditions led to the significantly higher a1/a2 of ch. 2 on day 6 (2.43±0.03 and 2.57±0.22, respectively), compared to the NG condition (2.15±0.18).
Our original setup is useful for measuring FLT of cultivated RPE cells while maintaining cell viability. The results suggest that glucose concentration-induced alterations of RPE metabolic status can result in change of the length and contribution of autofluorescence lifetime components, which can be detected by using FLIO.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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