Duration 5min, Q&A 3min
Two-Photon Microscopy and Fluorescence Lifetime Imaging of Retinal Pigment Epithelium and Photoreceptor Outer Segment Under Lipid Peroxidation
Two-photon microscopy (TPM) with fluorescence lifetime imaging (FLIM) enables the discrimination of different autofluorescence (AF) molecules. Purpose of this study is to investigate the AF of retinal pigment epithelial (RPE) cells and photoreceptor outer segments (POS) under physiological conditions and under lipid peroxidation with TPM and FLIM.
Porcine RPE-choroid tissue explants and neural retina explants were used for this study.
Porcine RPE-choroid tissue explants and neural retina explants were placed in the culture medium. Lipid peroxidation was induced by exposure of tissues to FeSO4 (0, 1, 10 mM) for 1h or by laser photocoagulation (0.1 s, 200 μm, 60-80 mW). At the indicated time point tissues were investigated with TPM and FLIM. Intracellular formation of reactive oxygen species (ROS) and nitric oxide (NO) were detected using chloromethyl-2,7-dichlorofluorescein diacetate (CM-H2DCFDA) and 4,5-diaminofluorescein diacetate (DAF-2), repectively. Melanosomes were isolated from RPE cells and their fluorescence properties were investigated under physiological conditions and under oxidative stress conditions induced by FeSO4 (10 mM) or H2O2 (1 mM).
Results and Conclusion
TPM-AF in RPE cells is mostly originated from the melanosomes under physiological conditions, which has a very short fluorescence lifetime (FLT) (τmean=117 ps). FeSO4 exposure and laser photocoagulation lead to the appearance of bright granular AF inside and around RPE cells, whose FLT is significantly longer (τmean =1388 ps) than the FLT of the melanosome-AF. Excitation and emission peaks are found at 710-750 nm and 450-500 nm, respectively. These bright AF appear mostly in the CM-H2DCFDA and DAF-2-positive RPE cells. Mean FLT (τmean) of the POS are 1400 ps and the FeSO4 exposure increases the fluorescence intensity of POS-AF and shortens their FLT down to 395 ps. Oxidative stress increases the fluorescence intensity of the melanosomes but does not change their FLT.
TPM and FLIM reveal oxidative stress-induced bright AF in and around the RPE cells and increase of the intensity of the POS-AF, with a significant alteration of their FLT. The appearance of the bright AF granules in RPE cells seems to be related to the formation of intracellular ROS and NO.
[ Keyword ]
RPE / two-photon microscopy / fluorescence lifetime imaging / oxidative stress / photoreceptor outer segment
[ Conflict of Interest ]