Fluorescence Lifetime Imaging Ophthalmoscopy of the Retinal Pigment Epithelium During Wound Healing After Laser Irradiation

PURPOSE: To investigate the change in fluorescence lifetime of retinal pigment epithelium (RPE) after laser irradiation by using an organ culture model. METHODS: Porcine RPE-choroid-sclera explants were irradiated with selective retina treatment laser (wavelength: 527 nm, beam diameter: 200 μm, energy: 80–150 μJ). At 24 and 72 hours after irradiation, the mean fluorescence lifetime (τ(m)) was measured with fluorescence lifetime imaging ophthalmoscopy (FLIO) (excitation wavelength: 473 nm, emission: short spectral channel: 498-560 nm, long spectral channel: 560–720 nm). For every laser spot, central damaged zone (zone 1: 120 × 120 μm), area including wound rim (280 × 280 μm except zone 1), and environmental zone (440 × 440 μm except zone 1 and 2) were analyzed. Peripheral zone at a distance from laser spots longer than 2000 μm was examined for comparison. Cell viability was evaluated with calcein-acetoxymethyl ester and morphology with fluorescence microscopy for filamentous-actin. RESULTS: The RPE defect after selective retina treatment was mostly closed within 72 hours. FLIO clearly demarcated the irradiated region, with prolonged τ(m) at the center of the defect decreasing with eccentricity. In short spectral channel, but not in long spectral channel, τ(m) in the environmental zone after 72 hours was still significantly longer than in the peripheral zone. CONCLUSIONS: FLIO may clearly demarcate the RPE defect, demonstrate its closure, and, moreover, indicate the induced metabolic changes of surrounding cells during wound healing. TRANSLATIONAL RELEVANCE: This ex vivo study showed that FLIO may be used to evaluate the extent and quality of restoration of the damaged RPE and to detect its metabolic change in human fundus noninvasively.