FA-DF imaging was performed in mice under general anesthesia using a 10:1 ketamine–xylazine mixture as above with the eyes kept moist during imaging using balanced salt solution (BSS; Alkon Laboratories Inc; Fort Worth, TX, USA) to avoid corneal dryness. The pupils were dilated prior to anesthesia with tropicamide ophthalmic solution 0.5% (Akorn Inc.; Lake Forest, IL, USA). Baseline images were acquired in each mouse within 1 min before (nominal time zero) intravenous injection of a mixture of two fluorescence-labelled dextrans of different molecular weight (M.W.) at 5 mg/mL each in saline solution, and time-lapse fluorescence imaging of the central and peripheral retina vasculature was initiated immediately after repositioning the mouse on the stage of a clinical-grade Heidelberg Engineering Multiline HRA + OCT SN 2884 imaging system (Heidelberg Engineering Inc.; Franklin, MA, USA). We used 500-kDa fluorescein isothiocyanate (FITC)-labeled dextran (Cat # MFCD00132418; Sigma Aldrich; St. Louis, MO, USA) and 2000-kDa tetramethyl rhodamine (TRITC)-labeled dextran (Cat #D7139; ThermoFisher Scientific; Waltham, MA USA). In each mouse, images were acquired every 5 min after the injection for 30 min. Image pairs (1536 × 1636 pixels, 96 dpi, 24 bit) were acquired in the green and red channels by alternating between the appropriate instrument’s filter cubes (FA-PB for green and LWP542 for red) at every time-point. The same instrument settings (e.g., gain, exposure time, etc.) were used during each imaging session and throughout the longitudinal studies. After imaging, the mouse eyes were covered with a thin layer of antibiotic ophthalmic ointment (neomycin, polymyxin B sulfates, and dexamethasone; Bausch & Lomb Pharmaceuticals; Tampa, FL, USA) to prevent eye infection and drying of the cornea during recovery from anesthesia.
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