The maximum visual angle of the fundus scan of the SLO was measured to be 51°. In converting visual angles to retinal distances, we initially adopted a conversion factor of 34 μm/deg, corresponding to a posterior nodal distance (PND) of 1.95 mm.15 (link) However, as the contact lens causes the nodal point of the combined contact lens and mouse eye optical system to shift to a position more anterior than the nodal point of the eye alone, we compared overlaid SLO fundus images (centered on the optic nerve) of the retinal vasculature with flat-mount explants of the retinas of the same eyes (Supplementary Material I ; Fig. 2 A). We determined the calibration factor with the contact lens to be 43 μm/deg, a 26% linear expansion of the image field over that calculated with the standard PND. The standard x-y scan mapped to 256 × 256 pixels, with a sampling duration of 6 μs/pixel and a total measured light exposure duration of 0.461 second. The latter value includes 17.2% “flyback” time during which the galvanometer mirror was returning to its initial scan position while scanning back across the tissue. Initial alignment of the dark-adapted mouse was performed with near-infrared (NIR) light (700 ± 20 nm, 5 μW). Before and after each experiment a reference SLO “background” image was recorded for each bleaching location with the imaging/bleaching lights on, but without the mouse present in the imaging system. These reference data were subtracted from the experimental data obtained with the mouse in place to ensure that no light backscattered from the optical elements of the imaging system affected the retina reflectance data.
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