Using NIR reflectance only, a retinal location was first chosen that had minimal retinal vasculature and lay near the peak density of the rod photoreceptors (~15° from the fovea). The animal was then dark adapted for 60 minutes, after which pairs of 2-second videos were recorded simultaneously, one at 514 nm and the other at 794 nm. The first video captured the retinal reflectance before photopigment bleaching, the second occurred during the bleach, and subsequent videos tracked the reflectance changes during 35 minutes of dark adaptation. These videos were recorded at 0, 2.5, 5.0, 7.5, 10.0, 15.0, 20.0, 25.0, 30.0, and 35.0 minutes after the bleach. Each video corresponded to a square 2° × 2° retinal region. The rhodopsin bleaching exposure (ArKr, 514 nm) was limited to a one-third degree by one-half degree rectangular region at the center of the 2° AOSLO raster scan. This was achieved by modulating the 514-nm laser intensity with an acousto-optic modulator (AOM; Brimrose [Sparks Glencoe, MD, USA] model TEM-200-50-485/635-02FP attached with an OZ-Optics [Ottawa, ON, Canada] fiber coupler model HPUC-2A3A-400/700-P-4.5AC-15) set to maximum transmission in the central region to be bleached and zero elsewhere.
Using this method, the time course of dark adaptation was measured four times in each of eight retinal locations in one eye of each primate, for a total of 64 bleach and recovery measurements.