Retinas were stained by DAPI (4,6 diamidino-2-phenylindole; Sigma-Aldrich, Poole, UK) solution (3 μg/mL in PBS) overnight at 4°C and wholemounted with the RGC side uppermost in a hard-set mounting medium for fluorescence (Vectashield; Vector Laboratories, Burlingame, CA). The specimen was placed on a motorized microscope stage that allowed the selection of any retinal location to an accuracy of 1 μm. DAPI molecules were excited with a Ti-Sapphire multiphoton laser at 843 nm (Tsunami; Newport SpectraPhysics GmBH, Darmstadt, Germany), and images were acquired (model TCS SP2; Leica, Milton Keynes, UK) with a confocal system (DMRE microscope with HCX PL Apo 40/1.25 NA oil CS objective lens; Leica). Laser power, gain, offset, and pinhole size of the confocal microscope were kept constant at each session, to facilitate comparisons between retinas.
To relate retinal disease to clinical data, a sampling grid, which retinotopically corresponded to the spacing stimuli in a 30-2 visual field analyser (Humphrey; Carl Zeiss Meditec, Oberkochen Germany) was used to define sample locations
(Fig. 1) .
15 16 To correlate anatomic positions on the retina to visual field, we converted the distance (in millimeters) to degrees of visual angle according to a nonlinear conversion of retinal magnification factor
17 (
y = 0.035
x 2 +3.4
x + 0.1, where
y is the eccentricity in arc degrees, and
x is in millimeters).