Autofluorescence of Visual Receptors

Abstract

SINCE 1878 when Kühne reported the faint bluish fluorescence of the dark adapted retina and its brighter greenish supplementation when the visual pigments were bleached¹, these observations have been little used or extended^2,3. Early in 1966, during another investigation, one of us noticed the interesting microscopic autofluorescence pattern shown in Fig. 1. This led us to similar observations on a number of retinas. Pieces of dark adapted retina (4 × 4 mm) placed receptor side up in a depression slide in Ringer's solution were examined in a fluorescence microscope equipped for infrared viewing; ultraviolet excitation was provided by a GE 1000 W AH6 Hg lamp plus a B and L 250 mm grating monochromator set to 366 nm giving a band pass of about 13 nm. Abbe illumination was provided through a 10 × 0.20 NA achromatic objective in place of a condenser. To avoid bleaching, the microscope was focused (at a magnification of × 50–100) using infrared illumination before exposure to ultraviolet light was begun. Several layers of ‘Wratten 2C’ filter served to block transmission of ultraviolet light to the observer's eye and film plane. Photography was performed directly through a 16 × 0.4 NA or a 25 × 0.60 NA objective without an ocular in order to maximize photon flux density at the film plane, thus minimizing exposure. Polaroid type 47, ASA 3000, black-and-white film and Kodak Ektochrome, ASA 125, colour film were used. A photographic shutter in the illumination optics could be opened for viewing fluorescence or to provide appropriate exposure times for photography. The shutter was closed at all other times to minimize ultraviolet bleaching of visual pigment or photo-inactivation of fluorescence. Photographic exposure times ranged from 2 s to 30 s.