Resonance Raman microscopy of rod and cone photoreceptors.

Abstract

A Raman microscope that has enabled resonance Raman vibrational spectral to be obtained from single photoreceptor cells was constructed. The laser beam which excites the Raman scattering is focused on the outer segment of the photoreceptor through the epiillumination system of a light microscope. Raman scattering from the visual pigment in the photoreceptor is collected by the objective and then dispersed onto a multichannel detector. High-quality spectra are recorded easily from individual outer segments that are 5 .times. 50 .mu.m in size; spectra from cells as small as 1 .times. 10 .mu.m were obtained. Photostationary steady-state mixtures in pigments from toad (Bufo marinus) and goldfish (Carassius auratus) photoreceptors frozen in glycerol glasses at 77.degree. K were studied. Comparison of toad red rod spectra with previously published spectra of bovine rod pigments demonstrates that the conformation of the chromophore in the 1st photointermediate, bathorodopsin, is sensitive to variations in protein structure. The first photointermediate in the goldfish rod photostationary steady-state was studied. This bathoporphyropsin has a much lower ethylenic stretching frequency (1507 cm-1) than that observed in the toad and bovine bathoproducts (.apprx. 1535 cm-1). Preliminary results on goldfish cone pigments are reported. These are the first vibrational studies on vertebrate photoreceptors responsible for color vision.