Resonance Raman microscopy of rod and cone photoreceptors

We have constructed a Raman microscope that has enabled us to obtain resonance Raman vibrational spectra from single photoreceptor cells. 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 x 50 micrometer in size, and we have obtained spectra from cells as small as 1 x 10 micrometer. We have used the Raman microscope to study photostationary steady-state mixtures in pigments from toad (Bufo marinus) and goldfish (Carassius auratus) photoreceptors; these photoreceptors were frozen in glycerol glasses at 77 degrees K. Comparison of our toad red rod spectra with previously published spectra of bovine rod pigments demonstrates that the conformation of the chromophore in the first photointermediate, bathorhodopsin, is sensitive to variations in protein structure. We have also studied the first photointermediate in the goldfish rod photostationary steady-state. This bathoporphyropsin has a much lower ethylenic stretching frequency (1,507 cm-1) than that observed in the toad and bovine bathoproducts (approximately 1,535 cm-1). Preliminary results of our work on goldfish cone pigments are also reported. These are the first vibrational studies on the vertebrate photoreceptors responsible for color vision.