Substructure in rod photoreceptor membranes

Frog retinae, fixed only in buffered glutaraldehyde, were embedded for sectioning in glutaraldehyde polymerized with urea. In suitably thin sections globular substructures were seen in negative contrast after ionic staining with uranyl acetate and lead citrate, or after staining with neutralized phosphotungstic acid. Efforts to extract at least some of the lipid from sections before ionic staining enhanced the visualization of the "globules". Exposure to KMnO4 solution, used as an oxidative section stain, also outlined globular substructure in negative contrast, but with the additional feature that positively stained surface "leaflets" associated with the aqueous compartment were well defined. Staining sections with OSO4 vapor resulted in positively stained membranes, but without any evident substructure. However, when sections which previously had been exposed to OSO4 vapor were secondarily stained with uranyl acetate and/or lead citrate, positively stained globular substructures then were revealed. The globular substructures always were centered in the hydrophobic core region of the disc membranes, and symmetrically spanned the full thickness of this layer. The diameter of individual particles approximated 50-55 A. Reasons are presented for the supposition that the evident globules incorporate at least hydrophobic components of rhodopsin molecules. Findings are discussed in relation to various models of disc membrane organization that have been proposed in recent years.