Protocol for 3-D visualization of molecules on mica via the quick-freeze, deep-etch technique

Abstract

Molecular imaging by freeze-drying of molecules adsorbed to a mica substrate often provides better images of molecules than those attainable with other methods; the images are easier to interpret than those obtained with frozen thin film or negative staining, and the 3-dimensional information content is greater. The complete procedure for the production and examination of platinum-carbon replicas of molecules is described. Topics include production of a mica flake suspension, chemical pretreatment of the flakes to enhance adsorption, quick-freezing of the samples on mica, optimal operation of the freeze-fracture equipment, and orientation of replica topography. The production of stereo micrographs is analyzed in detail, with emphasis on the photographic procedures necessary for interpretation and on the identification of correct micrograph orientation. Guidelines are provided for the extrapolation from observed molecular size in platinum replicas to expected molecular weight.