Studies on Cell Membrane Ultrastructure, and the use of the Freeze-Fracturing Technique in Electron Microscopy

Abstract

1) The Ultrastructural appearance of frozen-fractured mouse liver cell membranes, and consistent variations in the distribution of membrane-associated particles caused by various pretreatments as described by previous investigators, were confirmed in this study. 2) Freeze fracturing, was evaluated as a method for studying the ultrastructure of single celled microorganisms. Large prokaryotes with previously undescribed morphologies were investigated using freeze fracturing, thin sectioning, and negative staining techniques. 3) Structures thought to represent flagellar attachment sites were demonstrated in frozen fractured bacterial preparations for the first time. 4) A previously undescribed level of cell organization was discovered in a rumen organism classified morphologically as Selenomonas. 5) A technique was devised to retrieve replicas from both sides of the fracture of a single frozen-fractured specimen. The results obtained by its use support the theory that frozen cell membranes fracture along some interior plane rather than at the membrane-cytoplasm boundary. The technique also showed that the particles seen on frozen fractured membranes lie within the thickness of the membrane. 6) For determination of the third dimension in freeze fracture replicas, i.e. the heights of various features, an alternative method to stereoscopy was devised. This method, which can be made semiautomatic in execution, involves microdensitometry of electron micrograph negatives and certain mathematical manipulation of the optical density data.