Electron Microscopy

Douglas E. Chandler, William P. Sharp

Freeze fracture depends on the property of frozen tissues or cells, when cracked open, to split along the hydrophobic interior of membranes, thus revealing broad panoramas of membrane interior. These large panoramas reveal the three-dimensional contours of membranes making the methods well suited to studying changes in membrane architecture. Freshly split membrane faces are visualized by platinum or tungsten shadowing and carbon backing to form a replica that is then cleaned of tissue and imaged by TEM. Etching, i.e., removal of ice from the frozen fractured specimen by sublimation prior to shadowing, can also reveal the true surfaces of the membrane as well as the extracellular matrix and cytoskeletal networks that contact the membranes. Since the resolution of detail in the metal replicas formed is 1-2 nm, these methods can also be used to visualize macromolecules or macromolecular assemblies either in situ or displayed on a mica surface. These methods are available for either specimens that have been chemically fixed or specimens that have been rapidly frozen without chemical intervention.