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Fundamental Technical Elements of Freeze-fracture/Freeze-etch in Biological Electron Microscopy
Carson, J. Fundamental Technical Elements of Freeze-fracture/Freeze-etch in Biological Electron Microscopy. Journal of Visualized Experiments . JoVE, Somerville, MA, 91:51694, (2014).
SHORT ABSTRACT: Freeze-fracture processing of biological specimens for examination by transmission electron microscopy is a preferred method for investigation of specializations of biological membranes. Recently, this technique has enjoyed a resurgence of interest in its application as a means for ultrastructural investigation in the materials sciences and nanotephnology as well as its historic role in cell biology.
Freeze-fracture/freeze-etch describes a process whereby specimens, typically biological or nanomaterial in nature, are frozen, fractured, and replicated to generate a carbon/platinum "cast" intended for examination by transmission electron microscopy. Specimens are subjected to ultrarapid freezing rates, often in the presence of cryoprotective agents to limit ice crystal formation, with subsequent fracturing of the specimen at liquid nitrogen cooled temperatures under high vacuum. The resultant fractured surface is replicated and stabilized by evaporation of carbon and platinum from an angle that confers surface three-dimensional detail to the cast. This technique has proved particularly enlightening for the investigation of cell membranes and their specializations and has contributed considerably to the understanding of cellular form to related cell function. In this report, we survey the instrument requirements and technical protocol for performing freeze-fracture, the associated nomenclature and characteristics of fracture planes, variations on the conventional procedure, and criteria for interpretation of freeze-fracture images. This technique has been widely used for ultrastructural investigation in many areas of cell biology and holds promise as an emerging imaging technique for molecular, nanotechnology, and materials science studies.