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Scanning Microscopy

Abstract

Cryofixation is considered to be the best method for immobilizing biological material in its natural state. In jet-freezing, the specimen typically is sandwiched between two carriers and kept in place while a coolant is moved very rapidly against the opposite surfaces. The JFD 030 jet-freezing device has been used to optimize the operating parameters. The course of the temperature in place of a specimen was measured with thermocouples and recorded by an IBM-compatible personal computer using a specifically developed software program. Mean cooling rates, over the temperature range of 273K to 173K, achievable with different cryogens, including the non-flammable HCFC 124 (SUVA 124-CHClFCF3), were measured under a variety of conditions. The freezing capability of the JFD 030 was evaluated by analyzing transmission electron microscopic results obtained from freeze-substituted plant cells and freeze-fractured cosmetic emulsions. Jet-freezing, despite its limits in freezing thick specimens, can be applied to cell suspensions as well as to semi-thin sections by the use of thin Ti supports, a higher pressure of the nitrogen gas or by a brief pretreatment with a cryoprotectant (e.g., sucrose). In addition, with the non-flammable HCFC 124 in combination with the thin Ti supports, freezing rates similar to those achievable with propane and standard copper supports can be reached giving researchers the chance to use jet-freezing without the dangerous propane.

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