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Scanning Microscopy

Abstract

The preservation of cells for electron microscopy by chemical fixation is a lengthy process, requiring up to 30 minutes for cytoplasmic stabilisation. This time lag enables many changes to occur in specimens so that they may not reflect their living state when they are observed in electron microscopes. Many artefacts can be avoided by using cryofixation, which freezes specimens over a period that is measured in milliseconds, so that specimens are preserved by cryoimmobilisation. This time resolution can be used to study rapid processes in biology and chemistry because, although electron microscopes cannot observe dynamic cellular events directly, processes can be arrested after known time intervals so that transient stages are preserved and a series of time-lapse steps is acquired. Some experiments have involved freezing specimens which were maintained in controlled states and others have shown results after stimulation where structural differences are seen between one millisecond and the next. The experimental techniques that have been applied prior to freezing are electrical and chemical stimulation, electrophoresis, chemical relaxation after a temperature jump, electroporation, which is analogous to relaxation after applying a radio frequency electrical field, and flash photolysis methods. This review describes the origins and application of time-resolved freezing, which integrates electron microscopy with dynamic biochemical, physiological, and ultrastructural events.

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