TIME-RESOLVED CRYOFIXATION METHODS FOR THE STUDY OF DYNAMIC CELLULAR EVENTS BY ELECTRON-MICROSCOPY - A REVIEW

The preservation of cells for electron microscopy by chemical fixation is a lengthy process, requiring up to 30 minutes fbr cytoplasmic stab ilisation. This time lag enables many changes to occur in specimens so that they may not reflect their living state when they are observed i n electron microscopes, Many artefacts can be avoided by using cryofix ation, which freezes specimens over a period that is measured in milli seconds; so that specimens are preserved by cryoimmobilisation. This t ime resolution can be used to study rapid processes in biology and che mistry because, although electron microscopes cannot observe dynamic c ellular events directly, processes can be arrested after known time in tervals so that transient stages are preserved and a series of time-la pse steps is acquired. Some experiments have involved freezing specime ns which were maintained in controlled states and others have shown re sults after stimulation where structural differences are seen between one millisecond and the next. The experimental techniques that have be en applied prior to freezing are electrical and chemical stimulation, electrophoresis, chemical relaxation after a temperature jump, electro poration, which is analogous to relaxation after applying a radio freq uency electrical field, and flash photolysis methods. This review desc ribes the origins and application of time-resolved freezing, which int egrates electron microscopy with dynamic biochemical, physiological, a nd ultrastructural events.