A new method for the preparation of 'double-fixed', quick-frozen, freeze-substituted cells for whole-cell...

A method is described in which quick-frozen, freeze-substituted, cultured cells can be prepared for whole-cell transmission electron microscopy (WCTEM). This method is simple and reliable and can be carried out in most laboratories without special equipment. Cells grown of Formvar-carbon-coated nickel grids are quick-frozen in Freon 22, freeze-substituted in an ethanolic solution of glutaraldehyde, post-fixed in osmium tetroxide and critical-point-dried. The quality of ultrastructural preservation using this 'double fixation' protocol is comparable to that of conventional WCTEM. However, the combination of quick-freezing and WCTEM has the decided advantage over conventional WCTEM in that cellular activities are arrested almost instantaneously. Thus, this new method could potentially yield a more faithful representation of cytoarchitecture and is especially useful for studies on the structural basis of rapid cytoplasmic events which may remain undetected when using conventional fixation methods.