A new method for the preparation of ‘double‐fixed’, quick‐frozen, freeze‐substituted cells for whole‐cell transmission electron microscopy

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Abstract
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 on 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.