freeze substitution

Freeze substitution is a technique to replace amorphous ice with an organic solvent (acetone, etc.) (dehydration) in a biological specimen fixed by rapid freeze fixation, where the specimen is subject to rapid freeze fixation (high pressure freezing or metal mirror freezing (slam freezing)).  To prevent destruction of fine structures of the specimen, the substitution is carried out by raising temperature step-by-step from -80 ℃ to 4 °C over a few days.  In the course of the substitution, chemical fixation and electron staining are often performed by adding osmium tetroxide and/or uranium acetate.  Then, the substituted specimen is returned to room temperature and is subject to resin embedding.  A TEM specimen is made by ultrathin sectioning the specimen.

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