Freeze Substitution RMC FS-7500
Freeze substitution (FS) is a dehydratation technique by which the water is gently removed from a froen specimen. The technique ca serve as a link between cryofixation and conventional thin sectioning at room temperature. It is, therefore, a hybrid technique combining the advantages of the low temperature and the room temperature specimen preparation. With respect to the danger of artifacts, this procedure is much more obscure than ‘pure’ cryo-techniques, such as freeze etching or cryo-sectioning. FS is known from light microscopy and has been used in electron microscopy since its early days, but only during the last dozen years a breakthrough can be noticed, which is mainly due to improved cryo-fixation. As for any other cryo-techniques in biological electron microscopy, for successful FS the main prerequisite is also good cryofixation with as little freezing damage as possible.
FS dissolves the ice in a frozen specimen by an organic solvent, which usually also contains chemical fixatives. It has to be carried out at a temperature low enough to avoid secondary ice-crystal growth. After completion of FS, the temperature can be raised without risk, since water is now absent. Except for low temperature embedding, infiltration with the embedding resin is done at room temperature followed by heat polymerization. The result is a plastic-embedded specimen, which can be sectioned and stained in the routine way.