The problems of conventional EM-preparation techniques based on chemical fixation may be overcome to a considerable extent by freeze substitution techniques. Although at present substitution cannot be performed at sufficiently low temperatures to prevent the recrystallization of vitrified aqueous specimens, thin sectioned biological samples show an improved information density. If freeze-substitution is combined with conventional embedding above 273 K (Epon/Araldite, Spurr's resin) the substituting organic solvent must contain stabilizing agents such as osmiumtetroxide, glutaraldehyde or uranylions. In combination with low temperature embedding procedures (Lowicryl) completely unfixed samples are obtained, which are suitable for immunolabelling and electron spectroscopic experiments. Water in its different dynamic states is considered to be the most important factor in maintaining the structural and functional integrity. Thus, the main advantage of freeze substitution is a better control over the removal of the cellular water, necessary for subsequent plastic embedding.
Humbel, B. and Müller, M.
"Freeze Substitution and Low Temperature Embedding,"
Scanning Electron Microscopy: Vol. 4
, Article 19.
Available at: https://digitalcommons.usu.edu/electron/vol4/iss1/19