Freeze‐substitution and the preservation of diffusible ions

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Abstract
SUMMARY: Freeze‐substitution of biological material in pure acetone followed by low‐temperature embedding in the Lowicryls K11M and HM23 yields stable preparations well suited for sectioning and subsequent morphological and microanalytical studies. Transmission electron microscopy of dry‐cut sections shows that diffusible cellular thallium ions (Tl+) of Tl+‐loaded muscle are localized at similar protein sites in freeze‐substituted as in frozen‐hydrated preparations. A comparison of X‐ray micro‐analytical data obtained from freeze‐dried cryosections and sections of freeze‐substituted normal (potassium‐containing) muscle shows that K+ ion retention in the freeze‐substituted sample is highly dependent on the freeze‐substitution procedure used; so far, in the best case, about 67% of the cellular K+ is retained after freeze‐substitution in pure acetone and low‐temperature embedding. It is concluded that the retention of diffusible cellular ions is dependent on their interactions with cellular macromolecules during the preparative steps and that ion retention may be increased by further optimizing freeze‐substitution and low‐temperature embedding.