Ludwig Edelmann

Cryofixation Without Pretreatment at Ambient Pressure

Almost a century ago biological or medical objects were used in their frozen state in order to accelerate pathological diagnosis (immediate section) and to better maintain their chemical constitution (Altmann 1890). This alternative to chemical fixation gained in importance during the first half of this century due to the availability of liquefied air used as coolant (“cryogen”) as well as to the fact that effective cooling systems for cryostats had been developed (see e.g. Gersh 1932; Simpson 1941; Eranko 1954; Kulenkampff 1955; Neumann 1958). Attempts to freeze wet objects for electron microscopy revealed that at normal atmospheric pressure and under the most favourable conditions only an approx. 30- μm border zone can be perfectly frozen (see e.g. Sitte 1979; Plattner and Bachmann 1982; Robards and Sleytr 1985). At greater depths the mixed plasmatic phases segregate. The size of these segregation compartments formed by the growing ice crystals within the specimen increases so rapidly that deeper layers cannot be used for electron microscopy. The depth of the well-preserved border zone can be increased without chemical pretreatment to, at the most, 300 μm by applying high pressures of about 2100 bar (Muller and Moor 1984). These limits can be considerably extended by the use of anti-freezing agents (“cryoprotectants”): glycerol has proved to be an effective anti-freeze for the freeze-fracture/freeze-etch method (Moor and Muhlethaler 1963) and saccharose in cryoultramicrotomy (see e.g. Bernhard and Leduc 1967; Tokuyasu 1973; Griffiths et al. 1984).

Freeze-substitution and the preservation of diffusible ions

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.

A simple freeze-drying technique for preparing biological tissue without chemical fixation for electron microscopy

A simple technique for freeze‐drying and embedding of small pieces of biological tissue at low temperature is described, utilizing exclusively cryosorption pumping. Good preservation of chemically unfixed tissue is observed.

Safety Rules for Cryopreparation

As is often the case in every day life, the main risk in the routine use of cryopreparation methods is frequently due to inadequate knowledge or underestimation of the hazards. Vapourization of larger quantities of nitrogen in badly ventilated, small rooms or cool rooms may be lethal (Sect. 2). The same applies to explosive propane/air mixtures (Sect. 3). Eyes and skin may be severely damaged by secondary cryogen splashing, e.g. liquid propane, ethane or halogenated hydrocarbons (Sect. 4). The problems presented by primary cryogen splashing, e.g. liquid nitrogen (LN2) or liquid helium (LHe) are completely different (Sect. 5). Finally, precautions should be taken when working with inflammable, secondary cryogens and when using glass containers or vessels of materials which become brittle at cryogenic temperatures (Sects. 6 and 7). Transport and removal of cryogens require particular safety measures (Sect. 8).

Selective Accumulation of Alkali-Metal Ions at Cellular Protein Sites without Ion Pumps

The membrane theory maintains that the high K+ level inside a cell is dependent on a functioning Na/K pump and that the cell K is dissolved in the cell water. On the other hand, the association-induction hypothesis (AIH) maintains that the bulk of cell K+ is selectively adsorbed to fixed anions of cell proteins.1 Recent experimental findings strongly support this hypothesis: a) An effectively membrane-pumpless open-ended cell preparation continues to demonstrate K+ accumulation and Na+ exclusion much as a normal cell does.2 Independent measurements confirm the prediction of the AIH that cellular K+ (Rb+, Cs+) accumulation occurs at proteins carrying much β-and γ-carboxyl groups (myosin).3,4