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

Abstract

SUMMARY 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.

Freeze‐dried and resin‐embedded biological material is well suited for ultrastructure research

“…Van Harreveld & Crowell, 1964) or by sublimation of the water molecules in a suitable vacuum chamber (freeze‐drying, FD, e.g. Edelmann, 1978).…”

“…It was argued that short FD is possible because the vitrified water of well cryofixed biological material is probably sublimated much faster than predicted for hexagonal ice (for critique see Elder et al ., 1992). On the other hand, our own experiments carried out with the striated muscle of the frog led to the following conclusion (Edelmann, 1978, 1986b). The best structural preservation of chemically unfixed muscle, freeze‐dried and embedded in Spurr's resin, is obtained by long FD at low temperature (e.g.…”

Electron microscopy of high pressure frozen samples: bridging the gap between cellular ultrastructure and atomic resolution

“…Freeze fracturing (Moor and Mühletaler 1963) generates a replica of a fractured surface through the sample. Freeze substitution (Van Harreveld and Crowell 1964) and freeze drying (Edelmann 1978), respectively, result in resin-embedded samples that are suitable for conventional ultrathin sectioning. Last but not least, after mounting and trimming in a cryo-ultramicrotome the sample can be cryosectioned and the resulting frozen hydrated sections investigated on a cold stage in the electron microscope at ¡170°C (Al- Amoudi et al 2004;Dubochet et al 1983).…”

Cryotechniques in Biological Electron Microscopy

“…This is, however, not always essential for good morphological results (Edelmann 1986b); moreover, osmium might interfere with microanalysis. Although most authors perform resin infiltration under vacuum, Edelmann (1978) reported better preservation of muscle fine structure after infiltration at atmospheric pressure. Therefore, the admission of fixing vapours or resin monomer should be accomplished without venting the vacuum chamber unless dry nitrogen gas is used (Van Harreveld and Malhotra 1966;Edelmann 1978;Pfaller 1979).…”

“…Comparison of conventional chemical fixation (a, d), freeze-substitution (b, c) and freeze-etching (e). b Rapid freezing by impact on LN 2 -cooled Cu block, FD 3 days at 193 K, then 6 days at 213 K as described by Edelmann (1978Edelmann ( , 1986b; no chemical fixation, embedding into Spurr's resin and section staining as in a (Edelmann 1986 b) ), schedule as above. of the silk moth Bombyx mori, can be easily cryofixed without any chemical pretreatment by rapid immersion into liquid propane (b, c, e).…”