Summary
We evaluate a method for improved, ice-crystal-free cryofixation of various biological materials (macromolecules, subcellular fractions, whole cells in suspensions or as monolayers) without any chemical pretreatments. The principle of the method (as introduced byPscheid, Schudt, andPlattner 1981 for cell monolayer cultures) is as follows. Firstly, a thin sample layer is sandwiched between a thin copper object holder and a thermally insulating plastic sheet (Thermanox, which is also used as a cell culture substratum); secondly, a jet of melting propane is shot from a simple, inexpensive brass vessel onto the copper object holder. We now ascertained that this simple and inexpensive method allows cooling rates up to > 18,000 °C/sec and, thus, gives better results than comparable but more sophisticated and expensive procedures. The method also avoids all the disadvantages of some other rapid freezing methods. Specifically, the sample mass required is minimal, the sample remains fully hydrated and no changes of ionic or other conditions can occur. There is no hazard of passing a cold gas phase (cooling curves being very steep right at the beginning) or of impairment by mechanical impact (as it could be with “cold metal block freezing”). The expedient of using a thermal insulator as part of a sandwich sample allows cooling rates which are superior or at least equivalent to those obtained with a commercial double propane-jet device (and double copper sandwiches). The reason for this is that in practice a two-side propane jet onto a copper-copper sandwich can not reach both sides precisely at the very same time (as implied from our cooling rate measurements). The tentative transformation of our mono-jet into a double-jet variation (with a branched nozzle piece) did also not improve the results, when compared with a single propane-jet onto a copper-Thermanox sandwich.
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This work is taken in part from a Ph.D. thesis by G.Knoll.
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Knoll, G., Oebel, G. & Plattner, H. A simple sandwich-cryogen-jet procedure with high cooling rates for cryofixation of biological materials in the native state. Protoplasma 111, 161–176 (1982). https://doi.org/10.1007/BF01281964
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DOI: https://doi.org/10.1007/BF01281964