Chemical cryoprotection for structural studies

Abstract

KEY w o R D s. Cryoprotection, ultrastructure, freeze-fracture, microanalysis, glycerol, polymeric cryoprotectant, frozen sections. S U M M A R YWith the ascendency of techniques for ultrarapid cooling and the successful control of ice crystal damage by purely physical means, it has become necessary to make a case for the continued use of chemical antifreeze agents in any circumstances. These circumstances include the need to explore tissues deeper than those superficial layers, whose morphology can be preserved by ultrarapid cooling, the avoidance of superficial areas of damage inflicted by dissection and tissue slicing, and situations where the growth of ice crystals must be controlled throughout the specimen, as for example for the cutting of frozen sections.The control of ice crystal damage would ideally be by the avoidance of any freezing at all but in practice can be seen in terms of the control of ice crystal size, which in turn depends on the density of nuclei that develop in the specimen and the rate of accretion of water molecules to the crystallites that form on these nuclei. Chemical antifreeze agents act in a variety of ways to increase the density of nuclei and/or to reduce the rate of growth of ice crystals: by promoting nucleation, by enhancing subcooling and by increasing the viscosity of the extracellular medium. In this way, large numbers of small ice crystals are produced. The different types of cryofixative agent each have their own advocates, advantages, special applications and drawbacks and these must be taken into account when considering the range of methods available for the analysis of cryofixed material.

The Effects of Cryoprotection on the Structure and Activity of p21ras:Implications for Electron Spin-Echo Envelope Modulation Spectroscopy

“…Cryoprotectants retard the formation of the purified ice phase by lowering the 1 This work was supported in part by National Institutes of Health under Grant 5R 37 GM20168 (to A. G. Redfield) temperature of homogeneous nucleation, and thus facilitate the formation of a glassy phase in which the distribution of diffusible components, and thus the relevant interactions between components of the system, is maintained (12,14). The addition of glycerol or sucrose to protein solutions has been found to stabilize the protein (15) and to protect against inactivation during cold-storage, freezing, thawing, or lyophilization (16 -18).…”

Improved cryoprotection and freeze‐substitution of embryonic quail retina: A tem study on ultrastructural preservation

“…2) (Skaer, 1982;Verna, 1983;Volker et al, 1984). Even with the application of high-pressure freezing, the most sophisticated method available so far, "the formation of large crystals is prevented in tissue blocks up to 0.5 mm thick" (Moor et al, 1980).…”

“…Although, for example, glycerol "is extremely simple to use, the artifacts it causes are becoming very well domumented and results obtained can be compared with the wealth of data already accumulated with its use" (Skaer, 1982). Good documentation however, doesn't make the preservation of embryonic nerve tissue ultrastructure any better when glycerol is used for cryoprotection.…”

Preparation of Microfungi for Scanning Electron Microscopy

“…An examination of the advantages and disadvantages of chemical fixation and/or use of cryoprotectants prior to sample freezing is beyond the scope of this discussion and the reader is referred to a review by Sleytr and Robards (1982). Pretreatment of samples with cryoprotectants that do not penetrate into cells (Skaer, 1982) will prevent extracellular ice crystal formation and may delay intracellular nucleation, resulting in a general improvement of cryopreservation (Frederik, 1982).…”