The Physics of Rapid Cooling and Its Implications for Cryoimmobilization of Cells

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This chapter illustrates what happens when liquid water crystallizes on cooling, and the way crystal formation rearranges the surrounding biological material. It also discusses the phenomenon of vitrification and concludes that cryoimmobilization should always involve vitrification, whether or not the specimen remain vitreous for subsequent cryoelectron microscopy observation or is further processed to remove the water before being observed in the dry state. There is only one good way to achieve cryoimmobilization of biological samples—that is, through full vitrification. The result is suboptimal—an amiable euphemism—whenever cooling results in ice formation. It is not sufficient that vitrification is achieved in only some regions of the specimens because ice formation causes dehydration, and can thereby damage surrounding regions too. As for the verification of successful vitrification, it should not be left to the subjective evaluation of nice preservation. There is always a necessity of objectively testing the quality of cryoimmobilization by determining whether or not the sample has been vitrified.

Introduction

Nature has a long experience of crossing the 0°C barrier and knows how to deal with it. For a few decades now electron microscopists have also been learning to deal with this transition temperature, but they still have much progress to make. The problem is that freezing is quite a dramatic event. When water freezes, the molecules, which in a liquid state communicate exclusively with their nearest neighbor, suddenly adopt cooperative behavior. From being solitary, they become members of a demanding and exclusive group, an ice crystal. Once absorbed into the group, they lose part of their freedom and give up some of their energy. All other molecules become excluded from the resulting solid. The world is now divided into two, ice crystals and … all the rest.

Freezing is not good for life. Nature has adopted all kinds of strategies to avoid its deleterious effects. For large animals, a good method is to keep their temperatures well above freezing, but this is expensive; for small organisms it costs too much energy. Plants too have adopted ways of their own. Some have learned how to prevent ice crystal formation. Partial dehydration helps, as does the addition of a cryoprotectant. Indeed, there are proteins that prevent the formation or growth of ice. In other organisms, cryoresistance is achieved by letting ice crystals grow under such conditions that the organism survives. There are amphibians and reptiles that freeze hard in winter and then thaw out happily in the spring. For a long time these multiple facets of freezing have fascinated laymen and specialists. The literature is abundant (Ball 1999, Luyet 1940, Réaumur 1736).

Freezing is also important for human activity. Animal breeding relies on frozen cells (the gametes of prized livestock), a method that also works well with many human cells. The technical problems of preserving viability increase with the size of the sample. However, cryopreservation of tissues and organs is still in its infancy. The case of human embryo preservation may no longer present a limiting technical problem, but it will nevertheless for long remain a hot ethical question. As for the preservation of entire humans in a state of frozen “suspended life,” which has apparently become a lucrative business, this obviously relies on deception and ignorance.

Electron microscopists are interested in cryoimmobilization because it offers a way out of the water dilemma: water is the most abundant constituent of living material, but it cannot remain in the vacuum of an electron microscope except at very low temperatures. One avenue is cryospecimen preparation. It consists first of freezing the specimen and then removing the water from the frozen state. Once the specimen is dry, it can be brought to room temperature for observation, and the damage due to dehydration can be less severe than when water is removed from the liquid state.

The other avenue is cryo‐electron microscopy in which the water is kept at a temperature low enough to prevent evaporation. As long as cryoimmobilization was synonymous with freezing, and frozen water was ice, the advantage of cryo‐electron microscopy was questionable; the method was demanding and it did not eliminate freezing damage. The situation changed with vitrification. This is the process of solidifying water from its liquid state without crystallization. The idea is old, the expectations were high, and the amount of research carried out to achieve it has been considerable. However, a persistent lack of success led to the conclusion that it was fundamentally impossible. The breakthrough came in 1980 when Mayer 1980, Dubochet 1981 succeeded, against all expectations, in vitrifying small amounts of pure water. From this point, cryo‐electron microscopy of biological particles suspended in a thin layer of vitrified water was soon developed into a practical method, which is now widely used (Adrian 1984, Jiang 2005). It took 20 more years until this approach could be extended to larger samples, such as cells and tissues, by means of cryo‐electron microscopy of vitreous sections (CEMOVIS; Al‐Amoudi 2004, Dubochet 2006, Lucic 2005 and Chapter 15, this volume).

In the present chapter, we discuss what happens when liquid water crystallizes on cooling, and how crystal formation rearranges the surrounding biological material. We discuss vitrification, leaving aside the mysterious aspects of the phenomenon. We conclude that cryoimmobilization should always involve vitrification, whether or not the specimen will remain vitreous for subsequent cryo‐electron microscopy observation or will be further processed to remove the water before being observed in the dry state. Finally, we insist on the necessity of objectively testing the quality of cryoimmobilization by determining whether or not the sample has been vitrified. Further readings to complement this chapter are found in Angell 2004, Ball 1999, Dubochet 2006, Dubochet 1988, Dubochet 1991.

Section snippets

Freezing Water

In everyday life, we are familiar with three forms of water: vapor, liquid, and ice. At the molecular level we can feel comfortable with some of the simple, pertinent concepts. Vapor is the state in which the molecules are free to fly independently; ice is the state where each molecule has its place in the lattice of a crystal. Liquid water is more complicated. Situated somewhere between the order of crystals and the disorder of a gas, liquid water might be seen either as disordered ice or

Biological Material

In biology, we do not deal with pure water but with concentrated solutions of salts and macromolecules. In a cell, biological material is crowded (Goodsell, 1993). No water molecule deals exclusively with other water molecules for long, and none is far away from an ion or from the surface of a macromolecule. For example, we calculate that if the cell's content is modeled exclusively by spherical proteins of 20 kDa, then four layers of water on each protein would account for enough water to

Freezing Biological Material

What happens to a biological sample on freezing? The scene has been set: there is a physiological solution (ca. 200‐mM solutes) bathing the tissue and cells that are crowded with macromolecules. A biological membrane marks the border. In the cells most water molecules are moving 1012 times per second; most of the time they are not more than four neighbors away from the surface of the next macromolecule. Ions, soluble molecules, and macromolecules are also moving, although more slowly; they

Acknowledgments

I am thankful to Ms. P. Buhayer and S. Wauters for help with English and drawings.

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