§16§To preserve tissue by freezing is an ancient concept going back pre sumably to the practice of ice-age hunters. At first glance, it seems as simple as it is attractive: the dynamics of life are frozen in, nothing is added and nothing withdrawn except thermal energy. Thus, the result should be more life-like than after poisoning, tan ning and drying a living cell as we may rudely call the conventional preparation of specimens for electron microscopy. Countless mishaps, however, have taught electron microscopists that cryotechniques too are neither simple nor necessarily more life-like in their outcome. Not too long ago, experts in cryotechniques strictly denied that a cell could truly be vitrified, i.e. that all the solutes and macro molecules could be fixed within non-crystalline, glass-like solid water without the dramatic shifts and segregation effects caused by crystallization. We now know that vitrification is indeed pos sible. Growing insight into the fundamentals of the phys §16§ics of water and ice, as well as increasing experience of how to cool cells rapidly enough have enlivened the interest in cryofixation and pro duced a wealth of successful applications. §04§I Fundamentals.- 1 Physics of Water and Ice: Implications for Cryofixation.- 2 The Response of Biological Macromolecules and Supramolecular Structures to the Physics of Specimen Cryopreparation.- 3 Electron Beam Radiation Damage to Organic and Biological Cryospecimens.- II General Methodology.- 4 Cryofixation Without Pretreatment at Ambient Pressure.- 5 Cryoeleetron Microscopy of Vitrified Specimens.- 6 Cryoultramicrotomy.- 7 Freeze-Substitution and Freeze-Drying.- III Special Aspects.- 8 Theory and Practice of High Pressure Freezing.- 9 Freeze-Etching of Dispersions, Emulsions and Macromolecular Solutions of Biological Interest 192.- 10 High Resolution Metal Replication of Freeze-Dried Specimens.- 11 Immunogold Labelling of Cryosections and Cryofractures.- 12 Cryoultramicrotomy for Autoradiography and Enzyme Cytochemistry.- 13 Scanning Electron Microscopy and X-Ray Microanalysis of Frozen-Hydrated Bulk Samples.- 14 Cryofixation of Dynamic Processes in Cells and Organelles.- 15 Cr §04§yofixation of Diffusible Elements in Cells and Tissues for Electron Probe Microanalysis.- IV Appendix.- 16 Safety Rules for Cryopreparation.
