Practical Methods In Electron Microscopy Robards A W Sleytr U B Low Temperature Methods In Biological Electrn Microscopy

Practical Methods in Electron Microscopy: A.W. Robards and U.B. Sleytr. Low temperature methods in biological electron microscopy

Cryotechniques in Biological Electron Microscopy

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first glance, it seems as simple as it is attractive: the dynamics of life are frozen in, nothing is added and nothing withdrawn except thermal energy.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 physics 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.

Electron Microscopy Methods and Protocols

Electron Microscopy Methods and Protocols is designed for the established researcher as a manual for extending knowledge of the field. It is also for the newcomer who wishes to move into the field. A wide range of applications for the examination of cells, tissues, biological macromolecules, molecular structures, and their interactions are discussed. We have tried to gather together methods that we consider to be those most generally appli- ble to current research in both cell and molecular biology. Each chapter c- tains a set of related practical protocols with examples provided by experts who have first-hand knowledge of the techniques they describe. The individual chapters are grouped according to similarities in their specimen preparation and methodology. Methods are presented in detail, in a step-by-step fashion, using reproducible protocols the authors have personally checked. During the last decade, the scientific literature describing the use of colloidal gold as an immunocytochemical marker has increased at an ex- nential rate, and this trend is expected to continue. We have included a large number of variations on the immunogold labeling technique. In both the ne- tive staining and cryo chapters, authors emphasize the “immunological app- cations” in order to correlate as fully as possible with the emphasis on immunogold labeling in the other chapters. Electron Microscopy Methods and Protocols commences with the routine preparation of biological material for classical transmission electron microscopy involving tissue fixation, embedding, and sectioning (Chap. 1).