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first glance, it seems as simple as it is attractive: the dynamics of life are frozen in, nothing is added and nothing withdrawn except thermal energy.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

In this practical text, the author covers the fundamentals of biological electron microscopy - including fixation, instrumentation, and darkroom work - to provide an excellent introduction to the subject for the advanced undergraduate or graduate student.

The past decade has seen a remarkable increase in the use of electron microscopy as a researm tool in biology and medicine. Thus, most institu tions of higher learning now boast several electron optical laboratories having various levels of sophistication. Training in the routine use of elec tron optical equipment and interpretation of results is no longer restricted to a few prestigious centers. On the other hand, temniques utilized by researm workers in the ultrastructural domain have become extremely diverse and complex. Although a large number of quite excellent volumes of electron microscopic temnique are now dedicated to the basic elements available whim allow the novice to acquire a reasonable introduction to the field, relatively few books have been devoted to a discussion of more ad vanced temnical aspects of the art. It was with this view that the present volume was conceived as a handy reference for workers already having some background in the field, as an information source for those wishing to shift efforts into more promising temniques, or for use as an advanced course or seminar guide. Subject matter has been mosen particularly on the basis of pertinence to present researm activities in biological electron microscopy and emphasis has been given those areas whim seem destined to greatly expand in useful ness in the near future.

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).

Cryotechniques offer a number of advantages over conventional electron microscopy methods in biology, particularly with regard to preserving the morphology and composition of specimens. This handbook provides a practical guide for researchers, students, and technicians who wish to employ cryotechniques in their work. It covers those methods where the cryofixed specimen is sectioned or otherwise presented as a thin film for examination, giving special attention to topics of current interest. This guide will be of use to workers in a variety of biomedical disciplines, including cell biology, molecular biology, bacteriology, and pathology.

This volume is a continuation of two prior books on advanced electron microscope techniques. The purpose of this series has been to provide in depth analyses of methods which are considered to be at the leading edge of electron microscopic research procedures with applications in the biological sciences. The mission of the present volume remains that of a source book for the research practitioner or advanced student, especially one already well versed in basic electron optical methods. It is not meant to provide in troductory material, nor can this modest volume hope to cover the entire spectrum of advanced technology now available in electron microscopy. In the past decade, computers have found their way into many research laboratories thanks to the enormous increase in computing power and stor age available at a modest cost. The ultrastructural area has also benefited from this expansion in a number of ways which will be illustrated in this volume. Half of the contributions discuss technologies that either directly or indirectly make extensive use of computer methods.

Some Biological Techniques in Electron Microscopy discusses the problems that hinder or prevent progress in biological electron microscopy. This book explores the great potential of electron microscope for solving several pressing medical and biological research problems. Organized into five chapters, this book starts with an overview of the primary goal of biological electron microscopy, which is the visualization of atoms in biological molecules and structures. This text proceeds with a discussion of the freezing methods of electron microscopy in which some of the artifacts producing steps of environmental techniques are eliminated. Other chapters consider the objective of electron microscopy to resolve successively small objects, in the limiting instance, single atoms in amorphous structures. The final chapter deals with processing tissues for electron microscopy wherein temperature, agitation, pressure, and humidity can be controlled throughout the operation. Biologists, biophysicists, physicists, electron microscopists, and medical researchers will find this book extremely useful.