Low Temperature Methods In Biological Electron Microscopy

The frozen-hydrated specimen is the principal element that unifies the subject of low temperature microscopy, and frozen-hydrated specimens are what this book is all about. Freezing the sample as quickly as possible and then further preparing the specimen for microscopy or microanalysis, whether still embedded in ice or not: there seem to be as many variations on this theme as there are creative scientists with problems of structure and composition to investigate. Yet all share a body of com mon fact and theory upon which their work must be based. Low-Temperature Micros copy and Analysis provides, for the first time, a comprehensive treatment of all the elements to which one needs access. What is the appeal behind the use of frozen-hydrated specimens for biological electron microscopy, and why is it so important that such a book should now have been written? If one cannot observe dynamic events as they are in progress, rapid specimen freezing at least offers the possibility to trap structures, organelles, macro molecules, or ions and other solutes in a form that is identical to what the native structure was like at the moment of trapping. The pursuit of this ideal becomes all the more necessary in electron microscopy because of the enormous increase in resolution that is available with electron-optical instruments, compared to light optical microscopes.

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first glance, it seems as simple as it is attractive: the dynamics of life are frozen in, nothing is added and nothing withdrawn except thermal energy.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

This volume demonstrates how cellular and associated electron microscopy contributes to knowledge about biological structural information, primarily at the nanometer level. It presents how EM approaches complement both conventional structural biology (at the high end, angstrom level of resolution) and digital light microscopy (at the low end, 100-200 nanometers). *Basic techniques in transmission and scanning electron microscopy *Detailed chapters on how to use electron microscopy when dealing with specific cellular structures, such as the nucleus, cell membrane, and cytoskeleton *Discussion on electron microscopy of viruses and virus-cell interactions

This comprehensive reference illustrates optimal preparation methods in biological electron microscopy compared with common methodological problems. Not only will the basic methodologies of transmission electron microscopy like fixation, microtomy, and microscopy be presented, but the authors also endeavor to illustrate more specialized techniques such as negative staining, autoradiography, cytochemistry, immunoelectron microscopy, and computer-assisted image analysis. Authored by the key leaders in the biological electron microscopy field Illustrates both optimal and suboptimal or artifactual results in a variety of electron microscopy disciplines Introduces students on how to read and interpret electron micrographs

The use of the term "advanced" in the title of this book is somewhat ar bitrary and very much relative with respect to time. Many techniques which were considered at the "cutting edge" of ultrastructural methodology just a few years ago are now rou tin ely used in numerous laboratories. One could cite freeze-fracture, cryothin sectioning, or indeed most of the field of scan ning electron microscopy as concrete examples. Thus the use of the term "ad vanced techniques" must be interpreted with regard to the present state of the art, and is useful only in informing the potential reader that this volume is not a primer to be used as an initial introduction into basic biological elec tron microscopy. Many excellent volumes have filled that niche in the past few years, and it is not intended that this modest book be a complete com pendium of the field. Furthermore, any limited selection of papers on advanc ed techniques necessarily reflects the preferences and arbitrary whims of the editor, thereby excluding many equally important procedures which the knowledgeable reader will readily identify. The first volume of this series appeared approximately five years ago and illustrated techniques which were thought to represent advanced and yet ba sically morphological methods for gaining increased ultrastructural informa tion from biological specimens. The present volume, on the other hand, stresses techniques which provide specific physicochemical data on the speci mens in addition to the structural information.