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The aim of electron probe microanalysis of biological systems is to identify, localize, and quantify elements, mass, and water in cells and tissues. The method is based on the idea that all electrons and photons emerging from an electron beam irradiated specimen contain information on its structure and composition. In particular, energy spectroscopy of X-rays and electrons after interaction of the electron beam with the specimen is used for this purpose. However, the application of this method in biology and medicine has to overcome three specific problems: 1. The principle constituent of most cell samples is water. Since liquid water is not compatible with vacuum conditions in the electron microscope, specimens have to be prepared without disturbing the other components, in parti cular diffusible ions (elements). 2. Electron probe microanaly sis provides physical data on either dry specimens or fully hydrated, frozen specimens. This data usually has to be con verted into quantitative data meaningful to the cell biologist or physiologist. 3. Cells and tissues are not static but dynamic systems. Thus, for example, microanalysis of physiolo gical processes requires sampling techniques which are adapted to address specific biological or medical questions. During recent years, remarkable progress has been made to overcome these problems. Cryopreparation, image analysis, and electron energy loss spectroscopy are key areas which have solved some problems and offer promise for future improvements.
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first glance, it seems as simple as it is attractive: the dynamics of life are frozen in, nothing is added and nothing withdrawn except thermal energy.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."
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