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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frozen in, nothing is added and nothing withdrawn except thermal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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