Imaging of calcium in biological specimens: role of electron energy loss spectroscopy.

The regulation of intracytoplasmic calcium is an essential mechanism involved in many normal cytological functions. Disorders of calcium regulation are coupled to numerous pathological conditions. Calcium is such a universal participant that knowledge of its fine movements and sites of action is essential for the understanding of many biological phenomena at the cellular level. Techniques for the measurement of intracellular calcium have been dramatically improved, and we have now achieved great sensitivity to concentration changes as well as a very high time resolution. Recent advances in calcium research technology have concentrated on imaging methods permitting the visualization of calcium movements and localization of its cytoplasmic distribution. At the light microscopical level, it is now possible to trace the fluctuations of intracellular free calcium in a living cell, using sensitive calcium binding fluorescent indicators. Electron microscopic techniques are therefore necessary for the ultrastructural localization of cytoplasmic calcium stores. Electron energy loss spectroscopy is at present the most sensitive microanalytical mode in electron microscopy. It is capable of simultaneously producing high resolution images and elemental information. It therefore has application in calcium research if the specimens are prepared by an appropriate method. This review has two objectives: (1) To place electron energy loss imaging in its proper perspective in calcium research. (2) To suggest that freeze-dried embedded biological materials are appropriate for high resolution energy loss imaging of calcium.