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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.
List of contents
The history of electron probe microanalysis in biology.- 1. Specimen Preparation.- Specimen preparation and other limitations in quantitative electron probe X-ray microanalysis using ultrathin sections.- Freeze-substitution and low temperature embedding for analytical electron microscopy.- Ensuring the validity of results in biological X-ray microanalysis.- 2. Analytical Techniques.- a) X-ray microanalysis.- The subcellular accumulation of toxic heavy metals: Qualitative and quantitative X-ray microanalysis.- X-ray microanalysis of cryosections using image analysis.- Electron probe X-ray microanalysis in the silkmoth antenna - problems with quantification in ultrathin cryosections.- b) Electron energy loss spectroscopy.- Progress in electron energy loss spectroscopic imaging and analysing biological specimens with a field emission scanning transmission electron microscope.- Application of parallel-detection electron energy loss spectroscopy in biology.- Resin based standards for biological energy dispersive X-ray and electron energy loss microanalysis.- Imaging and microanalysis by electron spectroscopy.- 3. Biological Applications.- a) Intracellular element localization.- Application of X-ray microanalysis and electron energy loss spectroscopy to studies of secretory cell biology.- X-ray microanalysis of freshly isolated cells in suspension.- X-ray microanalysis and free calcium measurements in cultured neonatal rat ventricular myocytes.- b) Epithelial transport.- 1/um thick frozen hydrated/dried sections for analysing pericellular environment in transport epithelia; New results from old data.- Distribution of ions and water in epithelial cells and tissues.- Characterization of electrolyte transport mechanisms and compartments by the use of the markers Rb and Br.-Electron probe analysis of transport properties of cultured cells.- c) Dynamic processes.- Quantitative X-ray elemental mapping of dynamic physiologic events in skeletal muscle.- Single isolated cardiac myocytes frozen during voltage-camp pulses: A technique for correlating X-ray micro-analysis data on calcium distribution with calcium inward current in the same cell.- X-ray microanalysis of fast exocytotic processes.- 4. Medical Application.- Electron probe microanalysis in pathology.- Microprobe analysis in medicine - present practice and future trends.
