Electron-probe X-ray microanalysis: techniques and recent applications in biology

Abstract

Summary(1) X‐ray microanalysis is a powerful technique, allowing the quantitative measurement of many elements of physiological interest, at physiological concentrations and with a spatial resolution typically of a few micrometres in bulk specimens, and a few hundred nanometres in thin sections.(2) The basic requirements are a focussed, high‐energy electron beam, X‐ray spectrometers and a means of visualizing the specimen. These facilities are found in a number of different types of commercial microanalysers, which may be based on either the transmission electron microscope, or the scanning electron microscope. Scanning microanalysers offer a lower image resolution, but are considerably more versatile than instruments based on the transmission electron microscope.(3) Preparing the specimen in a form that will withstand electron bombardment under high vacuum, and yet in which the elements to be analysed are retained in their original locations, is clearly the most critical step. For analysing diffusible elements, especially water‐soluble electrolytes, the only reliable method is to freeze as rapidly as possible, and analyse the tissue without any chemical treatment.(4) The best results are obtained if frozen sections are cut and analysed on a cold‐stage with their water content retained as ice. Procedures have been worked out for doing this, and for quantitative interpretation of the results, so that original tissue concentrations of the common electrolytes, phosphorus, sulphur and other elements of interest can now be measured with confidence. The original water‐content of different cell and tissue compartments can also be estimated from the mass‐loss on drying.(5) A simpler alternative is to freeze‐dry the frozen sections before analysis. The distribution of diffusible elements is probably not too much disturbed, spatial resolution is improved, and the visual image becomes much clearer, but quantitation is made more difficult and less reliable. Nevertheless this technique is frequently used.(6) For precipitated materials and for fluid samples, much simpler methods of preparation can be used.(7) The technique is the subject of a large and rapidly expanding literature, and is providing new information on the sub‐cellular distribution of electrolytes and other elements in many different tissues from animals and plants.(8) Some of the earliest applications to the study of diffusible ions were in the analysis of micropuncture samples from kidney tubules, where it has been possible to analyse many very small samples, and for several elements at once. Some preliminary information has also been obtained on the intracellular ion concentrations in kidneys subjected to different physiological conditions.(9) A particularly successful field has been the study of transporting epithelia, including vertebrate and insect digestive and excretory tissues, where the distribution of ions along the intercellular spaces has been shown not to agree with that predicted from the ‘standing gradient’ theory of osmotic coupling. The regulatory mantle epithelium in a mollusc has also been investigated, and some new information obtained on intracellular ion distributions in the frog skin.(10) Studies on nervous tissue are still at a preliminary stage, because of the structural complexity of the tissue. In muscle, however, it has been possible to demonstrate the re‐uptake of calcium by intracellular structures, following its release into the cytoplasm during contraction.(11) Information has been obtained on the distribution of ions in the nuclei and chromosomes of cells at different stages in development and division. Nuclear sodium and potassium levels are generally similar to those in the cytoplasm: the very high sodium concentrations found in nuclei isolated anhydrously are shown to be artefactual.(12) A variety of plant cells has been investigated, problems of particular interest being the regulation of salt uptake by roots and leaves, and the role of potassium ions in causing opening of the stomata by osmotic swelling of the guard‐cells.(13) Some applications in pathology are briefly mentioned, including studies on the fate of accidentally or deliberately introduced minerals, and on differences in the ion content of normal and diseased muscle cells.(14) Numerous observations on the subcellular distribution of calcium are collected together. In general, measurable calcium uptake by mitochondria appears to occur only in damaged cells; in muscle the cell‐membrane and the sarcoplasmic reticulum seem to be the main organelles responsible for re‐uptake of calcium following a contraction. Direct involvement of calcium with a contractile system has been shown in the vorticellid protozoan Zoothamnium, and a connection between calcium and exocytosis has been demonstrated in digestive and other epithelia.