Electron probe microanalysis of biological soft tissues: principle and technique.

Electron probe microanalysis is a method based upon X-ray spectrometry used for analyzing the chemical element content of very small amounts of material. The concentration of electrolytes in the microenvironment of cells, in cells, and in intracellular organelles can be measured. The main difficulties in using this method in biological soft tissue lie in sample preparation and in proper interpretation of the data. Best tissue preparation seems to be to quench the sample and to analyze it either freeze dried in thin or ultrathin sections, or frozen hydrated in thin sections or bulk samples. In all cases analysis should be performed using a cold stage and an ultra clean vacuum in order to minimize mass loss due to beam damage and mass gain due to contamination trapping. Interpretation of the data relies upon the knowledge of both the localization of the volume excited by the electron beam and the origin of the continuum and characteristic X-ray signals received by the X-ray spectrometer. This knowledge can be complicated by two facts: 1) when the electron beam is used in an analytical mode, viewing of the analyzed microarea can be lost, and 2) the X-ray signals received by the spectrometer can originate not only from the volume directly excited by the electron beam but from areas that can be far apart, excited by the electron beam tail, scattered electrons or secondary fluorescence, particularly when using energy dispersive spectrometers. Theoretical quantitation of the results is well developed. Practical quantitation could be complicated by the possibility of mass gain, mass loss, standard inhomogeneity, non-uniformity of sample thickness, possibility of shrinkage during freeze drying and, when using energy dispersive spectrometry, by the low signal over background for low atomic number elements (Na), the possibility of overlap of characteristic X-ray lines, and the use of complex and empirical methods for background stripping and peak deconvolution. All these difficulties can be overcome, making electron probe microanalysis one of the most powerful tools available to the biologist.