Standards for X-ray microanalysis of calcified structures

The ability of electron probe X-ray microanalysis (EPMA) to solve biological problems often depends on the use of a quantitative approach. EPMA allows the quantitative determination of chemical elements of biological materials by using reference standards which resemble the specimen in the mode of interaction with the electron beam. Although there is a large experience in the quantification of elements in biological thin specimens, experience with standards for X-ray microanalysis of bulk specimens is limited, especially for calcified structures where the density of the specimen is difficult to estimate. The quality of the results in EPMA depends on obtaining accurate calibration curves which allow the establishment of the relationship between the signal measured and the concentration of the element of interest. The different methods for specimen preparation and the thickness of the specimen will also determine the precise nature of the standardization technique to be adopted. The physics of the electron beam-specimen interactions impose limitations upon the accuracy of calibration, and the choice of an unstable standard can result in large errors in the quantification of elements. We have reviewed the different types of compounds that have been used as standards for biological EPMA of thin and bulk specimens and discuss their potential use for quantitative analysis of mineralized tissues, with special reference to otoconia, the calcified structures of the vestibular system.