13] Electron probe x-ray microanalysis of Ca2+, Mg2+, and other ions in rapidly frozen cells

Abstract

Publisher Summary A unique contribution of electron probe X-ray microanalysis (EPMA) to biology is its capability to determine in situ and quantitatively the distribution of elements within organelles and in the cell cytoplasm. EPMA is also a truly microanalytic method as it can be used to quantitate the elemental composition of microdroplets and other very small samples, which is equivalent to or smaller than a single phage head or ferritin molecule. The subcellular compartmentalization and transport of elements within tissues in various functional states can be captured with this technique on a millisecond time scale using ultrarapid freezing. Meaningful quantitation requires that tissue preparatory techniques—including dissection, freezing, cryosectioning, and transfer to the microscope—as well as the analysis itself preserve the elemental composition as it exists in vivo . This precludes the use of chemical fixatives and stains. EPMA is based on the detection of signals due to atomic core-shell excitations that are, for practical purposes, insensitive to a chemical environment; therefore, it provides a measure of total elemental concentration. A single energy-dispersive X-ray spectrum contains information about all elements present from atomic number 11 (sodium) and higher as well as about the total mass in which these elements are contained. This chapter describes the tissue preparatory methods and the techniques for extracting quantitative information from X-ray spectra.