X-ray microanalysis of intracellular ions in epithelial tissues
Electron probe x-ray microanalysis provides a powerful tool in studying transepithelial ion transport mechanisms as it allows the quantitative detection of diffusible ions on a cellular and subcellular scale. The high spatial resolution afforded by this method is of particular advantage in epithelial tissues that are composed of different cell types and cases in which the transcellular transport pathway might involve different intracellular compartments. In the following, the methods applied in several studies are described, with emphasis on some recent modifications.The tissues are frozen by rapid immersion in liquified ethane kept at -180°C. Support systems for different epithelial preparations have been developed to facilitate freezing and maintain the functional state of the epithelium immediately up to the moment of freezing. A fluid-clamp technique is used to mount the tissue for sectioning (Figure 1). Sections are cut dry at -140°C and a nominal thickness of 200 nm using 40° glass knifes. The sections are picked up from the back of the knife by a fire-polished wire probe (1 μm tip diameter) and placed on a parlodion film which is suspended over a 2 mm opening in a special beryllium carrier disc.