CALCIUM DISTRIBUTION IN HIGH-PRESSURE FROZEN BONE-CELLS BY ELECTRON-ENERGY-LOSS SPECTROSCOPY AND ELECTRON SPECTROSCOPIC IMAGING

Subcellular localization of total calcium requires tissue processing t hat preserves the chemical composition of the samples and a highly sen sitive microanalytical technique. In this study rat fetal bone samples were submitted to high-pressure freezing and freeze substitution. Ult rastructural preservation was good in the superficial sections: osteob lasts near the bone mineral had clearly defined plasma and nuclear mem branes, dense mitochondria, and numerous ribosomes. Electron energy lo ss spectroscopy allowed high-resolution calcium-sensitive images to be obtained using ionization edge loss electrons. In biological samples, the Ca-L-2,L-3 signal is superimposed on the carbon edge and artifact s may result from thickness and scattering effects. Therefore the rela tive thickness was established for each area analyzed (t/lambda<0.5). Background was subtracted using the three-images method, allowing high resolution calcium-sensitive images of intramitochondrial granules an d of intracellular compartments, and semiquantitative data from the gr anules to be obtained. Calcium maps were confirmed by spectra collecte d on defined areas of the images and the shape of the net Ca-L-2,L-3 e dges was compared to the characteristic Ca-L-2,L-3 edge of bone crysta ls. These procedures will provide new information about total calcium localization in bone cells and the possibility of examining the distri bution of other elements.