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 that preserves the chemical composition of the samples and a highly sensitive microanalytical technique. In this study rat fetal bone samples were submitted to high-pressure freezing and freeze substitution. Ultrastructural preservation was good in the superficial sections: osteoblasts near the bone mineral had clearly defined plasma and nuclear membranes, dense mitochondria, and numerous ribosomes. Electron energy loss 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 artifacts may result from thickness and scattering effects. Therefore the relative 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 and of intracellular compartments, and semiquantitative data from the granules to be obtained. Calcium maps were confirmed by spectra collected on defined areas of the images and the shape of the net Ca-L-2,L-3 edges was compared to the characteristic Ca-L-2,L-3 edge of bone crystals. These procedures will provide new information about total calcium localization in bone cells and the possibility of examining the distribution of other elements.