Scanning Microscopy


Electron energy loss spectrometry - EELS, and energy dispersive X-ray microanalysis - XRMA, were used to study the elemental composition of mitochondrial dense granules - mdg. The study was performed on dry cut thin sections (80-200 nm) of freeze-dried and low temperature embedded cartilage. Results obtained by means of XRMA clearly showed high phosphorus and calcium content in the mdg. Using EELS at 100 kV primary voltage we found that small concentrations of elements (i.e. below typically 1% atomic weight) are difficult to analyze and map, this especially in sections thicker than 50-60 nm. Surprisingly, analysis of calcium can be successfully performed on thicker sections though the edge lies above the carbon K edge while this is not possible for the phosphorus edge which is located at lower energies. This is likely due to the edge shapes (sharp for calcium and delayed for phosphorus), and to the more intense contribution of multiple low loss scattering in the background for phosphorus between 100 and 130 eV. By means of EELS elemental mapping a centrally located core was found in numerous mdg. In the calcium map the signal was strongest in the middle of mdg which corresponds to the area of reduced carbon signal. We found that carbon maps might be used for high resolution structural studies of chemically unfixed and anhydrously processed biological tissues. As carbon is the main constituent of Lowicryl resin its distribution is reversed to the distribution of biological tissue in which the proportion of carbon is lower, but is proportional to water content in the specimen in vivo. Use of EELS in combination with electron microscope with accelerating voltages in range of 140-200 kV together with anhydrous techniques of the tissue preparation will provide a new type of information which might lead to better understanding of the etiology and function of small structures in the cell.

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