Manganese detected by electron spectroscopy imaging and electron energy loss spectroscopy in mitochondria of normal rat brain cells

Electron spectroscopy imaging (ESI) and electron energy loss spectroscopy (EELS) provide an opportunity to investigate the presence of different trace elements directly in the structure of fixed tissues. We evaluated the usefulness of such techniques in detecting the sub-cellular localization and relative content of manganese in the mitochondria of cells in the brain of normal rats. ESI provided a granular appearance to manganese ions aggregated at any site summed through the thickness of ultra-thin sections. EELS confirmed the presence of manganese in the organelles shown by ESI. To demonstrate the specificity of signals for manganese, a comparative study was also performed. Electron-spectroscopy analysis of manganese, iron and fluorine in the same mitochondria demonstrated the presence of strong and specific signals for manganese, but the absence of detectable iron, or of fluorine, which was expected to be normally absent. However, iron and manganese were both detected in myelin sheaths by ESI and the spectral analysis afforded by EELS revealed distinct peaks for manganese and iron in the same samples. The manganese concentration in mitochondria as measured by the relative optical density on photomicrographs was highly variable probably due to variability in organelle characteristics and subtle differences in section thickness. These results show that ESI and EELS are sensitive and valuable techniques to detect bound manganese within sub-cellular structures.