We have investigated the regulation of calcium in neurons by using electron energy-loss spectroscopic imaging to map elemental distributions in frog sympathetic ganglia at high spatial resolution in the scanning transmission electron microscope (STEM). The neurons which had been prepared by rapid freezing and cryosectioning were found to accumulate calcium within their mitochondria in the period of a few minutes after depolarization. Elemental maps, obtained by subtracting the background at each pixel, revealed that the peripheral regions of the neurons accumulated abundant small ten-nanometer sized calcium and phosphorus-rich deposits within their matrix, whereas in central regions of the cell the deposits were much more sparsely distributed. Quantitative analysis showed that the calcium concentration in the mitochondrial matrix was at a low level of approximately 2 mmol/kg. The small, mineral-like inclusions disappeared in neurons that were allowed to recover. This reversibility may reflect a high-capacity mechanism of intramitochondrial calcium sequestration. The radial dependence of depolarization-evoked elevations in mitochondrial calcium on distance from the plasma membrane, as well as its persistence, indicates that mitochondria can retain a record of early spatial differences in intracellular calcium long after such gradients have dissipated. - Compositional imaging, electron energy loss spectroscopy, STEM, elemental mapping, calcium, mitochondria, sympathetic neurons
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