Fig. 3.
Detection and quantitation of changes in [Ca]mito by elemental analysis. a, EDX spectra recorded from typical areas (<104nm2, size indicated by white dot on mitochondrion in Fig. 2b, inset) of cytoplasm (fine line) and mitochondrial matrix (heavy line) within cryosectioned neurons from a control ganglion. Spectra are plots of the number of binned x-ray counts versus energy (resolution 10 eV per channel). So-called characteristic peaks identify individual elements, and the integrated counts for each peak are proportional to the amount of that element. Elements corresponding to the major characteristic K-shell x-ray peaks are identified; in the case of K and Ca, two K-shell transitions arising from alternative electronic transitions are resolved, indicated asKα and Kβaccording to standard spectroscopic notation. Spectra ina, representing the sum of eight individual spectra each recorded for 100 sec at a beam current of ∼4.0 nA, illustrate typical distributions of major elements in living neurons. Note the approximately twofold difference in the slowly varying continuum radiation, e.g., between 1.50 and 1.75 keV, which reflects differences in the mass density of these cellular compartments. b, Mitochondrial EDX spectrum (sum of 10 individual spectra, each recorded for 100 sec) over the energy range 3.0–4.2 keV (dashed boxin a) recorded under control conditions. Superimposed are best-fit Gaussians for the K Kα plusKβ peaks (dotted curve) obtained as described in Materials and Methods. Vertical lines indicate energies of the K and Ca lines.c, Residual spectrum, obtained by subtracting the calculated K Kα andKβ counts from the experimental spectrum, indicates the absence of resolvable Ca peaks. d, Mitochondrial EDX spectrum (sum of 11 individual spectra, 100 sec each) recorded from a ganglion after a 45 sec exposure to 50 mmK+. Dotted and dashed traces show best-fit Gaussians for characteristic K and Ca peaks, respectively. e, Difference spectrum illustrating contributions from the Ca Kα andKβ peaks. The mean depolarization-induced elevation in [Ca]mito over these 11 mitochondria was ∼25 mmol/kg dry weight.