The goal of this project is to characterize the shape, molecular weight distribution and elemental composition of individual macromolecules and macromolecular assemblies, with emphasis on calcium binding by, and phosphorylation of, regulatory proteins. This project takes advantage of combining field-emission scanning transmission electron microscopy (STEM) with electron energy loss spectroscopy (EELS) ? a new technique known as spectrum imaging ? to analyze and map the distribution of physiologically important elements like P and Ca at a resolution of better than 10 nm. Application of this technology is illustrated by spectrum imaging of peripheral mitochondria in depolarized neurons of frog sympathetic ganglia. Intramitochondrial Ca maps reveal focal sites of Ca accumulation that are less than 10 nm in diameter; the Ca concentration within these sites is sufficiently high that a spectrum from even a single pixel exhibits a prominent Ca L23 EELS edge. Quantitation of this signal is consistent with a spatially averaged total Ca concentration of ~10 mmol/kg dry wt. These results show for the first time a quantitative elemental distribution map within an individual mitochondrion. Strategies for single atom detection at high resolution are illustrated by EELS mapping of a series of specimens including tobacco mosaic virus (TMV), isolated DNA plasmids, and phosphorylated proteins. Spectrum imaging of TMV yields P maps that correspond to 1-3 P atoms per pixel in the projected central region of the viral particles where the RNA strand lies, whereas outside the central region the number of P atoms is zero. The results demonstrate that single atoms of phosphorus are detectable within biological assemblies.
