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 components of the cytoskeleton. This project depends on our unique field-emission scanning transmission electron microscope (STEM), which is equipped for dark-field mass mapping of protein molecular weight distributions at 2 nm resolution and for analyzing and mapping by electron energy loss (EELS) spectroscopy the distribution of physiologically important elements such as P, Ca and S at a resolution of <10 nm. Application of this technology is well illustrated by the determination of the phosphorylation and subunit organization of individual squid axonal neurofilaments; recent refinement of these data have revealed a novel structure for intermediate filaments, consisting of eight coiled-coil heterodimers per cross section, each with one heavy and one light chain. The heavy chain carries at least 40 phosphate groups. This work, requiring a sensitivity of 0.5 P atoms/nm, represents the first biological application of """"""""nanoanalysis"""""""".
