Scanning transmission electron microscopy (STEM) provides a versatile method for determining the molecular mass and hence the arrangement of subunits in large protein assemblies. Macromolecules are adsorbed onto a thin support film and a nanometer-sized electron probe is scanned across the specimen while the elastic scattering signal is collected. The resulting digital image intensity is proportional to the local mass density of the specimen. Images are recorded at low electron dose by counting single pulses from an annular detector positioned after the specimen. STEM can be applied directly to individual macromolecules in a heterogeneous population and is not therefore subject to averaging artifacts. Mass determination can often be made to an accuracy of 1 or 2% based on analysis of only 1000 molecules, which corresponds to about 1 femtogram of material, although in practice several nanograms are currently required to prepare a suitable specimen.
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