Mass Mapping Of Macromolecular Assemblies

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Scanning transmission electron microscopy (STEM) provides a versatile method for determining the molecular mass and hence the arrangement of subunits in large protein assemblies. We have implemented STEM mass mapping in a 300 kV transmission electron microscope (FEI Tecnai TF30) equipped with a field-emission source and probe forming electron optics. A high-efficiency annular dark-field detector was installed in a port after the final projector lens of the microscope column. Addition of this capability to an advanced electron microscope will enable cryo-electron microscopy of frozen hydrated specimens, which can be subsequently dehydrated in situ and imaged by STEM to determine the mass distribution within the same structures. By adjusting the microscope?s projector lenses, it is possible to vary the camera length so that the range of scattering angles selected by the annular detector can be optimized. We have shown that it is possible to quantify the mass measurements by incorporating a theoretical model for the scattering cross sections (recent NIST Database). 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 can be recorded at low electron dose without significant radiation damage to the structures of interest. We have successfully tested the mass mapping system on the 300 kV FEI Tecnai electron microscope by analyzing the well known structures, tobacco mosaic virus particles and mollusk hemocyanin. We are now able to perform mass mapping with our 300 kV instrument as well as with our dedicated 100 kV VG Microscopes HB501 STEM. We have applied the technique to a number of projects including characterization of proteins associated with motility of the simple bacterium, Spiroplasma, and to determine the molecular mass of postsynaptic densities.