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Home > Research > Biotechnology Resource Centers
Stem Mass Mapping and Heavy Atom Labeling of BiomoleculesContents
Contact InformationBrookhaven National Laboratory
Brookhaven Science Associates, Inc. Biology Department, Building 463 Upton, NY 11973-5000 Principal Investigator/Contact
Grant NumberGrant No. EB002181
Research EmphasisThe scanning transmission electron microscope (STEM) produces an image one point at a time by moving a finely focused electron beam over a specimen. The numbers of scattered and unscattered electrons are measured at each point, giving a quantitative determination of the scattering power of the irradiated volume. For thin specimens (single biological molecules or complexes), this gives a direct measure of local mass; summing over an entire object gives its total mass. The spatial resolution of this mass map is typically 2-4 nm, limited by radiation damage to the freeze-dried specimen (probe size is only 0.25 nm). The contrast is high enough to "see" unstained DNA, proteins larger than 10 KDa, lipid layers, and single heavy atoms.
Current ResearchMost STEM studies use this quantitative imaging capability to determine the number and spatial arrangement of subunits (of known size) in a larger structure. Typically, 50 different projects are active at any given time.
Particularly active research areas are:
A wide variety of intermediates can be recognized and characterized in a single preparation. Intact structures from various sources can be compared in parallel preparations. Purified subcellular components in the size range 30 KDa to 500 MDa are the subject of many more studies. Tobacco mosaic virus is included in all STEM specimens as a control for specimen preparation quality, radiation damage, and mass calibration. Mass accuracy ranges from 10% for small particles to better than 1% for objects larger than 1 MDa.
In structures with cylindrical or spherical symmetry, the projected mass distribution (the direct STEM output) can be transformed to give a radial density profile. This usually requires aligning and averaging images of many similar particles in order to reduce noise. Specific labeling of interesting sites within complexes provides additional information about orientation of subunits or location of one type of unit in a mixed complex. Heavy atom clusters containing 11 or 67 gold atoms in a dense core provide adequate signal for low-dose visualization. These have been developed as monofunctional reagents that can be attached directly to a structure (e.g., accessible sulfhydryl or amino side chain in a protein) or through an intermediary such as an antibody fragment, biotin/avidin, or histidine tag. Localization is 2-4 nm, limited by radiation damage to the freeze-dried specimen. Higher spatial resolution can be obtained using the low-density negative stain methylamine vanadate to embed the specimen.
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