Comparative mass measurement of biological macromolecules by scanning transmission electron microscopy

A method is described for measuring the mass/length or mass of molecular assemblies by comparative electron scattering in the STEM. Standard particles whose mass is well established (e.g. TMV or fd bacteriophage) are deposited on the electron microscope grid together with the sample to be measured. Images containing at least one sample and standard and with a clean, contamination-free background are chosen and stored on computer disc and then directly integrated. Use of a comparative technique does not require accurate determination of scattering parameters or instrumental geometry and requires only that the limits of linearity be established. The results of the mass/length measurements on phage pf 1, pili, muscle thick filaments and actin are in good agreement with existing molecular weight data and generally have a standard deviation of about 10%. The results for the total mass measurement of the multisubunit enzymes glutamate dehydrogenase and glutamine synthetase are also close to the literature values for their molecular weights. The results for the spherical, Semliki forest and tomato bushy stunt viruses are lower than expected, possibly reflecting some dissociation during preparation.