Specificity of stain distribution in electron micrographs of protein molecules contrasted with uranyl acetate.
The mechanism of contrast enhancement of protein molecules by negative staining with uranyl acetate has been investigated by analysing electron micrographs of microcrystals of the human immunoglobulin Dob. Digitally filtered micrographs were compared systematically with idealized reference images which were constructed computationally, starting from knowledge of the primary sequence and three-dimensional crystal structure of this IgG molecule. By separately modelling negative staining as bulk exclusion of heavy metals, and positive staining as the specific decoration of charged amino acid residues, and then combining these simulated images, we were able to assess quantitatively the amount of positive staining present in micrographs of ostensibly 'negatively stained' proteins. At a resolution of 2 nm, we find that the experimental images do indeed exhibit predominantly negative staining, the best matches being obtained by simulations which also include a minor contribution (10-40%) of positive staining. We have also compared two independent measures for the significant resolution present in images of periodic biological specimens: (i) the outermost visible orders of optical diffraction patterns, and (ii) the band-limited resolutions of the idealized simulations when they most closely match the experimental images. These criteria observe close correspondence, thus vindicating the traditional practice of inferring resolution from the optical diffraction spectra of indirectly represented (stained) objects.
D003201:Computers; D003460:Crystallization; D006801:Humans; D007074:Immunoglobulin G / Q000032:analysis*; D008854:Microscopy, Electron; D009942:Organometallic Compounds; D013194:Staining and Labeling; D014501:Uranium
D007074:Immunoglobulin G; D009942:Organometallic Compounds; C005460:uranyl acetate; D014501:Uranium