The use of radial density plots to calibrate images of frozen-hydrated specimens

Abstract

Accurate magnification calibration for transmission electron microscopy is best achieved with appropriate external or internal standards. The use of the icosahedral polyoma virus as a standard in frozen-hydrated preparations was previously described. This method used the known diameter of polyoma (from x-ray measurements) as a reference to calibrate the diameters of other viruses. Measurements were made from circular averages computed from individual particle images or from an average image of many individual particle images. The measured diameters of several different spherical viruses are in good agreement with measurements of the same viruses made with x-ray crystallographic and solution scattering techniques.We found, however, that it is very difficult to accurately measure particle diameters in images taken from frozen-hydrated specimens. This difficulty is attributable to several factors including i) uncertainty about the solvent density level, ii) superposition of density features that occur in two-dimensional projections of three-dimensional objects, iii) uneven outer surfaces of virus particles, iv) characteristically high noise and low contrast levels in micrographs, and v) contrast transfer function (CTF) effects.

Adding the Third Dimension to Virus Life Cycles: Three-Dimensional Reconstruction of Icosahedral Viruses from Cryo-Electron Micrographs

“…This is not always straightforward, since magnetic lens hysteresis as well as changes in the height of the specimen in the objective lens causes small changes in magnification. One way to determine the absolute scale is comparison with a reconstruction of a standard virus whose precise dimensions have been determined from X-ray diffraction (26,30). Direct comparison in three dimensions is much more precise than attempting a similar comparison with the images, particularly for a virion whose aspect changes with orientation.…”