Magnification calibration and the determination of spherical virus diameters using cryo-microscopy

The diameters of several frozen-hydrated, spherical viruses were determined using polyoma virus as either an external or an internal calibration standard. The methods described provide a reproducible and accurate way to calibrate microscope magnification. The measured diameters are in excellent agreement with respective measurements previously reported for aqueous samples at room temperature using X-ray diffraction methods. These results indicate that the native morphology and dimensions of biological macromolecules are better preserved in frozen-hydrated samples when compared with more conventional electron microscopy techniques such as negative-staining, metal shadowing or thin-sectioning.