For single particle structure determination in cryo-EM, purified proteins are frozen (vitrified) in their native state. However, the imaging contrast in cryo EM is very weak due to the low density of the elements of life.

Negative staining can be used to increase contrast, but unstained native structural analysis is the method of choice.

During macromolecule purification, sample heterogeneity also often occurs because of degradation processes and protein complexes are destabilized by in vitro buffer solutions. These and other complications can seriously compromise high-resolution 3D reconstruction.