1984). By rapid cooling of a thin layer of an aqueous solution of macromolecules on an EM grid, a thin amorphous layer of ice is formed,

in which objects are visible without any staining agent. Ice-embedded specimens very much reflect cellular aqueous situations, and hence the method quickly became popular within the field. Because the contrast is only caused by the difference in density between amorphous ice (0.93 g/cm3) and protein (1.3–1.36 g/cm3), it is rather low in comparison to negative staining. It is obvious that for large objects such as symmetric virus molecules, cryo-EM is superior to negative staining. However, in the case of unstable protein complexes, which cannot be purified to AZD1152 manufacturer homogeneity (e.g., large, transient membrane complexes), unstained specimens can be a real problem. Due to the low contrast, the object of choice cannot be discriminated from all kinds of contaminants and PI3K inhibitor breakdown products. The low contrast is, however, likely to be improved in the near future by instrumental improvements, such as implementing phase plates in the microscopes, such as the Zernike phase plate (Yamaguchi et al. 2008). There are several advantages of cryo-EM of vitrified specimens: specimen flattening and other drying artifacts are circumvented. Moreover, cryo-images better reflect the true density of a protein, because the contrast directly originates from scattering

by the protein rather than from the surrounding stain. Also, the interaction of negative stain with the protein is often quite complex if the object is not fully embedded. In thinner stain layers,

the upper part of the protein could easily be less Rapamycin well embedded in the stain CHIR 99021 layer, as pointed out in Fig. 1. This means that the contributions of the upper- and lower half of a protein in the final recorded image do not have the same weighting. In contrast, the embedding in a full ice layer gives a more straightforward signal. Cryo-negative staining represents a complementary method for the conventional negative stain EM and a valuable alternative in particular for situations where cryo-EM reaches its limits in terms of visibility of the protein complexes (De Carlo et al. 2008). In cryo-negative staining, particles become embedded in a rather thick layer of stain which is not fully dehydrated, which may prevent flattening and preferential staining. Fig. 1 An example of the footprint effect of negative staining. a A part of a double-layered two-dimensional crystal containing about 1500 photosystem I monomers from a cyanobacterium (Böttcher et al. 1992). b, c Filtered images resulting from a crystallographic analysis in which the two layers could be separated. The crystal is composed of rows of monomers. Within the rows, the monomers are either up- or down-oriented, and there is a substantial difference in overall contrast between individual rows of monomers in the upper layer with respect to the lower layer.