Overview. Three-dimensional cryo-electron microscopy holds great potential for the investigation of macromolecular complexes, including viruses, the cytoskeleton, and the nanomachines that carry out many vital cellular functions. Such complexes often contain protein subunits of different kinds, with cumulative molecular weights running well into the megadalton range. To rationalize their functional assignments in structural terms, the first order of business is to map the locations of the component subunits of a given complex. A more detailed objective then becomes to understand the organization of their domains and the overall layout of the polypeptide chains. With current methodology, resolutions below 20Å are often achievable in cryo-EM, extending below 10 Å in favorable cases. Although resolutions of this order yield valuable morphological information, they do not usually suffice to identify individual subunits within a complex. Generalized difference imaging offers a solution to this problem of molecular mapping.