Single particle cryo-electron microscopy (cryo-EM) is a technique aimed at structure determination of large macromolecular complexes in their unconstrained, physiological conditions. The power of the method has been demonstrated in selected studies where for highly symmetric molecules the resolution attained permitted backbone tracing. However, most molecular complexes appear to exhibit intrinsic conformational variability necessary to perform their functions. Therefore, it is now increasingly recognized that sample heterogeneity constitutes a major methodological challenge for cryo-EM. To overcome it dedicated experimental and particularly computational multiparticle approaches have been developed. Their applications point to the future of cryo-EM as an experimental method uniquely suited to visualize the conformational modes of large macromolecular complexes and machines.