We describe a method, based on single-molecule imaging, that allows the real-time visualization of the infection pathway of single viruses in living cells, each labeled with only one fluorescent dye molecule. The tracking of single viruses removes ensemble averaging. Diffusion trajectories with high spatial and time resolution show various modes of motion of adeno-associated viruses (AAV) during their infection pathway into living HeLa cells: (i) consecutive virus touching at the cell surface and fast endocytosis; (ii) free and anomalous diffusion of the endosome and the virus in the cytoplasm and the nucleus; and (iii) directed motion by motor proteins in the cytoplasm and in nuclear tubular structures. The real-time visualization of the infection pathway of single AAVs shows a much faster infection than was generally observed so far.

Approach, contact, retreat; approach, contact, penetrate. With a number of fleeting subsecond contacts, the single virus moves across the cellular membrane (paths 1, 2 in the graphic) before it is suddenly engulfed (path 3). Although only a few viruses are successful, once inside they enter the nucleus in a matter of minutes (path 4), much faster than previously thought. Single virus tracing, the application of real‐time single‐molecule techniques to this biomedical problem, enables us to view the live scenes of viral infection with high spatial and temporal resolution and under ideal physiological conditions in a living cell. In this way, the “movie script” of a viral infection is obtained.

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