Single-Molecule Imaging Breaks Concentration Barrier

A new fluorescence method extends the concentration range of single-molecule imaging, enabling the technique to now work at physiologically relevant concentrations, Antoine M. van Oijen of the University of Groningen, Johannes C. Walter of Harvard Medical School, and coworkers report (Nat. Methods, DOI: 10.1038/nmeth.2174). The method combines photoswitchable fluorescent labels with total internal reflection microscopy, which restricts illumination to a thin layer at a glass-water interface. The combination works at micro­molar concentrations, whereas other single-molecule imaging methods are limited to nanomolar concentrations. The researchers first label a protein of interest with a photoswitchable protein and allow it to interact with an immobilized substrate. They then illuminate the protein with a wavelength of light that switches the label from one fluorescent form to another. After a few hundred milli-seconds, during which unbound proteins diffuse away, they excite the bound proteins with another wavelength and image the resulting fluorescence. To demonstrate the technique, the researchers tagged and imaged flap endonuclease, a protein involved in stitching together the discontinuous lagging strand of DNA during replication. The results suggest that origins of replication are closer together and more frequent than had been measured with other methods.