Biomed. Opt. Express 2, 2364–2371 (2011)

Credit: © 2011 OSA

Stimulated emission depletion (STED) microscopy — an imaging technique that offers diffraction-unlimited resolution — is usually restricted to the one- or two-colour imaging of fixed cells, thus significantly restricting its application in the study of protein dynamics. Patrina Pellett and colleagues from the USA have now developed an elegant solution for live-cell two-colour imaging. They first connected tags to target epidermal growth factors and epidermal growth factor receptors in living cells, thus allowing them to be labelled by separate fluorescent dyes. The difference in Stokes shift between the dyes allowed the two proteins to be distinguished. By alternating the excitation laser in a line-by-line manner, the team were able to record fluorescence from the two dyes quasi-simultaneously and hence obtain two-colour STED images. They report resolutions of 78 nm and 82 nm for 22 sequential two-colour scans. The researchers anticipate that future developments will allow STED microscopy with more colours and three-dimensional imaging.