Tønnesen, J. et al. Biophys J. 101, 2545–2552 (2011).

Super-resolution fluorescence imaging using stimulated emission depletion (STED) microscopy is experiencing increased application to living systems. But because of the need to both stimulate and deplete each fluorophore, extending STED imaging to multiple colors is more complex than it is with conventional fluorescence microscopy. Several methods for two-color STED imaging have been reported, but each has particular drawbacks. Tønnesen et al. now show that the use of fluorophores with similar spectral properties—such as YFP and GFP—coupled with conventional spectral unmixing methods, allows STED using a single pair of lasers—as in single-color STED—and leaves plenty of spectrum available for other dyes. They demonstrated the method's capabilities with time-lapse two-color super-resolution imaging of axonal boutons and dendritic spines in living brain slices labeled with YFP and GFP as well as other fluorophore pairs.