Multicolor fluorescence nanoscopy in fixed and living cells by exciting conventional fluorophores with a single wavelength
2010
Authors: Testa I, Wurm CA, Medda R, Rothermel E, von Middendorf C, Fölling J, Jakobs S, Schönle A, Hell SW, Eggeling C
CellNetworks People: Hell Stefan
Journal: Biophys J. 2010 Oct 20;99(8):2686-94. doi: 10.1016/j.bpj.2010.08.012

Current far-field fluorescence nanoscopes provide subdiffraction resolution by exploiting a mechanism of fluorescence inhibition. This mechanism is implemented such that features closer than the diffraction limit emit separately when simultaneously exposed to excitation light. A basic mechanism for such transient fluorescence inhibition is the depletion of the fluorophore ground state by transferring it (via a triplet) in a dark state, a mechanism which is workable in most standard dyes. Here we show that microscopy based on ground state depletion followed by individual molecule return (GSDIM) can effectively provide multicolor diffraction-unlimited resolution imaging of immunolabeled fixed and SNAP-tag labeled living cells. Implemented with standard labeling techniques, GSDIM is demonstrated to separate up to four different conventional fluorophores using just two detection channels and a single laser line. The method can be expanded to even more colors by choosing optimized dichroic mirrors and selecting marker molecules with negligible inhomogeneous emission broadening.