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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.

Original publication

DOI

10.1016/j.bpj.2010.08.012

Type

Journal article

Journal

Biophys J

Publication Date

20/10/2010

Volume

99

Pages

2686 - 2694

Keywords

Animals, Cell Line, Cell Survival, Color, Fluorescent Dyes, Microscopy, Atomic Force, Microscopy, Fluorescence, Nanotechnology, Potoroidae, Spectrometry, Fluorescence