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Biophys J. 2010 Oct 20;99(8):2686-94. doi: 10.1016/j.bpj.2010.08.012.

Multicolor fluorescence nanoscopy in fixed and living cells by exciting conventional fluorophores with a single wavelength.

Author information

  • 1Department of Nanobiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.

Abstract

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.

Copyright © 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

PMID:
20959110
[PubMed - indexed for MEDLINE]
PMCID:
PMC2956215
Free PMC Article

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