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Publication

Bibliographic Data

  • Authors: Testa, I.,Wurm, C.A.,Medda, R.,Rothermel, E.,von Middendorf, C.,Foelling, J.,Jakobs, S.,Schoenle, A.,Hell, S.W. and Eggeling, C.
  • Title: Multicolor Fluorescence Nanoscopy in Fixed and Living Cells by Exciting Conventional Fluorophores with a Single Wavelength
  • Journal: Biophys. J.
  • Volume: 99
  • Issue: 8
  • Volume: 2686-2694
  • DOI: 10.1016/j.bpj.2010.08.012

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.