Single-Molecule STED Microscopy with Photostable Organic Fluorophores

Authors

  • Robert Kasper,

    1. Applied Laser Physics and Laser Spectroscopy Bielefeld University Universitätsstr. 25, 33615 Bielefeld (Germany)
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    • These authors contributed equally to this work.

  • Benjamin Harke,

    1. Max Planck Institute for Biophysical Chemistry Am Faßberg 11, 37077 Göttingen (Germany)
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    • These authors contributed equally to this work.

  • Carsten Forthmann,

    1. Angewandte Physik–Biophysik and Center for NanoScience Ludwig-Maximilians-University Amalienstr. 54, 80799 Munich (Germany)
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  • Philip Tinnefeld,

    Corresponding author
    1. Angewandte Physik–Biophysik and Center for NanoScience Ludwig-Maximilians-University Amalienstr. 54, 80799 Munich (Germany)
    • Angewandte Physik–Biophysik and Center for NanoScience Ludwig-Maximilians-University Amalienstr. 54, 80799 Munich (Germany)
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  • Stefan W. Hell,

    Corresponding author
    1. Max Planck Institute for Biophysical Chemistry Am Faßberg 11, 37077 Göttingen (Germany)
    • Max Planck Institute for Biophysical Chemistry Am Faßberg 11, 37077 Göttingen (Germany).
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  • Markus Sauer

    Corresponding author
    1. Biotechnology and Biophysics Julius-Maximilians-University Wuerzburg Am Hubland, 97074 Wuerzburg (Germany)
    • Biotechnology and Biophysics Julius-Maximilians-University Wuerzburg Am Hubland, 97074 Wuerzburg (Germany).
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  • This work was supported by the Biophotonics Program of the BMBF/VDI (grant 13N9234) and the Nanosystems Initiative Munich (NIM). We thank R. Medda for providing labeled cells for bleaching experiments on microtubule networks. STED = stimulated emission depletion.

Abstract

original image

The combination of stimulated emission depletion (STED) microscopy with fluorophore stabilization through a reducing and oxidizing system (ROXS) enables the repetitive measurements necessary for 3D or dynamic STED imaging and resolution enhancement at the single-molecule level. A lateral resolution of <30 nm is obtained for raw image data recorded from single organic fluorophores immobilized in aqueous buffer.

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