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Bleaching protection and axial sectioning in fluorescence nanoscopy through two-photon activation at 515 nm

Author

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  • Jan-Erik Bredfeldt

    (Max Planck Institute for Multidisciplinary Sciences
    University of Göttingen)

  • Joanna Oracz

    (Max Planck Institute for Multidisciplinary Sciences)

  • Kamila A. Kiszka

    (Max Planck Institute for Multidisciplinary Sciences)

  • Thea Moosmayer

    (Max Planck Institute for Multidisciplinary Sciences
    University of Göttingen)

  • Michael Weber

    (Max Planck Institute for Multidisciplinary Sciences)

  • Steffen J. Sahl

    (Max Planck Institute for Multidisciplinary Sciences)

  • Stefan W. Hell

    (Max Planck Institute for Multidisciplinary Sciences
    Max Planck Institute for Medical Research)

Abstract

Activation of caged fluorophores in microscopy has mostly relied on the absorption of a single ultraviolet (UV) photon of ≲400 nm wavelength or on the simultaneous absorption of two near-infrared (NIR) photons >700 nm. Here, we show that two green photons (515 nm) can substitute for a single photon (~260 nm) to activate popular silicon-rhodamine (Si-R) dyes. Activation in the green range eliminates the chromatic aberrations that plague activation by UV or NIR light. Thus, in confocal fluorescence microscopy, the activation focal volume can be matched with that of confocal detection. Besides, detrimental losses of UV and NIR light in the optical system are avoided. We apply two-photon activation (2PA) of three Si-R dyes in different superresolution approaches. STED microscopy of thick samples is improved through optical sectioning and photobleaching reduced by confining active fluorophores to a thin layer. 2PA of individualized fluorophores enables MINSTED nanoscopy with nanometer-resolution.

Suggested Citation

  • Jan-Erik Bredfeldt & Joanna Oracz & Kamila A. Kiszka & Thea Moosmayer & Michael Weber & Steffen J. Sahl & Stefan W. Hell, 2024. "Bleaching protection and axial sectioning in fluorescence nanoscopy through two-photon activation at 515 nm," Nature Communications, Nature, vol. 15(1), pages 1-13, December.
    • Handle: RePEc:nat:natcom:v:15:y:2024:i:1:d:10.1038_s41467-024-51160-9
    DOI: 10.1038/s41467-024-51160-9
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    References listed on IDEAS

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    1. Michelle S. Frei & Philipp Hoess & Marko Lampe & Bianca Nijmeijer & Moritz Kueblbeck & Jan Ellenberg & Hubert Wadepohl & Jonas Ries & Stefan Pitsch & Luc Reymond & Kai Johnsson, 2019. "Photoactivation of silicon rhodamines via a light-induced protonation," Nature Communications, Nature, vol. 10(1), pages 1-10, December.
    2. Katrin I. Willig & Silvio O. Rizzoli & Volker Westphal & Reinhard Jahn & Stefan W. Hell, 2006. "STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis," Nature, Nature, vol. 440(7086), pages 935-939, April.
    3. Roman Schmidt & Tobias Weihs & Christian A. Wurm & Isabelle Jansen & Jasmin Rehman & Steffen J. Sahl & Stefan W. Hell, 2021. "MINFLUX nanometer-scale 3D imaging and microsecond-range tracking on a common fluorescence microscope," Nature Communications, Nature, vol. 12(1), pages 1-12, December.
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