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Wide-Field Fluorescence Lifetime Imaging of Single Molecules

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Download Hi-Res ImageDownload to MS-PowerPointCite This:J. Phys. Chem. A 2020, 124, 17, 3494-3500
    Article

    Wide-Field Fluorescence Lifetime Imaging of Single Molecules
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    • Nazar Oleksiievets
      Nazar Oleksiievets
      III. Institute of Physics−Biophysics, Georg August University, 37077 Göttingen, Germany
      More by Nazar Oleksiievets
    • Jan Christoph Thiele
      Jan Christoph Thiele
      III. Institute of Physics−Biophysics, Georg August University, 37077 Göttingen, Germany
      More by Jan Christoph Thiele
    • André Weber
      André Weber
      Special Laboratory for Electron and Laser Scanning Microscopy, Leibniz Institute for Neurobiology, 39118 Magdeburg, Germany
      More by André Weber
    • Ingo Gregor
      Ingo Gregor
      III. Institute of Physics−Biophysics, Georg August University, 37077 Göttingen, Germany
      More by Ingo Gregor
    • Oleksii Nevskyi
      Oleksii Nevskyi
      III. Institute of Physics−Biophysics, Georg August University, 37077 Göttingen, Germany
      More by Oleksii Nevskyi
    • Sebastian Isbaner
      Sebastian Isbaner
      III. Institute of Physics−Biophysics, Georg August University, 37077 Göttingen, Germany
      More by Sebastian Isbaner
    • Roman Tsukanov*
      Roman Tsukanov
      III. Institute of Physics−Biophysics, Georg August University, 37077 Göttingen, Germany
      *E-mail: [email protected]. Phone: +49 551 39 26911.
      More by Roman Tsukanov
      Orcidhttp://orcid.org/0000-0002-3967-1755
    • Jörg Enderlein*
      Jörg Enderlein
      III. Institute of Physics−Biophysics, Georg August University, 37077 Göttingen, Germany
      Cluster of Excellence “Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells” (MBExC), Georg August University, 37077 Göttingen, Germany
      *E-mail: [email protected]. Phone: +49 551 39 26908.
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      Orcidhttp://orcid.org/0000-0001-5091-7157
    Other Access OptionsSupporting Information (1)

    The Journal of Physical Chemistry A

    Cite this: J. Phys. Chem. A 2020, 124, 17, 3494–3500
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    https://doi.org/10.1021/acs.jpca.0c01513
    Published April 7, 2020
    Copyright © 2020 American Chemical Society
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    Abstract

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    Fluorescence lifetime imaging (FLIM) has become an important microscopy technique in bioimaging. The two most important of its applications are lifetime-multiplexing for imaging many different structures in parallel, and lifetime-based measurements of Förster resonance energy transfer. There are two principal FLIM techniques, one based on confocal-laser scanning microscopy (CLSM) and time-correlated single-photon counting (TCSPC) and the other based on wide-field microscopy and phase fluorometry. Although the first approach (CLSM-TCSPC) assures high sensitivity and allows one to detect single molecules, it is slow and has a small photon yield. The second allows, in principal, high frame rates (by 2–3 orders of magnitude faster than CLSM), but it suffers from low sensitivity, which precludes its application for single-molecule imaging. Here, we demonstrate that a novel wide-field TCSPC camera (LINCam25, Photonscore GmbH) can be successfully used for single-molecule FLIM, although its quantum yield of detection in the red spectral region is only ∼5%. This is due to the virtually absent background and readout noise of the camera, assuring high signal-to-noise ratio even at low detection efficiency. We performed single-molecule FLIM of different red fluorophores, and we use the lifetime information for successfully distinguishing between different molecular species. Finally, we demonstrate single-molecule metal-induced energy transfer (MIET) imaging which is a first step for three-dimensional single-molecule localization microscopy (SMLM) with nanometer resolution.

    Copyright © 2020 American Chemical Society

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    Supporting Information

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    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jpca.0c01513.

    • Estimation of the detected number of photons emitted by each type of fluorophore; cumulative TCSPC curves of surface-immobilized fluorophores; fluorescence lifetimes of freely diffusing fluorophores; comparison of lifetime values measured with the LINCam25 and with TCSPC-CLSM; comparison between lifetime values obtained in LINCam25 measurements of pure and mixed fluorophores samples; influence of virtual pixel size on resolution of the lifetime histograms; on/off-state-detection algorithm and example of improved peak separation in lifetime histograms after applying a on/off-state-detection algorithm; description and scheme of experimental setup; analysis of peak broadening of lifetime histogram for Atto 655; detailed DNA sequences and its modifications; analysis of the blinking behavior of Atto 655; and MIET experiment details and experimental and calculated lifetime and height values for the DNA-conjugated Atto 655 fluorophores (PDF)

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    Cited By

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    This article is cited by 38 publications.

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    The Journal of Physical Chemistry A

    Cite this: J. Phys. Chem. A 2020, 124, 17, 3494–3500
    Click to copy citationCitation copied!
    https://doi.org/10.1021/acs.jpca.0c01513
    Published April 7, 2020
    Copyright © 2020 American Chemical Society
    Request reuse permissions

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