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Correlative single-molecule and structured illumination microscopy of fast dynamics at the plasma membrane

Author

Listed:
  • Hauke Winkelmann

    (Osnabrück University)

  • Christian P. Richter

    (Osnabrück University)

  • Jasper Eising

    (Osnabrück University)

  • Jacob Piehler

    (Osnabrück University
    Osnabrück University)

  • Rainer Kurre

    (Osnabrück University
    Osnabrück University
    Osnabrück University)

Abstract

Total internal reflection fluorescence (TIRF) microscopy offers powerful means to uncover the functional organization of proteins in the plasma membrane with very high spatial and temporal resolution. Traditional TIRF illumination, however, shows a Gaussian intensity profile, which is typically deteriorated by overlaying interference fringes hampering precise quantification of intensities—an important requisite for quantitative analyses in single-molecule localization microscopy (SMLM). Here, we combine flat-field illumination by using a standard πShaper with multi-angular TIR illumination by incorporating a spatial light modulator compatible with fast super-resolution structured illumination microscopy (SIM). This distinct combination enables quantitative multi-color SMLM with a highly homogenous illumination. By using a dual camera setup with optimized image splitting optics, we achieve a versatile combination of SMLM and SIM with up to three channels. We deploy this setup for establishing robust detection of receptor stoichiometries based on single-molecule intensity analysis and single-molecule Förster resonance energy transfer (smFRET). Homogeneous illumination furthermore enables long-term tracking and localization microscopy (TALM) of cell surface receptors identifying spatial heterogeneity of mobility and accessibility in the plasma membrane. By combination of TALM and SIM, spatially and molecularly heterogenous diffusion properties can be correlated with nanoscale cytoskeletal organization and dynamics.

Suggested Citation

  • Hauke Winkelmann & Christian P. Richter & Jasper Eising & Jacob Piehler & Rainer Kurre, 2024. "Correlative single-molecule and structured illumination microscopy of fast dynamics at the plasma membrane," Nature Communications, Nature, vol. 15(1), pages 1-19, December.
    • Handle: RePEc:nat:natcom:v:15:y:2024:i:1:d:10.1038_s41467-024-49876-9
    DOI: 10.1038/s41467-024-49876-9
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    References listed on IDEAS

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    1. Hansjörg Götzke & Markus Kilisch & Markel Martínez-Carranza & Shama Sograte-Idrissi & Abirami Rajavel & Thomas Schlichthaerle & Niklas Engels & Ralf Jungmann & Pål Stenmark & Felipe Opazo & Steffen Fr, 2019. "The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications," Nature Communications, Nature, vol. 10(1), pages 1-12, December.
    2. Christian Eggeling & Christian Ringemann & Rebecca Medda & Günter Schwarzmann & Konrad Sandhoff & Svetlana Polyakova & Vladimir N. Belov & Birka Hein & Claas von Middendorff & Andreas Schönle & Stefan, 2009. "Direct observation of the nanoscale dynamics of membrane lipids in a living cell," Nature, Nature, vol. 457(7233), pages 1159-1162, February.
    3. Marcel Müller & Viola Mönkemöller & Simon Hennig & Wolfgang Hübner & Thomas Huser, 2016. "Open-source image reconstruction of super-resolution structured illumination microscopy data in ImageJ," Nature Communications, Nature, vol. 7(1), pages 1-6, April.
    4. Andreas Markwirth & Mario Lachetta & Viola Mönkemöller & Rainer Heintzmann & Wolfgang Hübner & Thomas Huser & Marcel Müller, 2019. "Video-rate multi-color structured illumination microscopy with simultaneous real-time reconstruction," Nature Communications, Nature, vol. 10(1), pages 1-11, December.
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