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Diffusion Measurements at the Nanoscale with STED-FCS

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Fluorescence Spectroscopy and Microscopy in Biology

Part of the book series: Springer Series on Fluorescence ((SS FLUOR,volume 20))

Abstract

The cellular plasma membrane, a seemingly chaotic structure composed of thousands of lipids and proteins, empowers cellular life and represents a versatile hub for signalling. Transient interactions of membrane components integrate to large-scale membrane properties such as packing, tension, curvature, or viscosity and reorganise the membrane in response to physiological needs. How do nanoscale interactions and heterogeneities determine cellular plasma membrane properties? This question is not trivial to address with technologies limited by the diffraction of light. Here, we discuss Stimulated Emission Depletion (STED) nanoscopy and its combination with fluorescence correlation spectroscopy (FCS, STED-FCS) as a technique to shed light onto the relevant molecular scales. While imaging with STED allows for tremendous insights into the spatial domain of organisation, the combination of STED with FCS provides increased temporal resolution and yields insights into the nature of the molecular motion on the relevant scales, allowing us to study nanoscale diffusion modes. We provide a comprehensive overview of the field, introduction to the technique and discuss common pitfalls with its implementation as well as alternative methodologies to measure the diffusion modes on the nanoscale.

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Acknowledgements

We thank SciLifeLab Advanced Light Microscopy facility and National Microscopy Infrastructure (VR-RFI 2016-00968) for their support on imaging. ES is supported by grants from Swedish Research Council Starting Grant (2020-02682), from Karolinska Institutet and from the SciLifeLab National COVID-19 Research Program, financed by the Knut and Alice Wallenberg Foundation. FS acknowledges support by EMBO (ALTF 849-2020) and HFSP (LT000404/2021-L).

Author Contributions

ES and FS wrote the manuscript and prepared the figures. Both authors contributed equally to the chapter.

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Authors and Affiliations

  1. Translational Imaging Center, University of Southern California, Los Angeles, CA, USA

    Falk Schneider

  2. Science for Life Laboratory, Department of Women’s and Children’s Health, Karolinska Institute, Solna, Sweden

    Erdinc Sezgin

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  1. Falk Schneider
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  2. Erdinc Sezgin
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Correspondence to Erdinc Sezgin .

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Editors and Affiliations

  1. J. Heyrovský Institute of Physical Chemistry, Prague, Czech Republic

    Radek Šachl

  2. J. Heyrovský Institute of Physical Chemistry, Prague, Czech Republic

    Mariana Amaro

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Schneider, F., Sezgin, E. (2022). Diffusion Measurements at the Nanoscale with STED-FCS. In: Šachl, R., Amaro, M. (eds) Fluorescence Spectroscopy and Microscopy in Biology. Springer Series on Fluorescence, vol 20. Springer, Cham. https://doi.org/10.1007/4243_2022_27

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