Spot Variation Fluorescence Correlation Spectroscopy for Analysis of Molecular Diffusion at the Plasma Membrane of Living Cells | Zendy

Sébastien Mailfert | Zendy; Karolina Wójtowicz | Zendy; Sophie Brustlein | Zendy; Ewa Błaszczak | Zendy; Nicolas Bertaux | Zendy; Marcin Łukaszewicz | Zendy; Didier Marguet | Zendy; Tomasz Trombik | Zendy

Open Access

Spot Variation Fluorescence Correlation Spectroscopy for Analysis of Molecular Diffusion at the Plasma Membrane of Living Cells

Author(s) -

Sébastien Mailfert,

Karolina Wójtowicz,

Sophie Brustlein,

Ewa Błaszczak,

Nicolas Bertaux,

Marcin Łukaszewicz,

Didier Marguet,

Tomasz Trombik

Publication year - 2020

Publication title -

journal of visualized experiments

Language(s) - English

Resource type - Journals

SCImago Journal Rank - 0.596

H-Index - 91

ISSN - 1940-087X

DOI - 10.3791/61823

Subject(s) - fluorescence correlation spectroscopy , membrane , plasma , fluorescence spectroscopy , microscopy , fluorescence microscope , fluorescence , raft , biophysics , materials science , chemistry , biology , optics , biochemistry , physics , polymer , quantum mechanics , copolymer , composite material

Dynamic biological processes in living cells, including those associated with plasma membrane organization, occur on various spatial and temporal scales, ranging from nanometers to micrometers and microseconds to minutes, respectively. Such a broad range of biological processes challenges conventional microscopy approaches. Here, we detail the procedure for implementing spot variation Fluorescence Correlation Spectroscopy (svFCS) measurements using a classical fluorescence microscope that has been customized. The protocol includes a specific performance check of the svFCS setup and the guidelines for molecular diffusion measurements by svFCS on the plasma membrane of living cells under physiological conditions. Additionally, we provide a procedure for disrupting plasma membrane raft nanodomains by cholesterol oxidase treatment and demonstrate how these changes in the lateral organization of the plasma membrane might be revealed by svFCS analysis. In conclusion, this fluorescence-based method can provide unprecedented details on the lateral organization of the plasma membrane with the appropriate spatial and temporal resolution.

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