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Nat Protoc. 2019 Apr;14(4):1054-1083. doi: 10.1038/s41596-019-0127-9. Epub 2019 Mar 6.

Measuring nanoscale diffusion dynamics in cellular membranes with super-resolution STED-FCS.

Author information

1
MRC Human Immunology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK. erdinc.sezgin@rdm.ox.ac.uk.
2
MRC Human Immunology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK.
3
Solid State Physics Department, Jožef Stefan Institute, Ljubljana, Slovenia.
4
Wolfson Imaging Centre, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK.
5
MRC Centre for Computational Biology, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK.
6
MRC Human Immunology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK. christian.eggeling@uni-jena.de.
7
Wolfson Imaging Centre, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK. christian.eggeling@uni-jena.de.
8
Institute of Applied Optics, Friedrich-Schiller-University Jena, Jena, Germany. christian.eggeling@uni-jena.de.
9
Department of Biophysical Imaging, Leibniz Institute of Photonic Technology e.V., Jena, Germany. christian.eggeling@uni-jena.de.

Abstract

Super-resolution microscopy techniques enable optical imaging in live cells with unprecedented spatial resolution. They unfortunately lack the temporal resolution required to directly investigate cellular dynamics at scales sufficient to measure molecular diffusion. These fast time scales are, on the other hand, routinely accessible by spectroscopic techniques such as fluorescence correlation spectroscopy (FCS). To enable the direct investigation of fast dynamics at the relevant spatial scales, FCS has been combined with super-resolution stimulated emission depletion (STED) microscopy. STED-FCS has been applied in point or scanning mode to reveal nanoscale diffusion behavior of molecules in live cells. In this protocol, we describe the technical details of performing point STED-FCS (pSTED-FCS) and scanning STED-FCS (sSTED-FCS) measurements, from calibration and sample preparation to data acquisition and analysis. We give particular emphasis to 2D diffusion dynamics in cellular membranes, using molecules tagged with organic fluorophores. These measurements can be accomplished within 4-6 h by those proficient in fluorescence imaging.

PMID:
30842616
DOI:
10.1038/s41596-019-0127-9
[Indexed for MEDLINE]

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