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

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.

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Fig. 1: Principles of STED–FCS.
Fig. 2: Schematic overview of the protocol.
Fig. 3: Alignment and calibration of the system.
Fig. 4: Artifacts in STED–FCS due to temporal misalignment of the excitation and STED laser (Time sync) and defocusing (Focus shift).
Fig. 5: Representative pSTED–FCS data for fluorescent phospholipids, sphingolipids and GPI-APs.
Fig. 6: Representative sSTED–FCS data for fluorescent phospholipids, sphingolipids and GPI-APs.
Fig. 7: Impact of photobleaching on sSTED–FCS and troubleshooting.
Fig. 8: Impact of bright signal bursts on sFCS and troubleshooting.

Data availability

The STED–FCS data are available upon request. The software used for data analysis is freely available (‘Equipment’).

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Acknowledgements

We thank the Wolfson Imaging Centre Oxford and the Micron Advanced Bioimaging Unit (Wellcome Trust Strategic Award 091911) for providing the microscope facility and financial support. We acknowledge funding by the Wolfson Foundation, the Medical Research Council (MRC, grant no. MC_UU_12010/unit programs G0902418 and MC_UU_12025), MRC/BBSRC/EPSRC (grant no. MR/K01577X/1), the Wellcome Trust (grant no. 104924/14/Z/14), the Deutsche Forschungsgemeinschaft (Research unit 1905 ‘Structure and function of the peroxisomal translocon’) and Oxford internal funds (John Fell Fund and EPA Cephalosporin Fund). E.S. is funded by a Newton-Katip Ҫelebi Institutional Links grant (352333122). I.U. is grateful for support from the European Commission through a Marie Skłodowska-Curie individual fellowship (707348). D.W. is funded by a URKI MRC grant (MR/S005382/1).

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E.S., F.S., S.G., I.U., D.W., B.C.L. and C.E. all took part in acquiring and analyzing the data and writing the manuscript.

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Correspondence to Erdinc Sezgin or Christian Eggeling.

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Key references using this protocol

Eggeling, C. et al. Nature 457, 1159–1162 (2009): https://www.nature.com/articles/nature07596

Honigmann, A. et al. Nat. Commun. 5, 5412 (2014): https://www.nature.com/articles/ncomms6412

Schneider, F. et al. Mol. Biol. Cell 28, 1507–1518 (2017): https://www.molbiolcell.org/doi/10.1091/mbc.e16-07-0536

Integrated supplementary information

Supplementary Figure 1

Loading the TCSPS data.

Supplementary Figure 2

Processing the TCSPS data.

Supplementary Figure 3

Fitting the correlated data.

Supplementary Figure 4

Navigating and saving the fit results.

Supplementary Figure 5

Loading the time mode data.

Supplementary Figure 6

Processing the sSTED–FCS data.

Supplementary Figure 7

Photobleaching correction for sSTED–FCS data.

Supplementary Figure 8

Spatial and temporal cropping of sSTED–FCS data.

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Sezgin, E., Schneider, F., Galiani, S. et al. Measuring nanoscale diffusion dynamics in cellular membranes with super-resolution STED–FCS. Nat Protoc 14, 1054–1083 (2019). https://doi.org/10.1038/s41596-019-0127-9

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