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Dissection of mechanical force in living cells by super-resolved traction force microscopy

Abstract

Cells continuously exert or respond to mechanical force. Measurement of these nanoscale forces is a major challenge in cell biology; yet such measurement is essential to the understanding of cell regulation and function. Current methods for examining mechanical force generation either necessitate dedicated equipment or limit themselves to coarse-grained force measurements on the micron scale. In this protocol, we describe stimulated emission depletion traction force microscopy—STED-TFM (STFM), which allows higher sampling of the forces generated by the cell than conventional TFM, leading to a twofold increase in spatial resolution (of up to 500 nm). The procedure involves the preparation of functionalized polyacrylamide gels loaded with fluorescent beads, as well as the acquisition of STED images and their analysis. We illustrate the approach using the example of HeLa cells expressing paxillin-EGFP to visualize focal adhesions. Our protocol uses widely available laser-scanning confocal microscopes equipped with a conventional STED laser, open-source software and common molecular biology techniques. The entire STFM experiment preparation, data acquisition and analysis require 2–3 d and could be completed by someone with minimal experience in molecular biology or biophysics.

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Figure 1: Outline of STFM.
Figure 2: Polyacrylamide gel fabrication and validation.
Figure 3: Relationship between bead density and traction resolution.
Figure 4: Microscope objective characterization.
Figure 5: STFM applied to HeLa cell focal adhesions.

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Acknowledgements

We gratefully acknowledge support from the Wolfson Imaging Centre (C. Lagerholm and E. Garcia) and funding from the Medical Research Council (MRC; grant no. MC_UU_12010/unit programme G0902418 and grant no. MC_UU_12025), the MRC/Biotechnology and Biological Sciences Research Council (BBSRC)/Engineering and Physical Sciences Research Council (EPSRC; grant no. MR/K01577X/1), the Wellcome Trust (grant ref. 104924/14/Z/14) and the Wolfson Foundation. We thank E. Sezgin for kindly reading the manuscript.

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H.C.-Y. conducted the experiments. H.C.-Y., M.F. and C.E. wrote the manuscript.

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Correspondence to Marco Fritzsche.

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Colin-York, H., Eggeling, C. & Fritzsche, M. Dissection of mechanical force in living cells by super-resolved traction force microscopy. Nat Protoc 12, 783–796 (2017). https://doi.org/10.1038/nprot.2017.009

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