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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. Access through

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TWO-DIMENSIONAL TIRF-SIM–TRACTION FORCE MICROSCOPY (2D TIRF-SIM-TFM) Article Open access 12 April 2021 HIGH-RESOLUTION ASSESSMENT OF MULTIDIMENSIONAL CELLULAR MECHANICS USING LABEL-FREE

REFRACTIVE-INDEX TRACTION FORCE MICROSCOPY Article Open access 20 January 2024 ASTIGMATIC TRACTION FORCE MICROSCOPY (ATFM) Article Open access 12 April 2021 REFERENCES * Moeendarbary, E.

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  Download references 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. AUTHOR INFORMATION

AUTHORS AND AFFILIATIONS * MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK Huw Colin-York, Christian Eggeling & Marco Fritzsche *

Kennedy Institute for Rheumatology, University of Oxford, Oxford, UK Marco Fritzsche Authors * Huw Colin-York View author publications You can also search for this author inPubMed Google

Scholar * Christian Eggeling View author publications You can also search for this author inPubMed Google Scholar * Marco Fritzsche View author publications You can also search for this

author inPubMed Google Scholar CONTRIBUTIONS H.C.-Y. conducted the experiments. H.C.-Y., M.F. and C.E. wrote the manuscript. CORRESPONDING AUTHOR Correspondence to Marco Fritzsche. ETHICS

DECLARATIONS COMPETING INTERESTS The authors declare no competing financial interests. RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE 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 Download citation * Published: 16 March 2017 * Issue Date: April 2017 * DOI: https://doi.org/10.1038/nprot.2017.009 SHARE THIS ARTICLE Anyone you share

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