title: 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.
authors.affiliation: MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK.
authors.forename: Huw
authors.identifier: 0000-0002-6585-3237
authors.initials: H
authors.lastname: Colin-York
authors.affiliation: MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK.
authors.forename: Christian
authors.initials: C
authors.lastname: Eggeling
authors.affiliation: MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK.|Kennedy Institute for Rheumatology, University of Oxford, Oxford, UK.
authors.forename: Marco
authors.initials: M
authors.lastname: Fritzsche
mesh_terms: D001696:Biomechanical Phenomena; D002470:Cell Survival; D005456:Fluorescent Dyes / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D055595:Mechanical Phenomena; D018625:Microscopy, Atomic Force / Q000379:methods*
chemical_list: D005456:Fluorescent Dyes