pyTFM: A tool for traction force and monolayer stress microscopy

Cellular force generation and force transmission are of fundamental importance for numerous biological processes and can be studied with the methods of Traction Force Microscopy (TFM) and Monolayer Stress Microscopy. Traction Force Microscopy and Monolayer Stress Microscopy solve the inverse problem of reconstructing cell-matrix tractions and inter- and intra-cellular stresses from the measured cell force-induced deformations of an adhesive substrate with known elasticity. Although several laboratories have developed software for Traction Force Microscopy and Monolayer Stress Microscopy computations, there is currently no software package available that allows non-expert users to perform a full evaluation of such experiments. Here we present pyTFM, a tool to perform Traction Force Microscopy and Monolayer Stress Microscopy on cell patches and cell layers grown in a 2-dimensional environment. pyTFM was optimized for ease-of-use; it is open-source and well documented (hosted at https://pytfm.readthedocs.io/) including usage examples and explanations of the theoretical background. pyTFM can be used as a standalone Python package or as an add-on to the image annotation tool ClickPoints. In combination with the ClickPoints environment, pyTFM allows the user to set all necessary analysis parameters, select regions of interest, examine the input data and intermediary results, and calculate a wide range of parameters describing forces, stresses, and their distribution. In this work, we also thoroughly analyze the accuracy and performance of the Traction Force Microscopy and Monolayer Stress Microscopy algorithms of pyTFM using synthetic and experimental data from epithelial cell patches.

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pyTFM: A tool for Traction Force and Monolayer Stress Microscopy

10.1101/2020.09.28.316430 ◽

2020 ◽

Cited By ~ 1

Author(s):

Andreas Bauer ◽

Magdalena Prechová ◽

Martin Gregor ◽

Ben Fabry

Keyword(s):

Image Annotation ◽

Single Cells ◽

Traction Force ◽

Theoretical Background ◽

Ease Of Use ◽

Traction Force Microscopy ◽

Force Microscopy ◽

Wide Range ◽

Cell Layers ◽

Cellular Stresses

AbstractCellular force generation and force transduction are of fundamental importance for numerous biological processes and can be studied with the methods of Traction Force Microscopy (TFM) and Monolayer Stress Microscopy. Traction Force Microscopy and Monolayer Stress Microscopy solve the inverse problem of reconstructing cell-matrix tractions and inter- and intra-cellular stresses from the measured cell force-induced deformations of an adhesive substrate with known elasticity. Although several laboratories have developed software for Traction Force Microscopy and Monolayer Stress Microscopy computations, there is currently no software package available that allows non-expert users to perform a full evaluation of such experiments. Here we present pyTFM, a tool to perform Traction Force Microscopy and Monolayer Stress Microscopy on single cells, cell patches and cell layers grown in a 2-dimensional environment. pyTFM was optimized for ease-of-use; it is open-source and well documented (hosted at https://pytfm.readthedocs.io/) including usage examples and explanations of the theoretical background. pyTFM can be used as a standalone Python package or as an add-on to the image annotation tool ClickPoints. In combination with the ClickPoints environment, pyTFM allows the user to set all necessary analysis parameters, select regions of interest, examine the input data and intermediary results, and calculate a wide range of parameters describing forces, stresses, and their distribution. The Monolayer Stress Microscopy implementation in pyTFM allows for the analysis of small cell patches and single cells; we analyze the accuracy and performance of Traction Force Microscopy and Monolayer Stress Microscopy algorithms using synthetic and experimental data from epithelial cell patches.

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Quantifying force transmission through fibroblasts: changes of traction forces under external shearing

European Biophysics Journal ◽

10.1007/s00249-021-01576-8 ◽

2021 ◽

Author(s):

Steven Huth ◽

Johannes W. Blumberg ◽

Dimitri Probst ◽

Jan Lammerding ◽

Ulrich S. Schwarz ◽

...

Keyword(s):

Cell Surface ◽

Mammalian Cells ◽

Shear Force ◽

Apical Cell ◽

Traction Force ◽

Traction Force Microscopy ◽

Force Transmission ◽

Traction Forces ◽

Force Microscopy ◽

Adhesion Sites

AbstractMammalian cells have evolved complex mechanical connections to their microenvironment, including focal adhesion clusters that physically connect the cytoskeleton and the extracellular matrix. This mechanical link is also part of the cellular machinery to transduce, sense and respond to external forces. Although methods to measure cell attachment and cellular traction forces are well established, these are not capable of quantifying force transmission through the cell body to adhesion sites. We here present a novel approach to quantify intracellular force transmission by combining microneedle shearing at the apical cell surface with traction force microscopy at the basal cell surface. The change of traction forces exerted by fibroblasts to underlying polyacrylamide substrates as a response to a known shear force exerted with a calibrated microneedle reveals that cells redistribute forces dynamically under external shearing and during sequential rupture of their adhesion sites. Our quantitative results demonstrate a transition from dipolar to monopolar traction patterns, an inhomogeneous distribution of the external shear force to the adhesion sites as well as dynamical changes in force loading prior to and after the rupture of single adhesion sites. Our strategy of combining traction force microscopy with external force application opens new perspectives for future studies of force transmission and mechanotransduction in cells.

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Microparticle traction force microscopy reveals subcellular force exertion patterns in immune cell–target interactions

Nature Communications ◽

10.1038/s41467-019-13804-z ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 9

Author(s):

Daan Vorselen ◽

Yifan Wang ◽

Miguel M. de Jesus ◽

Pavak K. Shah ◽

Matthew J. Footer ◽

...

Keyword(s):

Immune Cell ◽

Traction Force ◽

High Sensitivity ◽

Computational Method ◽

Cytotoxic T Cell ◽

Planar Geometry ◽

Traction Force Microscopy ◽

Cellular Interactions ◽

Force Microscopy ◽

Wide Range

AbstractForce exertion is an integral part of cellular behavior. Traction force microscopy (TFM) has been instrumental for studying such forces, providing spatial force measurements at subcellular resolution. However, the applications of classical TFM are restricted by the typical planar geometry. Here, we develop a particle-based force sensing strategy for studying cellular interactions. We establish a straightforward batch approach for synthesizing uniform, deformable and tuneable hydrogel particles, which can also be easily derivatized. The 3D shape of such particles can be resolved with superresolution (<50 nm) accuracy using conventional confocal microscopy. We introduce a reference-free computational method allowing inference of traction forces with high sensitivity directly from the particle shape. We illustrate the potential of this approach by revealing subcellular force patterns throughout phagocytic engulfment and force dynamics in the cytotoxic T-cell immunological synapse. This strategy can readily be adapted for studying cellular forces in a wide range of applications.

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Superresolved microparticle traction force microscopy reveals subcellular force patterns in immune cell-target interactions

10.1101/431221 ◽

2018 ◽

Cited By ~ 3

Author(s):

Daan Vorselen ◽

Yifan Wang ◽

Miguel M. de Jesus ◽

Pavak K. Shah ◽

Matthew J. Footer ◽

...

Keyword(s):

Immune Cell ◽

Traction Force ◽

Computational Method ◽

Cytotoxic T Cell ◽

Planar Geometry ◽

Traction Force Microscopy ◽

Cellular Interactions ◽

Cellular Behavior ◽

Force Microscopy ◽

Wide Range

Force exertion is an integral part of cellular behavior. Traction force microscopy (TFM) has been instrumental for studying such forces, providing both spatial and directional force measurements at subcellular resolution. However, the applications of classical TFM are restricted by the typical planar geometry. Here, we develop a particle-based force sensing strategy, specifically designed for studying ligand-dependent cellular interactions. We establish a straightforward batch approach for synthesizing highly uniform, deformable and tunable hydrogel particles, which can also be easily derivatized to trigger specific cellular behavior. The 3D shape of such particles can be resolved with superresolution (<50 nm) accuracy using conventional confocal microscopy. We introduce a computational method that allows inference of surface traction forces with high sensitivity (∼10 Pa) directly from the particle shape. We illustrate the potential and flexibility of this approach by revealing surprising subcellular force patterns throughout phagocytic engulfment and measuring dynamics of cytotoxic T cell force exertion in the immunological synapse. This strategy can readily be adapted for studying cellular forces in a wide range of applications.

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Two-dimensional TIRF-SIM–traction force microscopy (2D TIRF-SIM-TFM)

Nature Communications ◽

10.1038/s41467-021-22377-9 ◽

2021 ◽

Vol 12 (1) ◽

Cited By ~ 1

Author(s):

Liliana Barbieri ◽

Huw Colin-York ◽

Kseniya Korobchevskaya ◽

Di Li ◽

Deanna L. Wolfson ◽

...

Keyword(s):

Resolution Enhancement ◽

Traction Force ◽

Super Resolution ◽

Traction Force Microscopy ◽

Two Dimensional ◽

Structured Illumination ◽

Structured Illumination Microscopy ◽

Force Microscopy ◽

Health And Disease ◽

Spatio Temporal

AbstractQuantifying small, rapidly evolving forces generated by cells is a major challenge for the understanding of biomechanics and mechanobiology in health and disease. Traction force microscopy remains one of the most broadly applied force probing technologies but typically restricts itself to slow events over seconds and micron-scale displacements. Here, we improve >2-fold spatially and >10-fold temporally the resolution of planar cellular force probing compared to its related conventional modalities by combining fast two-dimensional total internal reflection fluorescence super-resolution structured illumination microscopy and traction force microscopy. This live-cell 2D TIRF-SIM-TFM methodology offers a combination of spatio-temporal resolution enhancement relevant to forces on the nano- and sub-second scales, opening up new aspects of mechanobiology to analysis.

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Different Cell Migration Modes: Insights from Traction Force Microscopy and Modeling

Biophysical Journal ◽

10.1016/j.bpj.2020.11.905 ◽

2021 ◽

Vol 120 (3) ◽

pp. 113a

Author(s):

Wouter-Jan Rappel ◽

Elisabeth Ghabache ◽

Yuansheng Cao ◽

Yuchuan Miao ◽

Alexander Groisman ◽

...

Keyword(s):

Cell Migration ◽

Traction Force ◽

Traction Force Microscopy ◽

Force Microscopy

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Epifluorescence-based three-dimensional traction force microscopy

Scientific Reports ◽

10.1038/s41598-020-72931-6 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Lauren Hazlett ◽

Alexander K. Landauer ◽

Mohak Patel ◽

Hadley A. Witt ◽

Jin Yang ◽

...

Keyword(s):

Spatial Frequency ◽

Open Source ◽

Single Particle ◽

Three Dimensional ◽

Traction Force ◽

High Spatial Frequency ◽

Epifluorescence Microscopy ◽

Traction Force Microscopy ◽

Force Microscopy ◽

Out Of Plane

Abstract We introduce a novel method to compute three-dimensional (3D) displacements and both in-plane and out-of-plane tractions on nominally planar transparent materials using standard epifluorescence microscopy. Despite the importance of out-of-plane components to fully understanding cell behavior, epifluorescence images are generally not used for 3D traction force microscopy (TFM) experiments due to limitations in spatial resolution and measuring out-of-plane motion. To extend an epifluorescence-based technique to 3D, we employ a topology-based single particle tracking algorithm to reconstruct high spatial-frequency 3D motion fields from densely seeded single-particle layer images. Using an open-source finite element (FE) based solver, we then compute the 3D full-field stress and strain and surface traction fields. We demonstrate this technique by measuring tractions generated by both single human neutrophils and multicellular monolayers of Madin–Darby canine kidney cells, highlighting its acuity in reconstructing both individual and collective cellular tractions. In summary, this represents a new, easily accessible method for calculating fully three-dimensional displacement and 3D surface tractions at high spatial frequency from epifluorescence images. We released and support the complete technique as a free and open-source code package.

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