Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Technical Report
  • Published:

Long-term imaging of cellular forces with high precision by elastic resonator interference stress microscopy

Nature Cell Biology volume 19, pages 864–872 (2017)Cite this article

Subjects

Abstract

Cellular forces are crucial for many biological processes but current methods to image them have limitations with respect to data analysis, resolution and throughput. Here, we present a robust approach to measure mechanical cell–substrate interactions in diverse biological systems by interferometrically detecting deformations of an elastic micro-cavity. Elastic resonator interference stress microscopy (ERISM) yields stress maps with exceptional precision and large dynamic range (2 nm displacement resolution over a >1 μm range, translating into 1 pN force sensitivity). This enables investigation of minute vertical stresses (<1 Pa) involved in podosome protrusion, protein-specific cell–substrate interaction and amoeboid migration through spatial confinement in real time. ERISM requires no zero-force reference and avoids phototoxic effects, which facilitates force monitoring over multiple days and at high frame rates and eliminates the need to detach cells after measurements. This allows observation of slow processes such as differentiation and further investigation of cells, for example, by immunostaining.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Elastic resonator interference stress microscopy (ERISM).
Figure 2: Validation of ERISM by AFM.
Figure 3: Investigation of podosome protrusions by ERISM.
Figure 4: ERISM for investigation of amoeboid migration in confined space and protein-specific cell–substrate interaction.
Figure 5: Analysis of the horizontal stress applied by adherent 3T3 fibroblasts using ERISM.
Figure 6: ERISM for long-term time-lapse studies of the mechanical activity of cells.

Similar content being viewed by others

References

  1. Geiger, B., Spatz, J. P. & Bershadsky, A. D. Environmental sensing through focal adhesions. Nat. Rev. Mol. Cell Biol. 10, 21–33 (2009).

    Article  CAS  Google Scholar 

  2. Koser, D. E. et al. Mechanosensing is critical for axon growth in the developing brain. Nat. Neurosci. 19, 1592–1598 (2016).

    Article  CAS  Google Scholar 

  3. Kshitiz et al. Control of stem cell fate and function by engineering physical microenvironments. Integr. Biol. 4, 1008–1018 (2012).

    Article  CAS  Google Scholar 

  4. Lo, C. M., Wang, H. B., Dembo, M. & Wang, Y. L. Cell movement is guided by the rigidity of the substrate. Biophys. J. 79, 144–152 (2000).

    Article  CAS  Google Scholar 

  5. Plotnikov, S. V., Pasapera, A. M., Sabass, B. & Waterman, C. M. Force fluctuations within focal adhesions mediate ECM-rigidity sensing to guide directed cell migration. Cell 151, 1513–1527 (2012).

    Article  CAS  Google Scholar 

  6. Paszek, M. J. et al. Tensional homeostasis and the malignant phenotype. Cancer Cell 8, 241–254 (2005).

    Article  CAS  Google Scholar 

  7. Jannat, R. A., Robbins, G. P., Ricart, B. G., Dembo, M. & Hammer, D. A. Neutrophil adhesion and chemotaxis depend on substrate mechanics. J. Phys. Condens. Matter 22, 194117 (2010).

    Article  Google Scholar 

  8. Ricart, B. G., Yang, M. T., Hunter, C. A., Chen, C. S. & Hammer, D. A. Measuring traction forces of motile dendritic cells on micropost arrays. Biophys. J. 101, 2620–2628 (2011).

    Article  CAS  Google Scholar 

  9. Betz, T., Koch, D., Lu, Y.-B., Franze, K. & Käs, J. A. Growth cones as soft and weak force generators. Proc. Natl Acad. Sci. USA 108, 13420–13425 (2011).

    Article  CAS  Google Scholar 

  10. Tomasek, J. J., Gabbiani, G., Hinz, B., Chaponnier, C. & Brown, R. A. Myofibroblasts and mechano-regulation of connective tissue remodelling. Nat. Rev. Mol. Cell Biol. 3, 349–363 (2002).

    Article  CAS  Google Scholar 

  11. Mertz, A. F. et al. Scaling of traction forces with the size of cohesive cell colonies. Phys. Rev. Lett. 108, 198101 (2012).

    Article  Google Scholar 

  12. Lemmon, C. A., Chen, C. S. & Romer, L. H. Cell traction forces direct fibronectin matrix assembly. Biophys. J. 96, 729–738 (2009).

    Article  CAS  Google Scholar 

  13. Brugués, A. et al. Forces driving epithelial wound healing. Nat. Phys. 10, 683–690 (2014).

    Article  Google Scholar 

  14. Tambe, D. T. et al. Collective cell guidance by cooperative intercellular forces. Nat. Mater. 10, 469–475 (2011).

    Article  CAS  Google Scholar 

  15. Case, L. B. & Waterman, C. M. Integration of actin dynamics and cell adhesion by a three-dimensional, mechanosensitive molecular clutch. Nat. Cell Biol. 17, 955–963 (2015).

    Article  CAS  Google Scholar 

  16. Labernadie, A. et al. Protrusion force microscopy reveals oscillatory force generation and mechanosensing activity of human macrophage podosomes. Nat. Commun. 5, 5343 (2014).

    Article  CAS  Google Scholar 

  17. Bergert, M. et al. Force transmission during adhesion-independent migration. Nat. Cell Biol. 17, 524–529 (2015).

    Article  CAS  Google Scholar 

  18. Liu, Y. J. et al. Confinement and low adhesion induce fast amoeboid migration of slow mesenchymal cells. Cell 160, 659–672 (2015).

    Article  CAS  Google Scholar 

  19. Toyjanova, J., Flores-Cortez, E., Reichner, J. S. & Franck, C. Matrix confinement plays a pivotal role in regulating neutrophil-generated tractions, speed, and integrin utilization. J. Biol. Chem. 290, 3752–3763 (2015).

    Article  CAS  Google Scholar 

  20. Harris, A. K., Wild, P. & Stopak, D. Silicone rubber substrata: a new wrinkle in the study of cell locomotion. Science 208, 177–179 (1980).

    Article  CAS  Google Scholar 

  21. Burton, K. & Taylor, D. L. Traction forces of cytokinesis measured with optically modified elastic substrata. Nature 385, 450–454 (1997).

    Article  CAS  Google Scholar 

  22. Dembo, M. & Wang, Y. L. Stresses at the cell-to-substrate interface during locomotion of fibroblasts. Biophys. J. 76, 2307–2316 (1999).

    Article  CAS  Google Scholar 

  23. Schwarz, U. S. et al. Calculation of forces at focal adhesions from elastic substrate data: the effect of localized force and the need for regularization. Biophys. J. 83, 1380–1394 (2002).

    Article  CAS  Google Scholar 

  24. Sabass, B., Gardel, M. L., Waterman, C. M. & Schwarz, U. S. High resolution traction force microscopy based on experimental and computational advances. Biophys. J. 94, 207–220 (2008).

    Article  CAS  Google Scholar 

  25. Hur, S. S., Zhao, Y., Li, Y. S., Botvinick, E. & Chien, S. Live cells exert 3-dimensional traction forces on their substrata. Cell. Mol. Bioeng. 2, 425–436 (2009).

    Article  Google Scholar 

  26. Legant, W. R. et al. Multidimensional traction force microscopy reveals out-of-plane rotational moments about focal adhesions. Proc. Natl Acad. Sci. USA 110, 881–886 (2013).

    Article  CAS  Google Scholar 

  27. Álvarez-González, B. Three-dimensional balance of cortical tension and axial contractility enables fast amoeboid migration. Biophys. J. 108, 821–832 (2015).

    Article  Google Scholar 

  28. Colin-York, H. et al. Super-resolved traction force microscopy (STFM). Nano Lett. 16, 2633–2638 (2016).

    Article  CAS  Google Scholar 

  29. Tan, J. L. et al. Cells lying on a bed of microneedles: an approach to isolate mechanical force. Proc. Natl Acad. Sci. USA 100, 1484–1489 (2003).

    Article  CAS  Google Scholar 

  30. Wolfenson, H. et al. Tropomyosin controls sarcomere-like contractions for rigidity sensing and suppressing growth on soft matrices. Nat. Cell Biol. 18, 33–42 (2015).

    Article  Google Scholar 

  31. Polacheck, W. J. & Chen, C. S. Measuring cell-generated forces: a guide to the available tools. Nat. Methods 13, 415–423 (2016).

    Article  CAS  Google Scholar 

  32. Polio, S. R., Rothenberg, K. E., Stamenovic, D. & Smith, M. L. A micropatterning and image processing approach to simplify measurement of cellular traction forces. Acta Biomater. 8, 82–88 (2012).

    Article  CAS  Google Scholar 

  33. Bergert, M. et al. Confocal reference free traction force microscopy. Nat. Commun. 7, 12814 (2016).

    Article  CAS  Google Scholar 

  34. Balaban, N. Q. et al. Force and focal adhesion assembly: a close relationship studied using elastic micropatterned substrates. Nat. Cell Biol. 3, 466–472 (2001).

    Article  CAS  Google Scholar 

  35. Dimitriadis, E. K., Horkay, F., Maresca, J., Kachar, B. & Chadwick, R. S. Determination of elastic moduli of thin layers of soft material using the atomic force microscope. Biophys. J. 82, 2798–2810 (2002).

    Article  CAS  Google Scholar 

  36. Levental, I., Georges, P. C. & Janmey, P. A. Soft biological materials and their impact on cell function. Soft Matter 2, 1–9 (2007).

    Google Scholar 

  37. Calle, Y., Burns, S., Thrasher, A. J. & Jones, G. E. The leukocyte podosome. Eur. J. Cell Biol. 85, 151–157 (2006).

    Article  CAS  Google Scholar 

  38. Charras, G. & Paluch, E. Blebs lead the way: how to migrate without lamellipodia. Nat. Rev. Mol. Cell Biol. 9, 730–736 (2008).

    Article  CAS  Google Scholar 

  39. Bastounis, E. et al. Both contractile axial and lateral traction force dynamics drive amoeboid cell motility. J. Cell Biol. 204, 1045–1061 (2014).

    Article  CAS  Google Scholar 

  40. Lämmermann, T. et al. Rapid leukocyte migration by integrin-independent flowing and squeezing. Nature 453, 51–55 (2008).

    Article  Google Scholar 

  41. Malawista, S. E. & de Boisfleury Chevance, A. Random locomotion and chemotaxis of human blood polymorphonuclear leukocytes (PMN) in the presence of EDTA: PMN in close quarters require neither leukocyte integrins nor external divalent cations. Proc. Natl Acad. Sci. USA 94, 11577–11582 (1997).

    Article  CAS  Google Scholar 

  42. Friedl, P. & Weigelin, B. Interstitial leukocyte migration and immune function. Nat. Immunol. 9, 960–969 (2008).

    Article  CAS  Google Scholar 

  43. Kinashi, T. Intracellular signalling controlling integrin activation in lymphocytes. Nat. Rev. Immunol. 5, 546–559 (2005).

    Article  CAS  Google Scholar 

  44. Chen, J., Li, H., SundarRaj, N. & Wang, J. H.-C. α-smooth muscle actin expression enhances cell traction force. Cell Motil. Cytoskeleton 64, 248–257 (2007).

    Article  CAS  Google Scholar 

  45. Delanoe-Ayari, H., Rieu, J. P. & Sano, M. 4D traction force microscopy reveals asymmetric cortical forces in migrating Dictyostelium cells. Phys. Rev. Lett. 105, 248103 (2010).

    Article  CAS  Google Scholar 

  46. Alamo, J. C. del et al. Three-dimensional quantification of cellular traction forces and mechanosensing of thin substrata by Fourier traction force microscopy. PLoS ONE 8, e69850 (2013).

    Article  Google Scholar 

  47. Renkawitz, J. et al. Adaptive force transmission in amoeboid cell migration. Nat. Cell Biol. 11, 1438–1443 (2009).

    Article  CAS  Google Scholar 

  48. Petrie, R. J., Gavara, N., Chadwick, R. S. & Yamada, K. M. Nonpolarized signaling reveals two distinct modes of 3D cell migration. J. Cell Biol. 197, 439–455 (2012).

    Article  CAS  Google Scholar 

  49. Yoshida, K. & Soldati, T. Dissection of amoeboid movement into two mechanically distinct modes. J. Cell Sci. 119, 3833–3844 (2006).

    Article  CAS  Google Scholar 

  50. Kronenberg, N. M., Liehm, P. & Gather, M. C. Fabrication of elastic micro-cavity chips for use in Elastic Resonator Interference Stress Microscopy. Nat. Protoc. Exch. http://dx.doi.org/10.1038/protex.2017.052 (2017).

  51. Graudejus, O., Görrn, P. & Wagner, S. Controlling the morphology of gold films on poly(dimethylsiloxane). ACS Appl. Mater. Interfaces 2, 1927–1933 (2010).

    Article  CAS  Google Scholar 

  52. Fritz, J. L. & Owen, M. J. Hydrophobic recovery of plasma-treated polydimethylsiloxane. J. Adhes. 54, 33–45 (1995).

    CAS  Google Scholar 

  53. Bowden, N., Brittain, S., Evans, A. G., Hutchinson, J. W. & Whitesides, G. M. Spontaneous formation of ordered structures in thin films of metals supported on an elastomeric polymer. Nature 393, 146–149 (1998).

    Article  CAS  Google Scholar 

  54. Li, B., Cao, Y.-P., Feng, X.-Q. & Gaoc, H. Mechanics of morphological instabilities and surface wrinkling in soft materials: a review. Soft Matter 8, 5728–5745 (2012).

    Article  CAS  Google Scholar 

  55. Yuffa, A. J. & Scales, J. A. Object-oriented electrodynamic S-matrix code with modern applications. J. Comput. Phys. 231, 4823–4835 (2012).

    Article  Google Scholar 

  56. Dimitriadis, E. K., Horkay, F., Maresca, J., Kachar, B. & Chadwick, R. S. Determination of elastic moduli of thin layers of soft material using the atomic force microscope. Biophys. J. 82, 2798–2810 (2002).

    Article  CAS  Google Scholar 

  57. Pritchard, R. H., Lava, P., Debruyne, D. & Terentjev, E. M. Precise determination of the Poisson ratio in soft materials with 2D digital image correlation. Soft Matter 9, 6037–6045 (2013).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors thank K. Venkatesan Iyer and P. A. Reynolds for fruitful discussion, R. Shahapure for TFM reference measurements, A. L. Sobiech for illustrations and the DictyoBase for provision of AX3 strain Dictyostelium discoideum. This project has received funding from the Human Frontiers Science Program (RGY0074/2013), the Scottish Funding Council (via SUPA), the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement no. 640012), the EPSRC DTP (EP/L505079/1), the RS MacDonald Charitable Trust and the MRC (G1100116 and G110312/1).

Author information

Author notes

  1. Jessica G. Borger

    Present address: Department of Immunology and Pathology, Monash University, Melbourne, Victoria, 3004, Australia

Authors and Affiliations

  1. SUPA, School of Physics and Astronomy, University of St Andrews, St Andrews, KY16 9SS, UK

    Nils M. Kronenberg, Philipp Liehm, Anja Steude & Malte C. Gather

  2. Institute of Immunology and Infection Research, University of Edinburgh, Edinburgh, EH9 3FL, UK

    Johanna A. Knipper & Jessica G. Borger

  3. Fischell Department of Bioengineering, University of Maryland, College Park, Maryland, 20742, USA

    Giuliano Scarcelli

  4. Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, CB2 3DY, UK

    Kristian Franze

  5. School of Medicine, University of St Andrews, St Andrews, KY16 9TF, UK

    Simon J. Powis

Authors
  1. Nils M. Kronenberg
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Philipp Liehm
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Anja Steude
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Johanna A. Knipper
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jessica G. Borger
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Giuliano Scarcelli
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Kristian Franze
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Simon J. Powis
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Malte C. Gather
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

N.M.K., P.L. and M.C.G. developed ERISM. N.M.K. fabricated and characterized the micro-cavity substrates and conducted measurements. P.L. developed the data analysis, stress map calculation and graphical data presentation. A.S. contributed to protein coating, staining and cell culture. J.A.K. designed and prepared the primary mouse fibroblast experiment. J.G.B. prepared and assisted in the T cell experiment. G.S. and K.F. performed rheometry and TFM, and contributed to AFM and general discussion. S.J.P. proposed and prepared the macrophage experiment. M.C.G. supervised the project. N.M.K. and M.C.G. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Malte C. Gather.

Ethics declarations

Competing interests

The University of St Andrews has filed a patent application related to the technique described here.

Integrated supplementary information

Supplementary Figure 1 Development and characterization of ERISM micro-cavities and data analysis.

(a–d) Oxygen plasma treatment of silicone elastomer surface. (a) Increase in water contact angle for different plasma treatment durations at plasma power of 80–90 W. (b) Reflectance of 15 nm-thick gold layers evaporated on glass (blue) and on untreated elastomer (black). (The oscillation for the black line is due to thin film interference.) (c) Reflectance at 650 nm (left) and apparent Young’s modulus (right, calculated for an indentation force of 2 nN) of surface-oxidized elastomer layers with 15 nm top gold mirror for different plasma durations t and plasma powers P (process pressure, 3 mtorr; gas flow, 20 sccm O2 and 5 sccm Ar). The apparent Young’s modulus was obtained by AFM indentation measurements with a 17 μm-diameter glass sphere. For comparison the reflectance and Young’s modulus, respectively, of a pristine non-oxidized elastomer sample without gold coating are shown at the origins of the t–P planes. (d) Plot of apparent Young’s modulus versus reflectance for all samples shown in (c). The region where low reflectance renders ERISM measurements unreliable is marked in red. (e) Wavelength-dependent intensity of the probe light used for the ERISM measurement when incident on the bottom glass side of the micro-cavity through a 4×, 10×, 20× and 40× objective, respectively. (f) Measurement of the Young’s modulus of the elastomer within the micro-cavity obtained by comparing AFM indentation forces to FEM predictions obtained from ERISM deformation profiles. See Methods for details.

Supplementary Figure 2 Flow cytometry of isolated human macrophages.

Adherent cells from isolated peripheral blood mononuclear cells were recovered and stained with anti-CD14-PE, shown shaded, compared to auto-fluorescence from cells without antibody staining (unshaded).

Supplementary Figure 3 Demonstration of the sensitivity of ERISM to horizontally applied forces.

(a) The needle of an AFM cantilever is slightly indented into the ERISM chip to make firm contact and the cantilever is then pulled to the side to apply a defined lateral force. This gives rise to an asymmetric ERISM signal that grows with increasing horizontal force. (b) Peak ERISM signal detected versus horizontal force applied by AFM. Red line, linear fit (R2 > 0.99). Dashed lines, displacement resolution of ERISM in the present configuration and resulting minimal horizontal force that can be detected.

Supplementary Figure 4 High frame rate ERISM (a–b), ERISM for long-term time-lapse studies of the mechanical activity of cells during mesenchymal migration (c–e) and differentiation of fibroblasts into myofibroblasts in situ on the ERISM micro-cavity (f–g).

(a) Phase contrast image and ERISM map of 3T3 fibroblast cells. Images from an 11.5 min-ERISM time-lapse measurement with a frame acquired every 2.3 s. (b) Evolution of mean displacement under the cell nucleus and the lamellum, respectively (areas circled in blue and red in (a)). The black trace shows the evolution for an area where no cell is present (black circle in (a)). Also see Supplementary Video 5. (c) Phase contrast image and ERISM map for primary mouse fibroblasts after cells migrated out of a tissue explant directly placed on the micro-cavity substrate. (d) ERISM time-lapse series of fibroblast migration for the cell highlighted in (c). The cell boundary is indicated by a black line. (e) Temporal evolution of (left) the force applied in each direction with respect to the cell centre as defined in the first panel of (d) and (right) the total force and speed of cell migration. Position and size of white circles indicate direction and speed of migration. [A similar analysis of the moment of the force orientation forces has been performed for Dictyostelium discoideum using TFM, see H. Tanimoto and M. Sano, Biophys. J. 106, 16–25 (2014).] (f) Representative phase contrast image (left) and ERISM stress map (right) of a cell after 66 h of TGF-β treatment. Cell outline is shown in black. (g) Fluorescence images of immune-labelled α-SMA (green) after fixation and permeabilisation of cells on the micro-cavity. Left: Cells stimulated with TGF-β for 66 h. Right: Non-stimulated control cells kept on the micro-cavity for the same time. While cells in the control group show weak and unspecific background fluorescence, parallel arranged α-SMA fibres arching through the cell bodies are clearly visible in differentiated cells. Scale bars, (a) 50 μm, (c) 100 μm, (d,f) 50 μm and (g) 200 μm.

Supplementary information

Supplementary Information

Supplementary Information (PDF 753 kb)

Illustration of mechanical stability of the micro-cavities used for ERISM.

Real time video of monochromatic reflectance of a micro-cavity, into which a 17 μm dimeter glass bead (glued to the tip of an AFM probe) was indented by 900 nm. While keeping the indentation constant, the bead is moved horizontally across the micro-cavity. (AVI 5296 kb)

Fourier-filtered ERISM time-lapse investigation of podosome protrusions of a human macrophage.

Phase contrast microscopy (left) and spatial Fourier-filtered stress map (right). (AVI 7940 kb)

Fourier-filtered ERISM time-lapse investigation of podosome protrusion of a human macrophage during cell migration.

Phase contrast microscopy (top) and spatial Fourier filtered substrate displacement (bottom). (AVI 4644 kb)

ERISM time-lapse investigation of amoeboid migration in spatial confinement.

Video shows the substrate displacement caused by a Dictyostelium discoideum amoeba migrating through a 5 μm-thick void formed above the micro-cavity substrate. The outline of the amoeba is shown in black. (AVI 1899 kb)

High-speed ERISM time-lapse investigation of 3T3 fibroblasts.

Phase contrast microscopy (left) and substrate displacement in nm (right). The inset shows the displacement for the region highlighted by a white box in the bright-field and displacement images with increased contrast to show displacements due to expansion of the cell edge in more detail. A frame was taken every 2.3 s. (AVI 2699 kb)

Long-term ERISM time-lapse investigation of 3T3 fibroblasts.

Phase-contrast microscopy (left) and substrate displacement in nm (right). The video was recorded over 5.5 days and contains more than 1,600 individual ERISM maps. (AVI 15341 kb)

ERISM time-lapse investigation of mesenchymal migration in a culture of primary mouse fibroblasts.

Video shows an overlay of substrate displacement with corresponding phase contrast microscopy image. Pulling is indicated in red, pushing is indicated in blue. (AVI 7125 kb)

Close-up ERISM time-lapse of mesenchymal migration of individual primary mouse fibroblast.

Phase contrast microscopy (left) and substrate displacement (right). (AVI 642 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kronenberg, N., Liehm, P., Steude, A. et al. Long-term imaging of cellular forces with high precision by elastic resonator interference stress microscopy. Nat Cell Biol 19, 864–872 (2017). https://doi.org/10.1038/ncb3561

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb3561

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing