Technical Report | Published:

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

Nature Cell Biology volume 19, pages 864872 (2017) | Download Citation

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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    , & Environmental sensing through focal adhesions. Nat. Rev. Mol. Cell Biol. 10, 21–33 (2009).

  2. 2.

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

  3. 3.

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

  4. 4.

    , , & Cell movement is guided by the rigidity of the substrate. Biophys. J. 79, 144–152 (2000).

  5. 5.

    , , & Force fluctuations within focal adhesions mediate ECM-rigidity sensing to guide directed cell migration. Cell 151, 1513–1527 (2012).

  6. 6.

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

  7. 7.

    , , , & Neutrophil adhesion and chemotaxis depend on substrate mechanics. J. Phys. Condens. Matter 22, 194117 (2010).

  8. 8.

    , , , & Measuring traction forces of motile dendritic cells on micropost arrays. Biophys. J. 101, 2620–2628 (2011).

  9. 9.

    , , , & Growth cones as soft and weak force generators. Proc. Natl Acad. Sci. USA 108, 13420–13425 (2011).

  10. 10.

    , , , & Myofibroblasts and mechano-regulation of connective tissue remodelling. Nat. Rev. Mol. Cell Biol. 3, 349–363 (2002).

  11. 11.

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

  12. 12.

    , & Cell traction forces direct fibronectin matrix assembly. Biophys. J. 96, 729–738 (2009).

  13. 13.

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

  14. 14.

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

  15. 15.

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

  16. 16.

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

  17. 17.

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

  18. 18.

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

  19. 19.

    , , & Matrix confinement plays a pivotal role in regulating neutrophil-generated tractions, speed, and integrin utilization. J. Biol. Chem. 290, 3752–3763 (2015).

  20. 20.

    , & Silicone rubber substrata: a new wrinkle in the study of cell locomotion. Science 208, 177–179 (1980).

  21. 21.

    & Traction forces of cytokinesis measured with optically modified elastic substrata. Nature 385, 450–454 (1997).

  22. 22.

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

  23. 23.

    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).

  24. 24.

    , , & High resolution traction force microscopy based on experimental and computational advances. Biophys. J. 94, 207–220 (2008).

  25. 25.

    , , , & Live cells exert 3-dimensional traction forces on their substrata. Cell. Mol. Bioeng. 2, 425–436 (2009).

  26. 26.

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

  27. 27.

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

  28. 28.

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

  29. 29.

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

  30. 30.

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

  31. 31.

    & Measuring cell-generated forces: a guide to the available tools. Nat. Methods 13, 415–423 (2016).

  32. 32.

    , , & A micropatterning and image processing approach to simplify measurement of cellular traction forces. Acta Biomater. 8, 82–88 (2012).

  33. 33.

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

  34. 34.

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

  35. 35.

    , , , & Determination of elastic moduli of thin layers of soft material using the atomic force microscope. Biophys. J. 82, 2798–2810 (2002).

  36. 36.

    , & Soft biological materials and their impact on cell function. Soft Matter 2, 1–9 (2007).

  37. 37.

    , , & The leukocyte podosome. Eur. J. Cell Biol. 85, 151–157 (2006).

  38. 38.

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

  39. 39.

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

  40. 40.

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

  41. 41.

    & 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).

  42. 42.

    & Interstitial leukocyte migration and immune function. Nat. Immunol. 9, 960–969 (2008).

  43. 43.

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

  44. 44.

    , , & α-smooth muscle actin expression enhances cell traction force. Cell Motil. Cytoskeleton 64, 248–257 (2007).

  45. 45.

    , & 4D traction force microscopy reveals asymmetric cortical forces in migrating Dictyostelium cells. Phys. Rev. Lett. 105, 248103 (2010).

  46. 46.

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

  47. 47.

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

  48. 48.

    , , & Nonpolarized signaling reveals two distinct modes of 3D cell migration. J. Cell Biol. 197, 439–455 (2012).

  49. 49.

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

  50. 50.

    , & Fabrication of elastic micro-cavity chips for use in Elastic Resonator Interference Stress Microscopy. Nat. Protoc. Exch. (2017).

  51. 51.

    , & Controlling the morphology of gold films on poly(dimethylsiloxane). ACS Appl. Mater. Interfaces 2, 1927–1933 (2010).

  52. 52.

    & Hydrophobic recovery of plasma-treated polydimethylsiloxane. J. Adhes. 54, 33–45 (1995).

  53. 53.

    , , , & Spontaneous formation of ordered structures in thin films of metals supported on an elastomeric polymer. Nature 393, 146–149 (1998).

  54. 54.

    , , & Mechanics of morphological instabilities and surface wrinkling in soft materials: a review. Soft Matter 8, 5728–5745 (2012).

  55. 55.

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

  56. 56.

    , , , & Determination of elastic moduli of thin layers of soft material using the atomic force microscope. Biophys. J. 82, 2798–2810 (2002).

  57. 57.

    , , & Precise determination of the Poisson ratio in soft materials with 2D digital image correlation. Soft Matter 9, 6037–6045 (2013).

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

    • Jessica G. Borger

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

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. Search for Nils M. Kronenberg in:

  2. Search for Philipp Liehm in:

  3. Search for Anja Steude in:

  4. Search for Johanna A. Knipper in:

  5. Search for Jessica G. Borger in:

  6. Search for Giuliano Scarcelli in:

  7. Search for Kristian Franze in:

  8. Search for Simon J. Powis in:

  9. Search for Malte C. Gather in:

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.

Competing interests

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

Corresponding author

Correspondence to Malte C. Gather.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    Supplementary Information

Videos

  1. 1.

    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.

  2. 2.

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

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

  3. 3.

    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).

  4. 4.

    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.

  5. 5.

    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.

  6. 6.

    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.

  7. 7.

    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.

  8. 8.

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

    Phase contrast microscopy (left) and substrate displacement (right).

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/ncb3561

Further reading