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Micropipette force sensors for in vivo force measurements on single cells and multicellular microorganisms

Abstract

Measuring forces from the piconewton to millinewton range is of great importance for the study of living systems from a biophysical perspective. The use of flexible micropipettes as highly sensitive force probes has become established in the biophysical community, advancing our understanding of cellular processes and microbial behavior. The micropipette force sensor (MFS) technique relies on measurement of the forces acting on a force-calibrated, hollow glass micropipette by optically detecting its deflections. The MFS technique covers a wide micro- and mesoscopic regime of detectable forces (tens of piconewtons to millinewtons) and sample sizes (micrometers to millimeters), does not require gluing of the sample to the cantilever, and allows simultaneous optical imaging of the sample throughout the experiment. Here, we provide a detailed protocol describing how to manufacture and calibrate the micropipettes, as well as how to successfully design, perform, and troubleshoot MFS experiments. We exemplify our approach using the model nematode Caenorhabditis elegans, but by following this protocol, a wide variety of living samples, ranging from single cells to multicellular aggregates and millimeter-sized organisms, can be studied in vivo, with a force resolution as low as 10 pN. A skilled (under)graduate student can master the technique in ~1–2 months. The whole protocol takes ~1–2 d to finish.

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Fig. 1: Principles of the MFS technique as exemplified with the nematode Caenorhabditis elegans.
Fig. 2: Basics of the micropipette design.
Fig. 3: The cross-correlation image analysis of the micropipette deflection.
Fig. 4: Equipment setup.
Fig. 5: Micropipette calibration approach as described in Step 9A.
Fig. 6: Micropipette calibration approach as described in Step 9B.
Fig. 7: Examples of typical experimental problems.
Fig. 8: Examples of two unsuccessful calibration attempts.
Fig. 9: Anticipated results from Caenorhabditis elegans swimming experiments.

Data availability

The data presented in this protocol are available from the corresponding authors upon request.

References

  1. 1.

    Kishino, A. & Yanagida, T. Force measurements by micromanipulation of a single actin filament by glass needle. Nature 334, 74–76 (1988).

    CAS  Article  Google Scholar 

  2. 2.

    Houchmandzadeh, B., Marko, J. F., Chatenay, D. & Libchaber, D. Elasticity and structure of eukaryote chromosomes studied by micromanipulation and micropipette aspiration. J. Cell Biol. 139, 1–12 (1997).

    CAS  Article  Google Scholar 

  3. 3.

    Hochmuth, R. M. Micropipette aspiration of living cells. J. Biomech. 33, 15–22 (2000).

    CAS  Article  Google Scholar 

  4. 4.

    Backholm, M., Ryu, W. S. & Dalnoki-Veress, K. Viscoelastic properties of the nematode Caenorhabditis elegans, a self-similar, shear-thinning worm. Proc. Natl Acad. Sci. USA 110, 4528–4533 (2013).

    CAS  Article  Google Scholar 

  5. 5.

    Kamimura, S. & Takahashi, K. Direct measurement of the force of microtubule sliding in flagella. Nature 293, 566–568 (1981).

    CAS  Article  Google Scholar 

  6. 6.

    Marcy, Y., Prost, J., Carlier, M.-F. & Sykes, C. Forces generated during actin-based propulsion: a direct measurement by micromanipulation. Proc. Natl Acad. Sci. USA 101, 5992–5997 (2004).

    CAS  Article  Google Scholar 

  7. 7.

    Schulman, R. D., Backholm, M., Ryu, W. S. & Dalnoki-Veress, K. Dynamic force patterns of an undulatory microswimmer. Phys. Rev. E Stat. Nonlin. Soft Matter Phys. 89, 050701 (2014).

    Article  Google Scholar 

  8. 8.

    Evans, E. A. Minimum energy analysis of membrane deformation applied to pipet aspiration and surface adhesion of red blood cells. Biophys. J. 30, 265–284 (1980).

    CAS  Article  Google Scholar 

  9. 9.

    Kreis, C. T., Le Blay, M., Linne, C., Makowski, M. M. & Bäumchen, O. Adhesion of Chlamydomonas microalgae to surfaces is switchable by light. Nat. Phys. 14, 45–49 (2018).

    CAS  Article  Google Scholar 

  10. 10.

    Rabets, Y., Backholm, M., Dalnoki-Veress, K. & Ryu, W. S. Direct measurements of drag forces in C. elegans crawling locomotion. Biophys. J. 107, 1980–1987 (2014).

    CAS  Article  Google Scholar 

  11. 11.

    Francis, G. W., Fisher, L. R., Gamble, R. A. & Gingell, D. Direct measurement of cell detachment force on single cells using a new electromechanical method. J. Cell Sci. 87, 519–523 (1987).

    PubMed  Google Scholar 

  12. 12.

    Colbert, M.-J., Brochard-Wyart, F., Fradin, C. & Dalnoki-Veress, K. Squeezing and detachment of living cells. Biophys. J. 99, 3555 (2010).

    CAS  Article  Google Scholar 

  13. 13.

    Backholm, M., Kasper, A. K. S., Schulman, R. D., Ryu, W. S. & Dalnoki-Veress, K. The effects of viscosity on the undulatory swimming dynamics of C. elegans. Phys. Fluids 27, 091901 (2015).

    Article  Google Scholar 

  14. 14.

    Schulman, R. D., Backholm, M., Ryu, W. S. & Dalnoki-Veress, K. Undulatory microswimming near solid boundaries. Phys. Fluids 26, 101902 (2014).

    Article  Google Scholar 

  15. 15.

    Florin, E.-L., Moy, V. & Gaub, H. E. Adhesion forces between individual ligand-receptor pairs. Science 264, 415–417 (1994).

    CAS  Article  Google Scholar 

  16. 16.

    Neuman, K. C. & Nagy, A. Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy. Nat. Methods 5, 491–505 (2008).

    CAS  Article  Google Scholar 

  17. 17.

    Dufrene, Y. F., Martinez-Martin, D., Medalsy, I., Alsteens, D. & Müller, D. J. Multiparametric imaging of biological systems by force-distance curve-based AFM. Nat. Methods 10, 847–854 (2013).

    CAS  Article  Google Scholar 

  18. 18.

    Beaussart, A. et al. Quantifying the forces guiding microbial cell adhesion using single-cell force spectroscopy. Nat. Protoc. 9, 1049–1055 (2014).

    CAS  Article  Google Scholar 

  19. 19.

    Iivarinen, J. T., Korhonen, R. K., Julkunen, P. & Jurvelin, J. S. Experimental and computational analysis of soft tissue stiffness in forearm using a manual indentation device. Med. Eng. Phys. 33, 1245–53 (2011).

    Article  Google Scholar 

  20. 20.

    Parker, D. et al. A device for characterizing the mechanical properties of the plantar soft tissue of the foot. Med. Eng. Phys. 37, 1098–1104 (2015).

    CAS  Article  Google Scholar 

  21. 21.

    Huber, G. et al. Evidence for capillarity contributions to gecko adhesion from single spatula nanomechanical measurements. Proc. Natl Acad. Sci. USA 102, 16293–16296 (2005).

    CAS  Article  Google Scholar 

  22. 22.

    Autumn, K. et al. Adhesive force of a single gecko foot-hair. Nature 405, 681–685 (2000).

    CAS  Article  Google Scholar 

  23. 23.

    Loskill, P. et al. Macroscale adhesion of gecko setae reflects nanoscale differences in subsurface contribution. J.R. Soc. Interface 10, 20120587 (2012).

    Article  Google Scholar 

  24. 24.

    Shimamoto, Y. & Kapoor, T. M. Microneedle-based analysis of the micromechanics of the metaphase spindle assembled in Xenopus laevis egg extracts. Nat. Protoc. 7, 959–969 (2012).

    CAS  Article  Google Scholar 

  25. 25.

    Sichel, F. J. M. The elasticity of isolated resting skeletal muscle fibers. J. Cell. Physiol. 5, 21–42 (1934).

    Article  Google Scholar 

  26. 26.

    Norris, C. H. The tension at the surface, and other physical properties of the nucleated erythrocyte. J. Cell. Physiol. 14, 117–133 (1939).

    CAS  Article  Google Scholar 

  27. 27.

    Coman, D. R. Decreased mutual adhesiveness, a property of cells from squamous cell carcinomas. Cancer Res. 4, 625–629 (1944).

    Google Scholar 

  28. 28.

    Coman, D. R. Adhesiveness and stickiness: two independent properties of cell surfaces. Cancer Res. 21, 1436–1438 (1961).

    CAS  PubMed  Google Scholar 

  29. 29.

    Yoneda, M. Force exerted by a single cilium of Mytilus edulis. I. J. Exp. Biol. 37, 461–468 (1960).

    Google Scholar 

  30. 30.

    Meyhofer, E. & Howard, J. The force generated by a single kinesin molecule against an elastic load. Proc. Natl Acad. Sci. USA 92, 574–578 (1995).

    CAS  Article  Google Scholar 

  31. 31.

    Suda, H. & Yamada, S. Force measurements for the movement of a water drop on a surface with a surface tension gradient. Langmuir 19, 529–531 (2002).

    Article  Google Scholar 

  32. 32.

    Lagubeau, G., Le Merrer, M., Clanet, C. & Quéré, D. Leidenfrost on a ratchet. Nat. Phys. 7, 395–398 (2011).

    CAS  Article  Google Scholar 

  33. 33.

    Pilat, D. W. et al. Dynamic measurement of the force required to move a liquid drop on a solid surface. Langmuir 28, 16812–16820 (2012).

    CAS  Article  Google Scholar 

  34. 34.

    Daniel, D., Timonen, J. V. I., Li, R., Velling, S. J. & Aizenberg, J. Oleoplaning droplets on lubricated surfaces. Nat. Phys. 13, 1020–1025 (2017).

    CAS  Article  Google Scholar 

  35. 35.

    Gao, N. et al. How drops start sliding over solid surfaces. Nat. Phys. 14, 191–196 (2018).

    CAS  Article  Google Scholar 

  36. 36.

    Mitrossilis, D. et al. Single-cell response to stiffness exhibits muscle-like behavior. Proc. Natl Acad. Sci. USA 106, 18243–18248 (2009).

    CAS  Article  Google Scholar 

  37. 37.

    Mitrossilis, D. et al. Real-time single-cell response to stiffness. Proc. Natl Acad. Sci. USA 107, 16518–16523 (2010).

    CAS  Article  Google Scholar 

  38. 38.

    Bowen, W. R., Lovitt, R. W. & Wright, C. J. Atomic force microscopy study of the adhesion of Saccharomyces cerevisiae. J. Colloid Interface Sci. 237, 54–61 (2001).

    CAS  Article  Google Scholar 

  39. 39.

    Mitchison, J. M. & Swann, M. M. The mechanical properties of the cell surface. J. Exp. Biol. 31, 443–460 (1954).

    Google Scholar 

  40. 40.

    Evans, E. A. Analysis of adhesion of large vesicles to surfaces. Biophys. J. 31, 425–431 (1980).

    CAS  Article  Google Scholar 

  41. 41.

    Neher, E. & Sakmann, S. Single-channel currents recorded from membrane of denervated frog muscle fibres. Nature 260, 799–802 (1976).

    CAS  Article  Google Scholar 

  42. 42.

    Sakmann, B. & Neher, E. Patch clamp techniques for studying ionic channels in excitable membranes. Annu. Rev. Physiol. 46, 455–472 (1984).

    CAS  Article  Google Scholar 

  43. 43.

    Basu, R. et al. Cytotoxic T cells use mechanical force to potentiate target cell killing. Cell 165, 100–110 (2016).

    CAS  Article  Google Scholar 

  44. 44.

    Guillou, L., Babataheri, A., Puech, P.-H., Barakat, A. I. & Husson, J. Dynamic monitoring of cell mechanical properties using profile microindentation. Sci. Rep. 6, 21529 (2016).

    CAS  Article  Google Scholar 

  45. 45.

    Sawicka, A. et al. Micropipette force probe to quantify single-cell force generation: application to T-cell activation. Mol. Biol. Cell 28, 3229–3239 (2017).

    CAS  Article  Google Scholar 

  46. 46.

    Houchmandzadeh, B. & Dimitrov, S. Elasticity measurements show the existence of thin rigid cores inside mitotic chromosomes. J. Cell Biol. 145, 215–223 (1999).

    CAS  Article  Google Scholar 

  47. 47.

    Poirier, M., Eroglu, S., Chatenay, D. & Marko, J. F. Reversible and irreversible unfolding of mitotic newt chromosomes by applied force. Mol. Biol. Cell 11, 269–276 (2000).

    CAS  Article  Google Scholar 

  48. 48.

    Poirier, M. G., Eroglu, S. & Marko, J. F. The bending rigidity of mitotic chromosomes. Mol. Biol. Cell 13, 2170–2179 (2002).

    CAS  Article  Google Scholar 

  49. 49.

    Moran, K., Yeung, A. & Masliyah, J. Measuring interfacial tensions of micrometer-sized droplets: a novel micromechanical technique. Langmuir 15, 8497–8504 (1999).

    CAS  Article  Google Scholar 

  50. 50.

    Yeung, A. K. C. & Pelton, R. Micromechanics: a new approach to studying the strength and breakup of flocs. J. Colloid Interface Sci. 184, 579–585 (1996).

    CAS  Article  Google Scholar 

  51. 51.

    Poppele, E. H. & Hozalski, R. M. Micro-cantilever method for measuring the tensile strength of biofilms and microbial flocs. J. Microbiol. Methods 55, 607–615 (2003).

    Article  Google Scholar 

  52. 52.

    Tsang, P. H., Li, G., Brun, Y. V., Freund, L. B. & Tang, J. X. Adhesion of single bacterial cells in the micronewton range. Proc. Natl Acad. Sci. USA 103, 5764–5768 (2006).

    CAS  Article  Google Scholar 

  53. 53.

    Schulman, R. D. & Dalnoki-Veress, K. Liquid droplets on a highly deformable membrane. Phys. Rev. Lett. 115, 206101 (2015).

    Article  Google Scholar 

  54. 54.

    ’t Mannetje, D. et al. Electrically tunable wetting defects characterized by a simple capillary force sensor. Langmuir 29, 9944–9949 (2013).

    Article  Google Scholar 

  55. 55.

    Frostad, J. M., Collins, M. C. & Leal, L. G. Cantilevered-capillary force apparatus for measuring multiphase fluid interactions. Langmuir 29, 4715–4725 (2013).

    CAS  Article  Google Scholar 

  56. 56.

    Frostad, J. M., Seth, M., Bernasek, S. M. & Leal, L. G. Direct measurement of interaction forces between charged multilamellar vesicles. Soft Matter 10, 7769–7780 (2014).

    CAS  Article  Google Scholar 

  57. 57.

    Frostad, J. M., Collins, M. C. & Leal, L. G. Direct measurement of the interaction of model food emulsion droplets adhering by arrested coalescence. Colloids Surf. A 441, 459–465 (2014).

    CAS  Article  Google Scholar 

  58. 58.

    Colbert, M.-J., Raegen, A. N., Fradin, C. & Dalnoki-Veress, K. Adhesion and membrane tension of single vesicles and living cells using a micropipette-based technique. Eur. Phys. J. E Soft Matter 30, 117 (2009).

    CAS  Article  Google Scholar 

  59. 59.

    Backholm, M., Ryu, W. S. & Dalnoki-Veress, K. The nematode C. elegans as a complex viscoelastic fluid. Eur. Phys. J. E Soft Matter 38, 36 (2015).

    Article  Google Scholar 

  60. 60.

    Backholm, M., Schulman, R. D., Ryu, W. S. & Dalnoki-Veress, K. Tangling of tethered swimmers: Interactions between two nematodes. Phys. Rev. Lett. 113, 138101 (2014).

    Article  Google Scholar 

  61. 61.

    Rüffer, U. & Nultsch, W. Comparison of the beating of cis- and trans-flagella of Chlamydomonas cells held on micropipettes. Cell Motil. Cytoskeleton 7, 87–93 (1987).

    Article  Google Scholar 

  62. 62.

    Rüffer, U. & Nultsch, W. Flagellar photoresponses of Chlamydomonas cells held on micropipettes: II. Change in flagellar beat pattern. Cell Motil. Cytoskeleton 18, 269–278 (1991).

    Article  Google Scholar 

  63. 63.

    Wan, K. Y. & Goldstein, R. E. Coordinated beating of algal flagella is mediated by basal coupling. Proc. Natl Acad. Sci. USA 113, E2784–E2793 (2016).

    CAS  Article  Google Scholar 

  64. 64.

    Drescher, K., Goldstein, R. E., Michel, N., Polin, M. & Tuval, I. Direct measurement of the flow field around swimming microorganisms. Phys. Rev. Lett. 105, 168101 (2010).

    Article  Google Scholar 

  65. 65.

    Harz, H. & Hegemann, P. Rhodopsin-regulated calcium currents in Chlamydomonas. Nature 351, 489–491 (1991).

    CAS  Article  Google Scholar 

  66. 66.

    Petit, J. et al. A modular approach for multifunctional polymersomes with controlled adhesive properties. Soft Matter 14, 894–900 (2018).

    CAS  Article  Google Scholar 

  67. 67.

    Kee, Y. S. & Robinson, D. N. Micropipette aspiration for studying cellular mechanosensory responses and mechanics. Methods Mol. Biol. 983, 367–382 (2013).

    CAS  Article  Google Scholar 

  68. 68.

    Biro, M. & Maître, J. L. Dual pipette aspiration: a unique tool for studying intercellular adhesion. Methods Cell Biol. 125, 255–267 (2015).

    Article  Google Scholar 

  69. 69.

    Guevorkian, K. & Maître, J. L. Micropipette aspiration: a unique tool for exploring cell and tissue mechanics in vivo. Methods Cell Biol. 139, 187–201 (2017).

    CAS  Article  Google Scholar 

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Acknowledgements

M.B. gratefully acknowledges support from the Academy of Finland (Centres of Excellence Programme (2014–2019, grant agreement no. 272361) and the Postdoctoral Researcher Project (grant agreement no. 309237)). O.B. acknowledges funding from the German Research Foundation (DFG) under grant BA3406/2. The authors are deeply grateful to K. Dalnoki-Veress for inspiring discussions and support. R.D. Schulman, C.T. Kreis, M.M. Makowski, T. Böddeker, and Q. Magdelaine are acknowledged for sharing their hands-on experiences and for valuable technical suggestions regarding improvements to the protocol.

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M.B. and O.B. developed the protocol and wrote the manuscript.

Corresponding authors

Correspondence to Matilda Backholm or Oliver Bäumchen.

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The authors declare no competing interests.

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Related links

Key reference using this protocol

Backholm, M., Ryu, W. S. & Dalnoki-Veress, K. Proc. Natl Acad. Sci. USA 110, 4528–4533 (2013): http://www.pnas.org/content/110/12/4528

Backholm, M., Kasper, A. K. S., Schulman, R. D., Ryu, W. S. & Dalnoki-Veress, K. Phys. Fluids 27, 091901 (2015): https://aip.scitation.org/doi/10.1063/1.4931795

Kreis, C. T., Le Blay, M., Linne, C., Makowski, M. M. & Bäumchen, O. Nat. Phys. 14, 45–49 (2018): https://www.nature.com/articles/nphys4258

Supplementary information

Supplementary Video 1

Real-time video from an MFS measurement of a swimming C. elegans nematode (in a 10% (wt/vol) PEO-M9 buffer solution) in which a three-dimensional micropipette is used to measure both lateral and propulsive drag forces.

Reporting Summary

Supplementary Software 1

Matlab code ‘calibration.m’ for determining the pipette spring constant in Calibration Option A.

Supplementary Software 2

Matlab code ‘deflection.m’ for analysing the pipette deflection via a cross-correlation approach.

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Backholm, M., Bäumchen, O. Micropipette force sensors for in vivo force measurements on single cells and multicellular microorganisms. Nat Protoc 14, 594–615 (2019). https://doi.org/10.1038/s41596-018-0110-x

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