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

Nature Protocols volume 14, pages 594–615 (2019)Cite this article

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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.

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Data availability

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

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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.

Author information

Authors and Affiliations

  1. Department of Applied Physics, Aalto University, Espoo, Finland

    Matilda Backholm

  2. Max Planck Institute for Dynamics and Self-Organization (MPIDS), Göttingen, Germany

    Oliver Bäumchen

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  1. Matilda Backholm
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M.B. and O.B. developed the protocol and wrote the manuscript.

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Correspondence to Matilda Backholm or Oliver Bäumchen.

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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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