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Tracking mechanics and volume of globular cells with atomic force microscopy using a constant-height clamp

Nature Protocols volume 7, pages 143–154 (2012)Cite this article

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Abstract

To understand the role of physical forces at a cellular level, it is necessary to track mechanical properties during cellular processes. Here we present a protocol that uses flat atomic force microscopy (AFM) cantilevers clamped at constant height, and light microscopy to measure the resistance force, mechanical stress and volume of globular animal cells under compression. We describe the AFM and cantilever setup, live cell culture in the AFM, how to ensure stability of AFM measurements during medium perfusion, integration of optical microscopy to measure parameters such as volume and track intracellular dynamics, and interpretation of the physical parameters measured. Although we use this protocol on trypsinized interphase and mitotic HeLa cells, it can also be applied to other cells with a relatively globular shape, especially animal cells in a low-adhesive environment. After a short setup phase, the protocol can be used to investigate approximately one cell per hour.

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Figure 1: Overview of the protocol.
Figure 2: AFM-based techniques for cellular mechanics and volume measurements.
Figure 3: Examples of spurious disturbance in force recorded by the AFM setup during exchange of medium with different refractive indices.
Figure 4: Representative force (black), cross-sectional area (green), rounding pressure (orange) and normalized volume (blue) data plots for a trypsinized cell transitioning through G2/M of the cell cycle.
Figure 5: Representative force (black), cross-sectional area (green), rounding pressure (orange) and normalized volume (blue) plots for cells.

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Acknowledgements

We thank J. Helenius for stimulatory discussions and advice, and J. Howard for advice on refractive index change during medium exchange. This work was supported by the Bundesministerium für Bildung und Forschung (BMBF), the Swiss National Center of Competence in Research (NCCR) “Nanoscale Science”, the Max Planck Society and the Japan Society for the Promotion of Science (JSPS).

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Authors and Affiliations

  1. Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland

    Martin P Stewart & Daniel J Müller

  2. Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany

    Martin P Stewart, Yusuke Toyoda & Anthony A Hyman

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  1. Martin P Stewart
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  2. Yusuke Toyoda
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  3. Anthony A Hyman
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  4. Daniel J Müller
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Contributions

M.P.S. and D.J.M. designed the AFM protocol, and Y.T. devised its application to mitotic cells. M.P.S. performed and optimized the experimental procedure. M.P.S., A.A.H., Y.T. and D.J.M. contributed to the manuscript.

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Correspondence to Daniel J Müller.

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Stewart, M., Toyoda, Y., Hyman, A. et al. Tracking mechanics and volume of globular cells with atomic force microscopy using a constant-height clamp. Nat Protoc 7, 143–154 (2012). https://doi.org/10.1038/nprot.2011.434

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