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Mapping heterogeneity of cellular mechanics by multi-harmonic atomic force microscopy

Nature Protocols volume 13, pages 2200–2216 (2018)Cite this article

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Abstract

The goal of mechanobiology is to understand the links between changes in the physical properties of living cells and normal physiology and disease. This requires mechanical measurements that have appropriate spatial and temporal resolution within a single cell. Conventional atomic force microscopy (AFM) methods that acquire force curves pointwise are used to map the heterogeneous mechanical properties of cells. However, the resulting map acquisition time is much longer than that required to study many dynamic cellular processes. Dynamic AFM (dAFM) methods using resonant microcantilevers are compatible with higher-speed, high-resolution scanning; however, they do not directly acquire force curves and they require the conversion of a limited number of instrument observables to local mechanical property maps. We have recently developed a technique that allows commercial AFM systems equipped with direct cantilever excitation to quantitatively map the viscoelastic properties of live cells. The properties can be obtained at several widely spaced frequencies with nanometer–range spatial resolution and with fast image acquisition times (tens of seconds). Here, we describe detailed procedures for quantitative mapping, including sample preparation, AFM calibration, and data analysis. The protocol can be applied to different biological samples, including cells and viruses. The transition from dAFM imaging to quantitative mapping should be easily achievable for experienced AFM users, who will be able to set up the protocol in <30 min.

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Fig. 1: Overview of the protocol.
Fig. 2: Typical dynamic approach curve obtained in the central region of a fibroblast cell (nuclear area) with a Lorentz excited cantilever.
Fig. 3: Bimodal AFM on a rat fibroblast.
Fig. 4: Comparison of AFM nanomechanical with fluorescence data in a growth cone developed by an Aplysia bag cell neuron.
Fig. 5: Tracking the fast temporal changes in nanomechanical heterogeneities of MDA-MB-231 breast cancer cells upon inhibition of Syk-AQL-EGFP protein tyrosine kinase with 1-NM-PP1.
Fig. 6: Property maps of ϕ29 bacteriophage mature virions.

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Acknowledgements

This work was supported by the Incentive Grant Program of the Office of the Executive Vice President for Research and Partnerships – Purdue University (D.M.S. and A.R.) and by NSF 1146944-IOS (D.M.S.). A.X.C.-R. was supported by intramural funding from the Division of Intramural Research Program at the National Institute on Deafness and Other Communication Disorders.

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Author notes

  1. These authors contributed equally: Yuri M. Efremov, Alexander X. Cartagena-Rivera

Authors and Affiliations

  1. School of Mechanical Engineering, Purdue University, West Lafayette, IN, USA

    Yuri M. Efremov & Arvind Raman

  2. Birck Nanotechnology Center, Purdue University, West Lafayette, IN, USA

    Yuri M. Efremov, Ahmad I. M. Athamneh, Daniel M. Suter & Arvind Raman

  3. Laboratory of Cellular Biology, Section on Auditory Mechanics, National Institute on Deafness and Other Communication Disorders, Bethesda, MD, USA

    Alexander X. Cartagena-Rivera

  4. Department of Biological Sciences, Purdue University, West Lafayette, IN, USA

    Ahmad I. M. Athamneh & Daniel M. Suter

  5. Bindley Bioscience Center, Purdue University, West Lafayette, IN, USA

    Daniel M. Suter

  6. Purdue Institute for Integrative Neuroscience, Purdue University, West Lafayette, IN, USA

    Daniel M. Suter

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Contributions

A.X.C.-R., Y.M.E., D.M.S., and A.R. conceived and designed the experiments. Y.M.E., A.X.C.-R., A.I.M.A., D.M.S., and A.R. developed experimental protocols for sample preparation. Y.M.E. and A.X.C.-R. performed all the research experiments, analyzed the data, and prepared the figures. Y.M.E., A.X.C.-R., D.M.S., and A.R. co-wrote the paper. All authors discussed the results and reviewed the paper.

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Correspondence to Arvind Raman.

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Key references using this protocol

Raman, A. et al. Nat. Nanotechnol. 6, 809–814 (2011) https://doi.org/10.1038/nnano.2011.186

Cartagena, A., Hernando-Pérez, M., Carrascosa, J. L., de Pablo, P. J. & Raman, A. Nanoscale 5, 4729–4736 (2013) https://doi.org/10.1039/C3NR34088K

Cartagena-Rivera, A. X., Wang, W.-H., Geahlen, R. L. & Raman, A. Sci. Rep. 5, 11692 (2015) https://doi.org/10.1038/srep11692

Integrated supplementary information

Supplementary Figure 1 Topography and maps of the multi-harmonic observables (amplitudes A0, A1, and phase φ1) for the growth cone presented in Fig. 4.

Scale bars are 5 µm, acquisition time of maps 5 min, 256X256 pixels.

Supplementary Figure 2 Tracking the fast temporal changes in nanomechanical heterogeneities of MDA-MB-231 breast cancer cells upon inhibition of Syk-AQL-EGFP protein tyrosine kinase with 1-NM-PP1.

The rapid loss of Syk activity was correlated with dramatic rearrangements in the cortical actin cytoskeleton observed as a retraction of the leading edge. Only the deflection channel is shown here. The acquisition time was 1 min 30 s (256X256 pixels), every second image in the series is shown. Scale bar is 5 µm, common for all the images.

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Efremov, Y.M., Cartagena-Rivera, A.X., Athamneh, A.I.M. et al. Mapping heterogeneity of cellular mechanics by multi-harmonic atomic force microscopy. Nat Protoc 13, 2200–2216 (2018). https://doi.org/10.1038/s41596-018-0031-8

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