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Fast and high-resolution mapping of elastic properties of biomolecules and polymers with bimodal AFM

Nature Protocols volume 13, pages 2890–2907 (2018)Cite this article

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Abstract

Fast, high-resolution mapping of heterogeneous interfaces with a wide elastic modulus range is a major goal of atomic force microscopy (AFM). This goal becomes more challenging when the nanomechanical mapping involves biomolecules in their native environment. Over the years, several AFM-based methods have been developed to address this goal. However, none of these methods combine sub-nanometer spatial resolution, quantitative accuracy, fast data acquisition speed, wide elastic modulus range and operation in physiological solutions. Here, we present detailed procedures for generating high-resolution maps of the elastic properties of biomolecules and polymers using bimodal AFM. This requires the simultaneous excitation of the first two eigenmodes of the cantilever. An amplitude modulation (AM) feedback acting on the first mode controls the tip–sample distance, and a frequency modulation (FM) feedback acts on the second mode. The method is fast because the elastic modulus, deformation and topography images are obtained simultaneously. The method is efficient because only a single data point per pixel is needed to generate the aforementioned images. The main stages of the bimodal imaging are sample preparation, calibration of the instrument, tuning of the microscope and generation of the nanomechanical maps. In addition, with knowledge of the deformation, bimodal AFM enables reconstruction of the true topography of the surface. It takes ~9 h to complete the whole procedure.

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Fig. 1: Bimodal AM–FM.
Fig. 2: Calibration of the spring constants and quality factors using the thermal noise spectra.
Fig. 3: Bimodal AFM images of a block copolymer.
Fig. 4: Cantilever excitation in bimodal AM–FM.
Fig. 5: Apparent and true topographic maps.
Fig. 6: Bimodal AFM maps of a purple membrane in buffer.
Fig. 7: Bimodal AFM maps of a 20S proteasome in liquid.
Fig. 8: Elastic modulus and deformation maps.

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Acknowledgements

We are grateful for financial support from the European Research Council (ERC–AdG–340177; 3DNanoMech) and grants CSD2010-00024 and MAT2016-76507-R from the Ministerio de Economía y Competitividad. This work received funding from the European Union’s Horizon 2020 Research and Innovation Programme under Marie Sklodowska-Curie grant agreement 721874 (SPM2.0). We also acknowledge fellowships FPU15/04622 (C.A.A.) and BES-2017-081907 (V.G.G.) from the Ministerio de Educación.

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  1. These authors contributed equally: Simone Benaglia, Victor G. Gisbert.

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  1. Materials Science Factory, Instituto de Ciencia de Materiales de Madrid (ICMM), CSIC, Madrid, Spain

    Simone Benaglia, Victor G. Gisbert, Alma P. Perrino, Carlos A. Amo & Ricardo Garcia

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Contributions

S.B., V.G.G. and A.P.P. performed the experiments. C.A.A. deduced the analytical expressions. S.B. and V.G.G. drafted the procedure. R.G. designed the experiments, supervised development of the theory, wrote the introduction and edited the manuscript. All the authors discussed the results and revised the manuscript.

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Correspondence to Ricardo Garcia.

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Competing interests

R.G. holds two US patents on bimodal AFM (7,921,466 B2 and 7,958,563 B2). The bimodal configuration presented in the Protocol is not described in those patents. The other authors declare no competing interests.

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Key references using this protocol

Amo, C. A., Perrino, A. P., Payam, A. F. & Garcia, R. ACS Nano 11, 8650–8659 (2017): https://pubs.acs.org/doi/10.1021/acsnano.7b04381

Herruzo, E. T., Perrino, A. P., & Garcia, R. Nat. Commun. 5, 3126 (2014): https://www.nature.com/articles/ncomms4126

Martinez-Martin, D., Herruzo, E. T., Dietz, C., Gomez-Herrero, J. & Garcia, R. Phys. Rev. Lett. 106, 198101 (2011): https://journals.aps.org/prl/abstract/10.1103/PhysRevLett.106.198101

Lozano, J. R. & Garcia, R. Phys. Rev. Lett. 100, 076102 (2008): https://journals.aps.org/prl/abstract/10.1103/PhysRevLett.100.076102

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Benaglia, S., Gisbert, V.G., Perrino, A.P. et al. Fast and high-resolution mapping of elastic properties of biomolecules and polymers with bimodal AFM. Nat Protoc 13, 2890–2907 (2018). https://doi.org/10.1038/s41596-018-0070-1

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