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Guide to video recording of structure dynamics and dynamic processes of proteins by high-speed atomic force microscopy

Nature Protocols volume 7, pages 1193–1206 (2012)Cite this article

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Abstract

High-speed atomic force microscopy (HS-AFM) allows direct visualization of dynamic structural changes and processes of functioning biological molecules in physiological solutions, at subsecond to sub-100-ms temporal and submolecular spatial resolution. Unlike fluorescence microscopy, wherein the subset of molecular events that you see is dependent on the site where the probe is placed, dynamic molecular events unselectively appear in detail in an AFM movie, facilitating our understanding of how biological molecules function. Here we present protocols for HS-AFM imaging of proteins in action, including preparation of cantilever tips, step-by-step procedures for HS-AFM imaging, and recycling of cantilevers and sample stages, together with precautions and troubleshooting advice for successful imaging. The protocols are adaptable in general for imaging many proteins and protein–nucleic acid complexes, and examples are described for looking at walking myosin, ATP-hydrolyzing rotorless F1-ATPase and cellulose-hydrolyzing cellulase. The entire protocol takes 10–15 h, depending mainly on the substrate surface to be used.

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Figure 1
Figure 2: Flowcharts showing overview of procedures before and during HS-AFM imaging and cleanup steps for the next imaging experiments.
Figure 3
Figure 4: Pictures and schematics of a container for growing EBD tips with SEM.
Figure 5: SEM photographs of small cantilevers with and without EBD tip.
Figure 6: Storage tools used to preserve a solution droplet on the sample stage without drying it.
Figure 7: Importance of firmly mounting the cantilever base and selecting the imaging region for successful imaging.
Figure 8: Optical microscopic views of a small cantilever.
Figure 9: Clipped high-speed AFM images showing relationship between applying tapping force and image quality.
Figure 10: High-speed AFM images of proteins in action.
Figure 11: Effect of AFM tip smear and its removal on image quality.

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Acknowledgements

We thank D. Yamamoto for technical assistance. This work was supported by the Core Research for Evolutionary Science and Technology (CREST) program of the Japan Science and Technology Agency (JST); a Grant-in-Aid for Basic Research (S) from the Japan Society for the Promotion of Science (JSPS) (no. 20221006); a Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed Research Area) from the Ministry of Education, Culture, Science, Sports and Technology (MEXT)-Japan; and the Knowledge Cluster Initiative/MEXT-Japan.

Author information

Authors and Affiliations

  1. Department of Physics, Kanazawa University, Kanazawa, Japan

    Takayuki Uchihashi & Toshio Ando

  2. Bio-AFM Frontier Research Center, Kanazawa University, Kanazawa, Japan

    Takayuki Uchihashi, Noriyuki Kodera & Toshio Ando

Authors
  1. Takayuki Uchihashi
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  2. Noriyuki Kodera
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Contributions

All the authors designed and discussed the experiments. T.U. and N.K. equally contributed to this work, conducted the experiments, prepared all figures and movies, and drafted the MATERIALS and PROCEDURE sections. T.A. wrote the introductory part of manuscript and edited the whole manuscript.

Corresponding author

Correspondence to Toshio Ando.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Video 1

Alignment of the laser position relative to the small cantilever (0 s – 12 s) and adjustment of the photodetector position (> 13 s). Inserted images on the right bottom is the 4-digit indicators for the total intensity of laser irradiated onto the four segments of the quadrant PIN photodiode (left) and difference between the laser intensities irradiated on the top two segments and the bottom two segments (right). (MOV 7097 kb)

Supplementary Video 2

Rinsing of sample or others placed on substrate disk attached to the top of a sample stage. (MOV 3561 kb)

Supplementary Video 3

Mounting of the scanner on the HS-AFM apparatus (0 s – 12 s) and position adjustment of the sample stage relative to the cantilever (> 13 s). (MOV 5127 kb)

Supplementary Video 4

HS-AFM imaging of myosin V-HMM moving on actin filament. (MOV 8683 kb)

Supplementary Video 5

Effect of dynamic PID control mode on parachuting. (MOV 8449 kb)

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Uchihashi, T., Kodera, N. & Ando, T. Guide to video recording of structure dynamics and dynamic processes of proteins by high-speed atomic force microscopy. Nat Protoc 7, 1193–1206 (2012). https://doi.org/10.1038/nprot.2012.047

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