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A guide to building a low-cost centrifuge force microscope module for single-molecule force experiments

Nature Protocols volume 20, pages 1951–1975 (2025)Cite this article

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Abstract

The ability to apply controlled forces to individual molecules or molecular complexes and observe their behaviors has led to many important discoveries in biology. Instruments capable of probing single-molecule forces typically cost >US$100,000, limiting the use of these techniques. The centrifuge force microscope (CFM) is a low-cost and easy-to-use instrument that enables high-throughput single-molecule studies. By combining the imaging capabilities of a microscope with the force application of a centrifuge, the CFM enables the simultaneous probing of hundreds to thousands of single-molecule interactions using tethered particles. Here we present a comprehensive set of instructions for building a CFM module that fits within a commercial benchtop centrifuge. The CFM module uses a 3D-printed housing, relies on off-the-shelf optical and electrical components, and can be built for less than US$1,000 in about 1 day. We also provide detailed instructions for setting up and running an experiment to measure force-dependent shearing of a short DNA duplex, as well as the software for CFM control and data analysis. The protocol is suitable for users with basic experience in analytical biochemistry and biophysics. The protocol enables the use of CFM-based experiments and may facilitate access to the single-molecule research field.

Key points

  • The protocol covers the build and setup of a centrifuge force microscope that uses 3D-printed components and can be accommodated within an existing centrifuge, and the experimental procedure to probe the force-dependent dissociation of a short DNA duplex in the low piconewton force range.

  • Alternative methods include optical and magnetic tweezers, atomic force microscopy and acoustic forces, fluid flow or DNA-based probes.

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Fig. 1: Conceptual overview of the CFM and its basic elements.
Fig. 2: Visual overview of the main steps of the procedure.
Fig. 3: Details and construction of the CFM module.
Fig. 4: Preparing materials for a CFM experiment.
Fig. 5: Running the experiment and analyzing data.

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Data availability

The main data discussed in this protocol (including raw source data that appear in Figs. 2 and 5) are available in the supporting primary research paper (https://doi.org/10.1038/s41467-023-36373-8). As mentioned in the data availability statement of the primary research paper, the raw videos are too large to be publicly shared (~1 TB) but are available for research purposes from the corresponding author upon reasonable request.

Code availability

Analysis software is available in the supporting primary research paper (https://doi.org/10.1038/s41467-023-36373-8). Our image acquisition software in LabView is provided in Supplementary Files.

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Acknowledgements

The authors thank A. R. Chandrasekaran for useful conversations about the manuscript and figures, and B. Halvorsen for feedback to improve the CFM build instructions. Research reported in this publication was supported by the National Institutes of Health (NIH) through the National Institute of General Medical Sciences under awards R35GM124720 to K.H. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

Author information

Authors and Affiliations

  1. The RNA Institute, University at Albany, State University of New York, Albany, NY, USA

    Jibin Abraham Punnoose, Andrew Hayden, Chai S. Kam & Ken Halvorsen

  2. Department of Biological Sciences, University at Albany, State University of New York, Albany, NY, USA

    Chai S. Kam

Authors
  1. Jibin Abraham Punnoose
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  2. Andrew Hayden
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  3. Chai S. Kam
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  4. Ken Halvorsen
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Contributions

This protocol was conceived and planned by K.H. All authors were involved in different aspects of protocol development, primarily J.A.P. for experiment design and data analysis, C.S.K. for experiment preparation and running, A.H. for electrical design and 3D modeling, and K.H. for mechanical design and assembly. The main text was written by J.A.P. and K.H. with input and editing from all authors.

Corresponding author

Correspondence to Ken Halvorsen.

Ethics declarations

Competing interests

K.H. has several issued patents and pending patent applications related to the CFM and has previously earned licensing royalties related to CFM.

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Peer review information

Nature Protocols thanks Hans Bergal, Wesley Wong and the other, anonymous reviewer(s) for their contribution to the peer review of this work.

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Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Key references

Abraham Punnoose, J. et al. High-throughput single-molecule quantification of individual base stacking energies in nucleic acids. Nat. Commun. 14.1, 631 (2023): https://doi.org/10.1038/s41467-023-36373-8

Abraham Punnoose, J. et al. Wi-Fi live-streaming centrifuge force microscope for benchtop single-molecule experiments. Biophys. J. 119.11, 2231–2239 (2020): https://doi.org/10.1016/j.bpj.2020.10.017

Supplementary information

Supplementary Information

Supplementary Notes 1–4, Figs. 1 and 2, Tables 1 and 2.

Reporting Summary

Supplementary Video 1

Video tutorial Steps 5–22.

Supplementary Video 2

Video tutorial Steps 23–38.

Supplementary Files

Fusion 3D model, individual .stl 3D models for printing, LabVIEW control software, MATLAB analysis code.

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Abraham Punnoose, J., Hayden, A., Kam, C.S. et al. A guide to building a low-cost centrifuge force microscope module for single-molecule force experiments. Nat Protoc 20, 1951–1975 (2025). https://doi.org/10.1038/s41596-024-01102-y

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