Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Acoustic tweezers for high-throughput single-cell analysis

Nature Protocols volume 18, pages 2441–2458 (2023)Cite this article

Subjects

Abstract

Acoustic tweezers provide an effective means for manipulating single cells and particles in a high-throughput, precise, selective and contact-free manner. The adoption of acoustic tweezers in next-generation cellular assays may advance our understanding of biological systems. Here we present a comprehensive set of instructions that guide users through device fabrication, instrumentation setup and data acquisition to study single cells with an experimental throughput that surpasses traditional methods, such as atomic force microscopy and micropipette aspiration, by several orders of magnitude. With acoustic tweezers, users can conduct versatile experiments that require the trapping, patterning, pairing and separation of single cells in a myriad of applications ranging across the biological and biomedical sciences. This procedure is widely generalizable and adaptable for investigations in materials and physical sciences, such as the spinning motion of colloids or the development of acoustic-based quantum simulations. Overall, the device fabrication requires ~12 h, the experimental setup of the acoustic tweezers requires 1–2 h and the cell manipulation experiment requires ~30 min to complete. Our protocol is suitable for use by interdisciplinary researchers in biology, medicine, engineering and physics.

Key points

  • The procedural steps cover the fabrication of the microfluidic chamber, the interdigital transducer and the acoustic tweezers, followed by the experimental setup, the culturing of cells and their chemically induced perturbations and the manipulation of single cells and particles, data acquisition and analysis.

  • Acoustic tweezers enable the fast, high-throughput handling of single cells and particles, and outperforms alternative methods such as atomic force microscopy, optical tweezers or micropipettes.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Flow diagram of the workflow.
Fig. 2: Device fabrication and experimental setup of the acoustic tweezers.
Fig. 3: Cell culture and chemical perturbations.
Fig. 4: Anticipated results with the acoustic tweezers.

Similar content being viewed by others

Data availability

The main data discussed in this protocol are available in the supporting primary research paper30. The raw datasets are available for research purposes from the corresponding authors upon reasonable request.

References

  1. Barrow, A. D. et al. Natural killer cells control tumor growth by sensing a growth factor. Cell 172, 534–548. e519 (2018).

    Article  CAS  PubMed  Google Scholar 

  2. Landsberg, J. et al. Melanomas resist T-cell therapy through inflammation-induced reversible dedifferentiation. Nature 490, 412–416 (2012).

    Article  CAS  PubMed  Google Scholar 

  3. Galbraith, C. G., Yamada, K. M. & Galbraith, J. A. Polymerizing actin fibers position integrins primed to probe for adhesion sites. Science 315, 992–995 (2007).

    Article  CAS  PubMed  Google Scholar 

  4. Cunha, L. D. et al. LC3-associated phagocytosis in myeloid cells promotes tumor immune tolerance. Cell 175, 429–441. e416 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Kawai, T. & Akira, S. The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat. Immunol. 11, 373 (2010).

    Article  CAS  PubMed  Google Scholar 

  6. Hosseini, B. H. et al. Immune synapse formation determines interaction forces between T cells and antigen-presenting cells measured by atomic force microscopy. Proc. Natl Acad. Sci. USA 106, 17852–17857 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Friedrichs, J., Helenius, J. & Muller, D. J. Quantifying cellular adhesion to extracellular matrix components by single-cell force spectroscopy. Nat. Protoc. 5, 1353–1361 (2010).

    Article  CAS  PubMed  Google Scholar 

  8. Chu, Y. S. et al. Force measurements in E-cadherin-mediated cell doublets reveal rapid adhesion strengthened by actin cytoskeleton remodeling through Rac and Cdc42. J. Cell Biol. 167, 1183–1194 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Liu, B., Chen, W., Evavold, B. D. & Zhu, C. Accumulation of dynamic catch bonds between TCR and agonist peptide-MHC triggers T cell signaling. Cell 157, 357–368 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Dholakia, K. & Reece, P. Optical micromanipulation takes hold. Nano Today 1, 18–27 (2006).

    Article  Google Scholar 

  11. Feng, Y. et al. Mechanosensing drives acuity of αβ T-cell recognition. Proc. Natl Acad. Sci. USA 114, E8204–E8213 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Guillaume-Gentil, O. et al. Force-controlled manipulation of single cells: from AFM to FluidFM. Trends Biotechnol. 32, 381–388 (2014).

    Article  CAS  PubMed  Google Scholar 

  13. Aimon, S. et al. Membrane shape modulates transmembrane protein distribution. Dev. Cell 28, 212–218 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Ashkin, A., Dziedzic, J. M. & Yamane, T. Optical trapping and manipulation of single cells using infrared laser beams. Nature 330, 769–771 (1987).

    Article  CAS  PubMed  Google Scholar 

  15. Guck, J. et al. The optical stretcher: a novel laser tool to micromanipulate cells. Biophys. J. 81, 767–784 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Dholakia, K., Drinkwater, B. W. & Ritsch-Marte, M. Comparing acoustic and optical forces for biomedical research. Nat. Rev. Phys. 2, 480–491 (2020).

    Article  Google Scholar 

  17. Zhang, Z. & Ahmed, D. Light-driven high-precision cell adhesion kinetics. Light Sci. Appl. 11, 1–2 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  18. Shi, Y. et al. Nanometer-precision linear sorting with synchronized optofluidic dual barriers. Sci. Adv. 4, eaao0773 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  19. Shi, Y. et al. Sculpting nanoparticle dynamics for single-bacteria-level screening and direct binding-efficiency measurement. Nat. Commun. 9, 815 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Ozcelik, A. et al. Acoustic tweezers for the life sciences. Nat. Methods 15, 1021–1028 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Guo, F. et al. Controlling cell–cell interactions using surface acoustic waves. Proc. Natl Acad. Sci. USA 112, 43–48 (2015).

    Article  CAS  PubMed  Google Scholar 

  22. Collins, D. J. et al. Two-dimensional single-cell patterning with one cell per well driven by surface acoustic waves. Nat. Commun. 6, 8686 (2015).

    Article  CAS  PubMed  Google Scholar 

  23. Collins, D. J. et al. Acoustic tweezers via sub–time-of-flight regime surface acoustic waves. Sci. Adv. 2, e1600089 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  24. Muller, P. B., Barnkob, R., Jensen, M. J. H. & Bruus, H. A numerical study of microparticle acoustophoresis driven by acoustic radiation forces and streaming-induced drag forces. Lab Chip 12, 4617–4627 (2012).

    Article  CAS  PubMed  Google Scholar 

  25. Shpak, O. et al. Acoustic droplet vaporization is initiated by superharmonic focusing. Proc. Natl Acad. Sci. USA 111, 1697–1702 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Wang, Y. et al. A rapid and controllable acoustothermal microheater using thin film surface acoustic waves. Sens. Actuator A Phys. 318, 112508 (2021).

    Article  CAS  Google Scholar 

  27. Tian, Z. et al. Wave number–spiral acoustic tweezers for dynamic and reconfigurable manipulation of particles and cells. Sci. Adv. 5, eaau6062 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Rufo, J., Cai, F., Friend, J., Wiklund, M. & Huang, T. J. Acoustofluidics for biomedical applications. Nat. Rev. Methods Prim. 2, 30 (2022).

    Article  CAS  Google Scholar 

  29. Sitters, G. et al. Acoustic force spectroscopy. Nat. Methods 12, 47–50 (2015).

    Article  CAS  PubMed  Google Scholar 

  30. Yang, S. et al. Harmonic acoustics for dynamic and selective particle manipulation. Nat. Mater. 21, 540–546 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Skelley, A. M., Kirak, O., Suh, H., Jaenisch, R. & Voldman, J. Microfluidic control of cell pairing and fusion. Nat. Methods 6, 147–152 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Cermak, N. et al. High-throughput measurement of single-cell growth rates using serial microfluidic mass sensor arrays. Nat. Biotechnol. 34, 1052–1059 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Zhang, S.-Q. et al. High-throughput determination of the antigen specificities of T cell receptors in single cells. Nat. Biotechnol. 36, 1156–1159 (2018).

    Article  CAS  Google Scholar 

  34. Alapan, Y. et al. Microrobotics and microorganisms: biohybrid autonomous cellular robots. Annu. Rev. Control Robot. Auton. Syst. 2, 205–230 (2019).

    Article  Google Scholar 

  35. Mulvana, H., Cochran, S. & Hill, M. Ultrasound assisted particle and cell manipulation on-chip. Adv. Drug Deliv. Rev. 65, 1600–1610 (2013).

    Article  CAS  PubMed  Google Scholar 

  36. Neužil, P., Giselbrecht, S., Länge, K., Huang, T. J. & Manz, A. Revisiting lab-on-a-chip technology for drug discovery. Nat. Rev. Drug Discov. 11, 620–632 (2012).

    Article  PubMed Central  Google Scholar 

  37. Franke, T., Braunmüller, S., Schmid, L., Wixforth, A. & Weitz, D. Surface acoustic wave actuated cell sorting (SAWACS). Lab Chip 10, 789–794 (2010).

    Article  CAS  PubMed  Google Scholar 

  38. Nilsson, J., Evander, M., Hammarström, B. & Laurell, T. Review of cell and particle trapping in microfluidic systems. Anal. Chim. Acta 649, 141–157 (2009).

    Article  CAS  PubMed  Google Scholar 

  39. Wiklund, M. & Hertz, H. M. Ultrasonic enhancement of bead-based bioaffinity assays. Lab Chip 6, 1279–1292 (2006).

    Article  CAS  PubMed  Google Scholar 

  40. Reboud, J. et al. Shaping acoustic fields as a toolset for microfluidic manipulations in diagnostic technologies. Proc. Natl Acad. Sci. USA 109, 15162–15167 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Garg, N. et al. Whole-blood sorting, enrichment and in situ immunolabeling of cellular subsets using acoustic microstreaming. Microsyst. Nanoeng. 4, 1–9 (2018).

    Article  Google Scholar 

  42. Baudoin, M. et al. Folding a focalized acoustical vortex on a flat holographic transducer: miniaturized selective acoustical tweezers. Sci. Adv. 5, eaav1967 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Yeo, L. Y. & Friend, J. R. Surface acoustic wave microfluidics. Annu. Rev. Fluid Mech. 46, 379–406 (2014).

    Article  Google Scholar 

  44. Huang, P. H. et al. Acoustofluidic synthesis of particulate nanomaterials. Adv. Sci. 6, 1900913 (2019).

    Article  CAS  Google Scholar 

  45. Gu, Y. et al. Acoustofluidic centrifuge for nanoparticle enrichment and separation. Sci. Adv. 7, eabc0467 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Melde, K., Mark, A. G., Qiu, T. & Fischer, P. Holograms for acoustics. Nature 537, 518 (2016).

    Article  CAS  PubMed  Google Scholar 

  47. Ahmed, D. et al. Neutrophil-inspired propulsion in a combined acoustic and magnetic field. Nat. Commun. 8, 1–8 (2017).

    Article  CAS  Google Scholar 

  48. Fan, X.-D., Zou, Z. & Zhang, L. Acoustic vortices in inhomogeneous media. Phys. Rev. Res. 1, 032014 (2019).

    Article  CAS  Google Scholar 

  49. Glynne-Jones, P., Boltryk, R. J., Harris, N. R., Cranny, A. W. & Hill, M. Mode-switching: a new technique for electronically varying the agglomeration position in an acoustic particle manipulator. Ultrasonics 50, 68–75 (2010).

    Article  CAS  PubMed  Google Scholar 

  50. Marzo, A., Caleap, M. & Drinkwater, B. W. Acoustic virtual vortices with tunable orbital angular momentum for trapping of Mie particles. Phys. Rev. Lett. 120, 044301 (2018).

    Article  CAS  PubMed  Google Scholar 

  51. Wang, Z. et al. Acoustofluidic salivary exosome isolation: a liquid biopsy compatible approach for human papillomavirus-associated oropharyngeal cancer detection. J. Mol. Diagn. 22, 50–59 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Hao, N. et al. Acoustofluidic multimodal diagnostic system for Alzheimer’s disease. Biosens. Bioelectron. 196, 113730 (2022).

    Article  CAS  PubMed  Google Scholar 

  53. Ren, L. et al. Standing surface acoustic wave (SSAW)‐based fluorescence‐activated cell sorter. Small 14, 1801996 (2018).

    Article  Google Scholar 

  54. Zhang, J. et al. Surface acoustic waves enable rotational manipulation of Caenorhabditis elegans. Lab Chip 19, 984–992 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Schuetz, M. J. et al. Acoustic traps and lattices for electrons in semiconductors. Phys. Rev. X 7, 041019 (2017).

    Google Scholar 

  56. Schülein, F. J. et al. Fourier synthesis of radiofrequency nanomechanical pulses with different shapes. Nat. Nanotechnol. 10, 512–516 (2015).

    Article  PubMed  Google Scholar 

  57. Zhao, P. et al. Acoustically induced giant synthetic hall voltages in graphene. Phys. Rev. Lett. 128, 256601 (2022).

    Article  CAS  PubMed  Google Scholar 

  58. Krenner, H. J. & Westerhausen, C. Handy nanoquakes. Nat. Mater. 21, 499–501 (2022).

    Article  CAS  PubMed  Google Scholar 

  59. Sun, W., Gao, X., Lei, H., Wang, W. & Cao, Y. Biophysical approaches for applying and measuring biological forces. Adv. Sci. 9, 2105254 (2022).

    Article  Google Scholar 

  60. Neuman, K. C. & Nagy, A. Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy. Nat. Methods 5, 491–505 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Maître, J.-L. et al. Adhesion functions in cell sorting by mechanically coupling the cortices of adhering cells. Science 338, 253–256 (2012).

    Article  PubMed  Google Scholar 

  62. González-Bermúdez, B., Guinea, G. V. & Plaza, G. R. Advances in micropipette aspiration: applications in cell biomechanics, models, and extended studies. Biophys. J. 116, 587–594 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  63. Hochmuth, R. M. Micropipette aspiration of living cells. J. Biomech. 33, 15–22 (2000).

    Article  CAS  PubMed  Google Scholar 

  64. Zhang, J. et al. A solution to the biophysical fractionation of extracellular vesicles: Acoustic Nanoscale Separation via Wave-pillar Excitation Resonance (ANSWER). Sci. Adv. 8, eade0640 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Leach, J. et al. Comparison of Faxén’s correction for a microsphere translating or rotating near a surface. Phys. Rev. E 79, 026301 (2009).

    Article  CAS  Google Scholar 

  66. Yang, Z. et al. Light sheet microscopy with acoustic sample confinement. Nat. Commun. 10, 1–8 (2019).

    Google Scholar 

  67. Baudoin, M. et al. Spatially selective manipulation of cells with single-beam acoustical tweezers. Nat. Commun. 11, 1–10 (2020).

    Article  Google Scholar 

  68. Thalhammer, G., McDougall, C., MacDonald, M. P. & Ritsch-Marte, M. Acoustic force mapping in a hybrid acoustic-optical micromanipulation device supporting high resolution optical imaging. Lab Chip 16, 1523–1532 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Li, B., Zhou, D. & Han, Y. Assembly and phase transitions of colloidal crystals. Nat. Rev. Mater. 1, 15011 (2016).

    Article  CAS  Google Scholar 

  71. Hou, J., Li, M. & Song, Y. Recent advances in colloidal photonic crystal sensors: materials, structures and analysis methods. Nano Today 22, 132–144 (2018).

    Article  CAS  Google Scholar 

  72. Lim, M. X., Souslov, A., Vitelli, V. & Jaeger, H. M. Cluster formation by acoustic forces and active fluctuations in levitated granular matter. Nat. Phys. 15, 460–464 (2019).

    Article  CAS  Google Scholar 

  73. Manoharan, V. N. Colloidal matter: packing, geometry, and entropy. Science 349, 1253751 (2015).

    Article  PubMed  Google Scholar 

Download references

Acknowledgements

We acknowledge support from the Shared Materials Instrumentation Facility at Duke University. We acknowledge support from the National Institutes of Health (R01GM145960 (L.P.L), R01GM141055 (T.J.H.), R01GM132603 (T.J.H.), R01HD103727 (T.J.H.), R01GM143439 (T.J.H.), R01GM135486 (T.J.H.), R44AG063643 (T.J.H.), R44OD024963 (T.J.H.), R44HL140800 (T.J.H.), R21HD102790 (T.J.H.), U18TR003778 (T.J.H.) and UH3TR002978 (T.J.H.)), the National Science Foundation (ECCS-1807601 (T.J.H.), MCB-2042704 (T.J.H.) and CMMI-2104295 (T.J.H.)) and the National Science Foundation Graduate Research Fellowship (1644868 (J.R.)).

Author information

Authors and Affiliations

  1. Thomas Lord Department of Mechanical Engineering and Materials Science, Duke University, Durham, NC, USA

    Shujie Yang, Joseph Rufo, Ruoyu Zhong, Zeyu Wang & Tony Jun Huang

  2. Department of Biomedical Engineering, Duke University, Durham, NC, USA

    Joseph Rich

  3. Renal Division and Division of Engineering in Medicine, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA

    Luke P. Lee

  4. Department of Bioengineering, Department of Electrical Engineering and Computer Science, University of California, Berkeley, Berkeley, CA, USA

    Luke P. Lee

  5. Institute of Quantum Biophysics, Department of Biophysics, Sungkyunkwan University, Suwon, South Korea

    Luke P. Lee

Authors
  1. Shujie Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Joseph Rufo
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ruoyu Zhong
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Joseph Rich
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Zeyu Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Luke P. Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Tony Jun Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

S.Y., L.P.L. and T.J.H. designed the research. S.Y. performed the research. Z.W. performed the western blot analysis. S.Y., J. Rufo, R.Z., J. Rich, L.P.L., Z. W. and T.J.H. analyzed data. S.Y., R.Z. and L.P.L. drew the figures. S.Y., J. Rufo, J. Rich, L.P.L. and T.J.H. wrote the paper. S.Y., J. Rich, L.P.L. and T.J.H. revised the manuscript.

Corresponding authors

Correspondence to Luke P. Lee or Tony Jun Huang.

Ethics declarations

Competing interests

T.J.H. has cofounded a start-up company, Ascent Bio-Nano Technologies Inc., to commercialize technologies involving acoustofluidics and acoustic tweezers. The remaining authors declare no competing interests.

Peer review

Peer review information

Nature Protocols thanks Weiwei Cui, Kishan Dholakia, Tiziano Serra and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

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

Related links

Yang, S. et al. Nat. Mater. 21, 540–546 (2022): https://doi.org/10.1038/s41563-022-01210-8

Supplementary information

Supplementary Information

Supplementary Figs. 1–4, Table 1, Notes 1 and 2 and Appendix 1.

Reporting Summary

Supplementary Video 1

Fabrication process of the acoustic tweezers.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yang, S., Rufo, J., Zhong, R. et al. Acoustic tweezers for high-throughput single-cell analysis. Nat Protoc 18, 2441–2458 (2023). https://doi.org/10.1038/s41596-023-00844-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41596-023-00844-5

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing