High-resolution, long-term characterization of bacterial motility using optical tweezers

Abstract

We present a single-cell motility assay, which allows the quantification of bacterial swimming in a well-controlled environment, for durations of up to an hour and with a temporal resolution greater than the flagellar rotation rates of 100 Hz. The assay is based on an instrument combining optical tweezers, light and fluorescence microscopy, and a microfluidic chamber. Using this device we characterized the long-term statistics of the run-tumble time series in individual Escherichia coli cells. We also quantified higher-order features of bacterial swimming, such as changes in velocity and reversals of swimming direction.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Combined optical trap and fluorescence microscope setup.
Figure 2: Direct observation of tumbles in an optically trapped cell.
Figure 3: Run-tumble phenotyping using the optical trapping assay.
Figure 4: Run duration statistics in individual bacteria.
Figure 5: Higher-order features in cell motility.

References

  1. 1

    Berg, H.C. E. coli in motion. (Springer, New York, 2004).

    Google Scholar 

  2. 2

    Alon, U. An introduction to systems biology: design principles of biological circuits. (Chapman & Hall/CRC, Boca Raton, Florida, USA, 2007).

    Google Scholar 

  3. 3

    Brown, D.A. & Berg, H.C. Temporal stimulation of chemotaxis in Escherichia coli. Proc. Natl. Acad. Sci. USA 71, 1388–1392 (1974).

    CAS  Article  Google Scholar 

  4. 4

    Khan, S. et al. Excitatory signaling in bacteria probed by caged chemoeffectors. Biophys. J. 65, 2368–2382 (1993).

    CAS  Article  Google Scholar 

  5. 5

    Block, S.M., Segall, J.E. & Berg, H.C. Impulse responses in bacterial chemotaxis. Cell 31, 215–226 (1982).

    CAS  Article  Google Scholar 

  6. 6

    Staropoli, J.F. & Alon, U. Computerized analysis of chemotaxis at different stages of bacterial growth. Biophys. J. 78, 513–519 (2000).

    CAS  Article  Google Scholar 

  7. 7

    Berg, H.C. & Brown, D.A. Chemotaxis in Escherichia coli analyzed by 3-dimensional tracking. Nature 239, 500–504 (1972).

    CAS  Article  Google Scholar 

  8. 8

    Silverman, M. & Simon, M. Flagellar rotation and the mechanism of bacterial motility. Nature 249, 73–74 (1974).

    CAS  Article  Google Scholar 

  9. 9

    Sowa, Y. et al. Direct observation of steps in rotation of the bacterial flagellar motor. Nature 437, 916–919 (2005).

    CAS  Article  Google Scholar 

  10. 10

    Bustamante, C., Chemla, Y.R. & Moffitt, J.R. High resolution dual trap optical tweezers with differential detection. in Single-Molecule Techniques: A Laboratory Manual (eds., P. Selvin & T. Ha) 297–324 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA, 2009).

  11. 11

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

    CAS  Article  Google Scholar 

  12. 12

    Rowe, A.D., Leake, M.C., Morgan, H. & Berry, R.M. Rapid rotation of micron and submicron dielectric particles measured using optical tweezers. J. Mod. Opt. 50, 1539–1554 (2003).

    CAS  Article  Google Scholar 

  13. 13

    Chattopadhyay, S., Moldovan, R., Yeung, C. & Wu, X.L. Swimming efficiency of bacterium Escherichia coli. Proc. Natl. Acad. Sci. USA 103, 13712–13717 (2006).

    CAS  Article  Google Scholar 

  14. 14

    Darnton, N.C., Turner, L., Rojevsky, S. & Berg, H.C. On torque and tumbling in swimming Escherichia coli. J. Bacteriol. 189, 1756–1764 (2007).

    CAS  Article  Google Scholar 

  15. 15

    Parkinson, J.S. & Houts, S.E. Isolation and behavior of Escherichia coli deletion mutants lacking chemotaxis functions. J. Bacteriol. 151, 106–113 (1982).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16

    Neuman, K.C., Chadd, E.H., Liou, G.F., Bergman, K. & Block, S.M. Characterization of photodamage to Escherichia coli in optical traps. Biophys. J. 77, 2856–2863 (1999).

    CAS  Article  Google Scholar 

  17. 17

    Rasmussen, M.B., Oddershede, L.B. & Siegumfeldt, H. Optical tweezers cause physiological damage to Escherichia coli and Listeria bacteria. Appl. Environ. Microbiol. 74, 2441–2446 (2008).

    CAS  Article  Google Scholar 

  18. 18

    Neidhardt, F.C., Ingraham, J.L. & Schaechter, M. Physiology of the bacterial cell: a molecular approach (Sinauer Associates, Sunderland, Massachusetts, USA, 1990).

  19. 19

    Turner, L., Ryu, W.S. & Berg, H.C. Real-time imaging of fluorescent flagellar filaments. J. Bacteriol. 182, 2793–2801 (2000).

    CAS  Article  Google Scholar 

  20. 20

    Alon, U. et al. Response regulator output in bacterial chemotaxis. EMBO J. 17, 4238–4248 (1998).

    Article  Google Scholar 

  21. 21

    Teolis, A. Computational signal processing with wavelets. (Birkhäuser, Boston, 1998).

    Google Scholar 

  22. 22

    Berg, H.C. & Turner, L. Cells of Escherichia coli swim either end forward. Proc. Natl. Acad. Sci. USA 92, 477–479 (1995).

    CAS  Article  Google Scholar 

  23. 23

    Korobkova, E., Emonet, T., Vilar, J.M., Shimizu, T.S. & Cluzel, P. From molecular noise to behavioural variability in a single bacterium. Nature 428, 574–578 (2004).

    CAS  Article  Google Scholar 

  24. 24

    Cisneros, L., Dombrowski, C., Goldstein, R.E. & Kessler, J.O. Reversal of bacterial locomotion at an obstacle. Phys. Rev. E 73, 030901 (2006).

    Article  Google Scholar 

  25. 25

    Korobkova, E.A., Emonet, T., Park, H. & Cluzel, P. Hidden stochastic nature of a single bacterial motor. Phys. Rev. Lett. 96, 058105 (2006).

    Article  Google Scholar 

  26. 26

    Saini, S., Brown, J.D., Aldridge, P.D. & Rao, C.V. Fli Z. is a posttranslational activator of FlhD4C2-dependent flagellar gene expression. J. Bacteriol. 190, 4979–4988 (2008).

    CAS  Article  Google Scholar 

  27. 27

    Selvin, P.R. et al. In vitro and in vivo FIONA and other acronyms for watching molecular motors walk. in Single-Molecule Techniques: A Laboratory Manual (eds., Selvin P. and Ha T.) 37–71 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA, 2008).

  28. 28

    Adler, J. & Templeton, B. The effect of environmental conditions on the motility of Escherichia coli. J. Gen. Microbiol. 46, 175–184 (1967).

    CAS  Article  Google Scholar 

  29. 29

    Berg, H.C. & Turner, L. Chemotaxis of bacteria in glass capillary arrays. Escherichia coli, motility, microchannel plate, and light scattering. Biophys. J. 58, 919–930 (1990).

    CAS  Article  Google Scholar 

  30. 30

    Joo, C. & Ha, T. Single-molecule FRET with total internal reflection microscopy. in Single-Molecule Techniques: A Laboratory Manual (eds., Selvin P. and Ha T.) 3–36 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA, 2008).

  31. 31

    Dijk, M.A., Kapitein, L.C., Mameren, J., Schmidt, C.F. & Peterman, E.J. Combining optical trapping and single-molecule fluorescence spectroscopy: enhanced photobleaching of fluorophores. J. Phys. Chem. B 108, 6479–6484 (2004).

    Article  Google Scholar 

  32. 32

    Datsenko, K.A. & Wanner, B.L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA 97, 6640–6645 (2000).

    CAS  Article  Google Scholar 

  33. 33

    Gonzalez, R.C., Woods, R.E. & Eddins, S.L. Digital Image Processing Using MATLAB (Pearson/Prentice Hall, Upper Saddle River, New Jersey, USA, 2004).

    Google Scholar 

  34. 34

    Moffitt, J.R., Chemla, Y.R., Izhaky, D. & Bustamante, C. Differential detection of dual traps improves the spatial resolution of optical tweezers. Proc. Natl. Acad. Sci. USA 103, 9006–9011 (2006).

    CAS  Article  Google Scholar 

  35. 35

    Amsler, C.D. Use of computer-assisted motion analysis for quantitative measurements of swimming behavior in peritrichously flagellated bacteria. Anal. Biochem. 235, 20–25 (1996).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank P. Cluzel (Harvard University) for the gift of the PS2001-pMS164 strain and the following people for their generous advice: C. Guet, H. Park, M. McLachlan, K. Neuman, S. Chattopadhyay, W. Ryu, T. Shimizu, R. Segev, G. Ordal, I. Nemenman, T. Emonet and all members of the Golding, Chemla, Rao, Selvin and Ha laboratories. The work was supported by the US National Science Foundation (grant 082265, Center for the Physics of Living Cells). Y.R.C. is supported by Burroughs-Wellcome Fund Career Awards at the Scientific Interface. T.L.M. was supported by National Institutes of Health Institutional National Research Service Award in Molecular Biophysics (PHS 5 T32 GM08276). C.V.R. is supported by National Institutes of Health grant GM054365.

Author information

Affiliations

Authors

Contributions

Y.R.C. and I.G. conceived the cell-trapping project. T.L.M. developed the cell-trapping assay. T.L.M. and P.J.M. developed the measurement protocols, performed the experiments and analyzed the data. L.M.C. and C.V.R. constructed and tested bacterial strains used in this study. C.V.R. provided expertise on bacterial physiology and chemotaxis. P.J.M., T.L.M., I.G. and Y.R.C. wrote the paper.

Corresponding author

Correspondence to Ido Golding.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7, Supplementary Tables 1–3 and Supplementary Notes 1–7 (PDF 774 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Min, T., Mears, P., Chubiz, L. et al. High-resolution, long-term characterization of bacterial motility using optical tweezers. Nat Methods 6, 831–835 (2009). https://doi.org/10.1038/nmeth.1380

Download citation

Further reading

Search

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