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:

Construction and calibration of an optical trap on a fluorescence optical microscope

Abstract

The application of optical traps has come to the fore in the last three decades. They provide a powerful, sterile and noninvasive tool for the manipulation of cells, single biological macromolecules, colloidal microparticles and nanoparticles. An optically trapped microsphere may act as a force transducer that is used to measure forces in the piconewton regime. By setting up a well-calibrated single-beam optical trap within a fluorescence microscope system, one can measure forces and collect fluorescence signals upon biological systems simultaneously. In this protocol, we aim to provide a clear exposition of the methodology of assembling and operating a single-beam gradient force trap (optical tweezers) on an inverted fluorescence microscope. A step-by-step guide is given for alignment and operation, with discussion of common pitfalls.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

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

Figure 1: Experimental setup.
Figure 2: Beam steering lens relay system.
Figure 3: Airy disk/aberration and power transmission measurements.
Figure 4: Three-dimensional trapping of a 1 μm polystyrene sphere.
Figure 5: Phase contrast ring and back focal plane interference.
Figure 6: Trap stiffness for 1 μm red fluorescent sphere.
Figure 7: Linear dependence of trap stiffness determined from the roll-off frequency with optical power of the trap.
Figure 8: Voltage response versus y displacement and particle position histogram for y axis.

Similar content being viewed by others

References

  1. Ashkin, A. Optical trapping and manipulation of neutral particles using lasers. Proc. Natl. Acad. Sci. USA 94, 4853–4860 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Ashkin, A., Dziedzic, J.M., Bjorkholm, J.E. & Chu, S. Observation of a single-beam gradient force optical trap for dielectric particles. Opt. Lett. 11, 288–290 (1986).

    Article  CAS  PubMed  Google Scholar 

  3. Rohrbach, A. et al. Trapping and tracking a local probe with a photonic force microscope. Rev. Sci. Instrum. 75, 2197–2210 (2004).

    Article  CAS  Google Scholar 

  4. Fallman, E. & Axner, O. Design for fully steerable dual-trap optical tweezers. Appl. Opt. 36, 2107–2113 (1997).

    Article  CAS  PubMed  Google Scholar 

  5. Li, Z.W. et al. Membrane tether formation from outer hair cells with optical tweezers. Biophys. J. 82, 1386–1395 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Bao, X.Y.R., Lee, H.J. & Quake, S.R. Behavior of complex knots in single DNA molecules. Phys. Rev. Lett. 91, 265506 (2003).

    Article  PubMed  Google Scholar 

  7. Svoboda, K., Schmidt, C.F., Schnapp, B.J. & Block, S.M. Direct observation of kinesin stepping by optical trapping interferometry. Nature 365, 721–727 (1993).

    Article  CAS  PubMed  Google Scholar 

  8. Finer, J.T., Simmons, R.M. & Spudich, J.A. Single myosin molecule mechanics—piconewton forces and nanometre steps. Nature 368, 113–119 (1994).

    Article  CAS  PubMed  Google Scholar 

  9. Lang, M.J. et al. Simultaneous, coincident optical trapping and single-molecule fluorescence. Nat. Methods 1, 133–139 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Tarsa, P.B. et al. Detecting force-induced molecular transitions with fluorescence resonant energy transfer. Angew. Chem. Int. Ed. Engl. 46, 1999–2001 (2007).

    Article  CAS  PubMed  Google Scholar 

  11. Bustamante, C., Bryant, Z. & Smith, S.B. Ten years of tension: single-molecule DNA mechanics. Nature 421, 423–427 (2003).

    Article  PubMed  Google Scholar 

  12. Bustamante, C., Smith, S.B., Liphardt, J. & Smith, D. Single-molecule studies of DNA mechanics. Curr. Opin. Struct. Biol. 10, 279–285 (2000).

    Article  CAS  PubMed  Google Scholar 

  13. Svoboda, K. & Block, S.M. Biological applications of optical forces. Annu. Rev. Biophys. Biomol. Struct. 23, 247–285 (1994).

    Article  CAS  PubMed  Google Scholar 

  14. Visscher, K., Gross, S.P. & Block, S.M. Construction of multiple-beam optical traps with nanometer-resolution position sensing. IEEE J. Select. Top. Quant. Electron. 2, 1066–1076 (1996).

    Article  CAS  Google Scholar 

  15. Lang, M.J. & Block, S.M. Resource letter: LBOT-1: LASER-based optical tweezers. Am. J. Phys. 71, 201–215 (2003).

    Article  PubMed  PubMed Central  Google Scholar 

  16. Neuman, K.C. & Block, S.M. Optical trapping. Rev. Sci. Instrum. 75, 2787–2809 (2004).

    Article  CAS  PubMed  Google Scholar 

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

    Article  Google Scholar 

  18. Smith, S.P. et al. Inexpensive optical tweezers for undergraduate laboratories. Am. J. Phys. 67, 26–35 (1999).

    Article  Google Scholar 

  19. Bechhoefer, J. & Wilson, S. Faster, cheaper, safer optical tweezers for the undergraduate laboratory. Am. J. Phys. 70, 393–400 (2002).

    Article  CAS  Google Scholar 

  20. Appleyard, D.C., Vandermeulen, K.Y., Lee, H. & Lang, M.J. Optical trapping for undergraduates. Am. J. Phys. 75, 5–14 (2007).

    Article  CAS  Google Scholar 

  21. Polin, M. et al. Optimized holographic optical traps. Opt. Express 13, 5831–5845 (2005).

    Article  PubMed  Google Scholar 

  22. Svoboda, K. & Block, S.M. Optical trapping of metallic Rayleigh particles. Opt. Lett. 19, 930–932 (1994).

    Article  CAS  PubMed  Google Scholar 

  23. Gittes, F. & Schmidt, C.F. Interference model for back-focal-plane displacement detection in optical tweezers. Opt. Lett. 23, 7–9 (1998).

    Article  CAS  PubMed  Google Scholar 

  24. Moothoo, D.N. et al. Beth's experiment using optical tweezers. Am. J. Phys. 69, 271–276 (2001).

    Article  Google Scholar 

  25. Berg-Sorensen, K. & Flyvbjerg, H. Power spectrum analysis for optical tweezers. Rev. Sci. Instrum. 75, 594–612 (2004).

    Article  CAS  Google Scholar 

  26. Mao, H.B. et al. Temperature control methods in a laser tweezers system. Biophys. J. 89, 1308–1316 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Huisstede, J.H.G., van der Werf, K.O., Bennink, M.L. & Subramaniam, V. Force detection in optical tweezers using backscattered light. Opt. Express 13, 1113–1123 (2005).

    Article  CAS  PubMed  Google Scholar 

  28. Neuman, K.C. et al. Characterization of photodamage to Escherichia coli in optical traps. Biophys. J. 77, 2856–2863 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. O'Neill, A.T. & Padgett, M.J. Axial and lateral trapping efficiency of Laguerre–Gaussian modes in inverted optical tweezers. Opt. Commun. 193, 45–50 (2001).

    Article  Google Scholar 

  30. Tolic-Norrelykke, I.M., Berg-Sorensen, K. & Flyvbjerg, H. MatLab program for precision calibration of optical tweezers. Comput. Phys. Commun. 159, 225–240 (2004).

    Article  CAS  Google Scholar 

  31. Greenleaf, W.J., Woodside, M.T., Abbondanzieri, E.A. & Block, S.M. Passive all-optical force clamp for high-resolution laser trapping. Phys. Rev. Lett. 95, 208102 (2005).

    Article  PubMed  PubMed Central  Google Scholar 

  32. Jonas, A., Zemanek, P. & Florin, E.L. Single-beam trapping in front of reflective surfaces. Opt. Lett. 26, 1466–1468 (2001).

    Article  Google Scholar 

Download references

Acknowledgements

This work is supported by the UK Engineering and Physical Sciences Research Council. We acknowledge several useful discussions with Daniel Burnham.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Kishan Dholakia.

Supplementary information

Supplementary Figure 1

Sample Chamber (PDF 17 kb)

Supplementary Note 1

Dual beam optical tweezers system (PDF 90 kb)

Supplementary Note 2

Sample preparation (PDF 78 kb)

Supplementary Note 3

Q values (PDF 259 kb)

Supplementary Note 4

Trap stiffness (PDF 373 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lee, W., Reece, P., Marchington, R. et al. Construction and calibration of an optical trap on a fluorescence optical microscope. Nat Protoc 2, 3226–3238 (2007). https://doi.org/10.1038/nprot.2007.446

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2007.446

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

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