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.

  • Review Article
  • Published:

Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy

Abstract

Single-molecule force spectroscopy has emerged as a powerful tool to investigate the forces and motions associated with biological molecules and enzymatic activity. The most common force spectroscopy techniques are optical tweezers, magnetic tweezers and atomic force microscopy. Here we describe these techniques and illustrate them with examples highlighting current capabilities and limitations.

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

Access options

Buy this article

Purchase on Springer Link

Instant access to full article PDF

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

Figure 1: Schematics of optical tweezers–based assays.
Figure 2: Influence of probe size, stiffness and measurement bandwidth on spatial resolution.
Figure 3: Magnetic tweezers.
Figure 4: Atomic force microscopy.

Similar content being viewed by others

References

  1. Cluzel, P. et al. DNA: An extensible molecule. Science 271, 792–794 (1996).

    CAS  PubMed  Google Scholar 

  2. Evans, E., Ritchie, K. & Merkel, R. Sensitive force technique to probe molecular adhesion and structural linkages at biological interfaces. Biophys. J. 68, 2580–2587 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Smith, S.B., Finzi, L. & Bustamante, C. Direct mechanical measurements of the elasticity of single DNA molecules by using magnetic beads. Science 258, 1122–1126 (1992).

    CAS  PubMed  Google Scholar 

  4. Kim, S.J., Blainey, P.C., Schroeder, C.M. & Xie, X.S. Multiplexed single-molecule assay for enzymatic activity on flow-stretched DNA. Nat. Methods 4, 397–399 (2007).

    CAS  PubMed  Google Scholar 

  5. Greenleaf, W.J., Woodside, M.T. & Block, S.M. High-resolution, single-molecule measurements of biomolecular motion. Annu. Rev. Biophys. Biomol. Struct. 36, 171–190 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Neuman, K.C. & Block, S.M. Optical trapping. Rev. Sci. Instrum. 75, 2787–2809 (2004). A detailed and thorough technical review of optical trapping.

    CAS  PubMed  Google Scholar 

  7. Tanase, M., Biais, N. & Sheetz, M. Magnetic tweezers in cell biology. Methods Cell Biol. 83, 473–493 (2007).

    CAS  PubMed  Google Scholar 

  8. Zlatanova, J., Lindsay, S.M. & Leuba, S.H. Single molecule force spectroscopy in biology using the atomic force microscope. Prog. Biophys. Mol. Biol. 74, 37–61 (2000).

    CAS  PubMed  Google Scholar 

  9. Lee, C.K., Wang, Y.M., Huang, L.S. & Lin, S. Atomic force microscopy: determination of unbinding force, off rate and energy barrier for protein-ligand interaction. Micron 38, 446–461 (2007).

    CAS  PubMed  Google Scholar 

  10. Lang, M.J., Asbury, C.L., Shaevitz, J.W. & Block, S.M. An automated two-dimensional optical force clamp for single molecule studies. Biophys. J. 83, 491–501 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Neuman, K.C., Lionnet, T. & Allemand, J.F. Single-molecule micromanipulation techniques. Annu. Rev. Mater. Res. 37, 33–67 (2007).

    CAS  Google Scholar 

  12. Abbondanzieri, E.A., Greenleaf, W.J., Shaevitz, J.W., Landick, R. & Block, S.M. Direct observation of base-pair stepping by RNA polymerase. Nature 438, 460–465 (2005). This is an experimental tour de force in which individual 0.34 nm base-pair steps of transcribing RNA polymerases were directly measured with optical tweezers.

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Grandbois, M., Beyer, M., Rief, M., Clausen-Schaumann, H. & Gaub, H.E. How strong is a covalent bond? Science 283, 1727–1730 (1999).

    CAS  PubMed  Google Scholar 

  14. Hohng, S. et al. Fluorescence-force spectroscopy maps two-dimensional reaction landscape of the holliday junction. Science 318, 279–283 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Litvinov, R.I., Shuman, H., Bennett, J.S. & Weisel, J.W. Binding strength and activation state of single fibrinogen-integrin pairs on living cells. Proc. Natl. Acad. Sci. USA 99, 7426–7431 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Prass, M., Jacobson, K., Mogilner, A. & Radmacher, M. Direct measurement of the lamellipodial protrusive force in a migrating cell. J. Cell Biol. 174, 767–772 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. MacKintosh, F.C. & Schmidt, C.F. Microrheology. Curr. Opin. Colloid Interface Sci. 4, 300–307 (1999).

    CAS  Google Scholar 

  18. Daniels, B.R., Masi, B.C. & Wirtz, D. Probing single-cell micromechanics in vivo: the microrheology of C. elegans developing embryos. Biophys. J. 90, 4712–4719 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Block, S.M., Goldstein, L.S. & Schnapp, B.J. Bead movement by single kinesin molecules studied with optical tweezers. Nature 348, 348–352 (1990).

    CAS  PubMed  Google Scholar 

  20. Svoboda, K. & Block, S.M. Force and velocity measured for single kinesin molecules. Cell 77, 773–784 (1994).

    CAS  PubMed  Google Scholar 

  21. Simmons, R.M. et al. Force on single actin filaments in a motility assay measured with an optical trap. Adv. Exp. Med. Biol. 332, 331–336 (1993).

    CAS  PubMed  Google Scholar 

  22. Tinoco, I. Jr & Bustamante, C. The effect of force on thermodynamics and kinetics of single molecule reactions. Biophys. Chem. 101–102, 513–533 (2002). A detailed and comprehensive treatment of the effects of force on single-molecule reactions; the concepts and analysis presented form the underpinning of single-molecule force spectroscopy.

    PubMed  Google Scholar 

  23. Tskhovrebova, L., Trinick, J., Sleep, J.A. & Simmons, R.M. Elasticity and unfolding of single molecules of the giant muscle protein titin. Nature 387, 308–312 (1997).

    CAS  PubMed  Google Scholar 

  24. Kellermayer, M.S.Z., Smith, S.B., Granzier, H.L. & Bustamante, C. Folding-unfolding transitions in single titin molecules characterized with laser tweezers. Science 276, 1112–1116 (1997).

    CAS  PubMed  Google Scholar 

  25. Rief, M., Gautel, M., Oesterhelt, F., Fernandez, J.M. & Gaub, H.E. Reversible unfolding of individual titin immunoglobulin domains by AFM. Science 276, 1109–1112 (1997). This paper and the one above (ref. 24 ) appeared together and described the first demonstrations of the mechanical unfolding of an individual protein.

    CAS  PubMed  Google Scholar 

  26. Hummer, G. & Szabo, A. Free energy surfaces from single-molecule force spectroscopy. Acc. Chem. Res. 38, 504–513 (2005).

    CAS  PubMed  Google Scholar 

  27. Woodside, M.T. et al. Nanomechanical measurements of the sequence-dependent folding landscapes of single nucleic acid hairpins. Proc. Natl. Acad. Sci. USA 103, 6190–6195 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Lim, C.T., Zhou, E.H., Li, A., Vedula, S.R.K. & Fu, H.X. Experimental techniques for single cell and single molecule biomechanics. Mater. Sci. Eng. C-Biomimetic Supramol. Syst. 26, 1278–1288 (2006).

    CAS  Google Scholar 

  29. Zhuang, X.W. & Rief, M. Single-molecule folding. Curr. Opin. Struct. Biol. 13, 88–97 (2003).

    CAS  PubMed  Google Scholar 

  30. Evans, E. Probing the relation between force–lifetime–and chemistry in single molecular bonds. Annu. Rev. Biophys. Biomol. Struct. 30, 105–128 (2001).

    Article  CAS  PubMed  Google Scholar 

  31. Jarzynski, C. Nonequilibrium equality for free energy differences. Phys. Rev. Lett. 78, 2690–2693 (1997). This seminal paper presents the remarkable Jarzynski equality that relates the equilibrium free energy difference to non-equilibrium measurements of work. The Jarzynski equality and subsequent relations based on the equality permit the extraction of folding free energies from out-of-equilibrium mechanical unfolding experiments.

    CAS  Google Scholar 

  32. Lu, H.P., Xun, L. & Xie, X.S. Single-molecule enzymatic dynamics. Science 282, 1877–1882 (1998).

    CAS  PubMed  Google Scholar 

  33. Xie, X.S. & Lu, H.P. Single-molecule enzymology. J. Biol. Chem. 274, 15967–15970 (1999).

    CAS  PubMed  Google Scholar 

  34. Hua, W., Young, E.C., Fleming, M.L. & Gelles, J. Coupling of kinesin steps to ATP hydrolysis. Nature 388, 390–393 (1997).

    CAS  PubMed  Google Scholar 

  35. Schnitzer, M.J. & Block, S.M. Kinesin hydrolyses one ATP per 8-nm step. Nature 388, 386–390 (1997).

    CAS  PubMed  Google Scholar 

  36. Hermanson, G. Bioconjugate Techniques. (Academic Press, San Diego 1996).

    Google Scholar 

  37. Hinterdorfer, P. & Dufrene, Y.F. Detection and localization of single molecular recognition events using atomic force microscopy. Nat. Methods 3, 347–355 (2006).

    CAS  PubMed  Google Scholar 

  38. 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). This classic paper is the first experimental demonstration of the single-beam gradient trap, or optical tweezers.

    CAS  PubMed  Google Scholar 

  39. 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  PubMed  PubMed Central  Google Scholar 

  40. Sacconi, L., Tolic-Norrelykke, I.M., Stringari, C., Antolini, R. & Pavone, F.S. Optical micromanipulations inside yeast cells. Appl. Opt. 44, 2001–2007 (2005).

    PubMed  Google Scholar 

  41. Cherney, D.P., Bridges, T.E. & Harris, J.M. Optical trapping of unilamellar phospholipid vesicles: Investigation of the effect of optical forces on the lipid membrane shape by confocal-raman microscopy. Anal. Chem. 76, 4920–4928 (2004).

    CAS  PubMed  Google Scholar 

  42. Lee, W.M., Reece, P.J., Marchington, R.F., Metzger, N.K. & Dholakia, K. Construction and calibration of an optical trap on a fluorescence optical microscope. Nat. Protocols 2, 3226–3238 (2007).

    CAS  PubMed  Google Scholar 

  43. Neuman, K.C., Abbondanzieri, E.A. & Block, S.M. Measurement of the effective focal shift in an optical trap. Opt. Lett. 30, 1318–1320 (2005).

    PubMed  PubMed Central  Google Scholar 

  44. Fallman, E. & Axner, O. Influence of a glass-water interface on the on-axis trapping of micrometer-sized spherical objects by optical tweezers. Appl. Opt. 42, 3915–3926 (2003).

    PubMed  Google Scholar 

  45. Viana, N.B., Rocha, M.S., Mesquita, O.N., Mazolli, A. & Neto, P.A.M. Characterization of objective transmittance for optical tweezers. Appl. Opt. 45, 4263–4269 (2006).

    CAS  PubMed  Google Scholar 

  46. Misawa, H., Koshioka, M., Sasaki, K., Kitamura, N. & Masuhara, H. Three-dimensional optical trapping and laser ablation of a single polymer latex particle in water. J. Appl. Phys. 70, 3829–3836 (1991).

    CAS  Google Scholar 

  47. Rohrbach, A. & Stelzer, E.H.K. Three-dimensional position detection of optically trapped dielectric particles. J. Appl. Phys. 91, 5474–5488 (2002).

    CAS  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  49. Peterman, E.J.G., van Dijk, M.A., Kapitein, L.C. & Schmidt, C.F. Extending the bandwidth of optical-tweezers interferometry. Rev. Sci. Instrum. 74, 3246–3249 (2003).

    CAS  Google Scholar 

  50. 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).

    PubMed  PubMed Central  Google Scholar 

  51. Carter, A.R., King, G.M. & Perkins, T.T. Back-scattered detection provides atomic-scale localization precision, stability, and registration in 3D. Opt. Express 15, 13434–13445 (2007).

    PubMed  Google Scholar 

  52. Carter, A.R. et al. Stabilization of an optical microscope to 0.1 nm in three dimensions. Appl. Opt. 46, 421–427 (2007).

    PubMed  Google Scholar 

  53. Vermeulen, K.C. et al. Calibrating bead displacements in optical tweezers using acousto-optic deflectors. Rev. Sci. Instrum. 77, 013704 (2006).

    Google Scholar 

  54. Tolic-Norrelykke, S.F. et al. Calibration of optical tweezers with positional detection in the back focal plane. Rev. Sci. Instrum. 77, 103101 (2006).

    Google Scholar 

  55. 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). The first direct measurement of the steps taken by individual kinesin.

    CAS  PubMed  Google Scholar 

  56. Mammen, M. et al. Optically controlled collisions of biological objects to evaluate potent polyvalent inhibitors of virus-cell adhesion. Chem. Biol. 3, 757–763 (1996).

    CAS  PubMed  Google Scholar 

  57. Kerssemakers, J.W.J. et al. Assembly dynamics of microtubules at molecular resolution. Nature 442, 709–712 (2006).

    CAS  PubMed  Google Scholar 

  58. Wang, M.D. et al. Force and velocity measured for single molecules of RNA polymerase. Science 282, 902–907 (1998).

    CAS  PubMed  Google Scholar 

  59. Herbert, K.M. et al. Sequence-resolved detecton of pausing by single RNA polymerase molecules. Cell 125, 1083–1094 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  60. Neuman, K.C., Abbondanzieri, E.A., Landick, R., Gelles, J. & Block, S.M. Ubiquitous transcriptional pausing is independent of RNA polymerase backtracking. Cell 115, 437–447 (2003).

    CAS  PubMed  Google Scholar 

  61. Shaevitz, J.W., Abbondanzieri, E.A., Landick, R. & Block, S.M. Backtracking by single RNA polymerase molecules observed at near-base-pair resolution. Nature 426, 684–687 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  62. 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  PubMed  PubMed Central  Google Scholar 

  63. Dame, R.T., Noom, M.C. & Wuite, G.J. Bacterial chromatin organization by H-NS protein unravelled using dual DNA manipulation. Nature 444, 387–390 (2006).

    CAS  PubMed  Google Scholar 

  64. Smith, D.E. et al. The bacteriophage phi 29 portal motor can package DNA against a large internal force. Nature 413, 748–752 (2001).

    CAS  PubMed  Google Scholar 

  65. Liphardt, J., Onoa, B., Smith, S.B., Tinoco, I. & Bustamante, C. Reversible unfolding of single RNA molecules by mechanical force. Science 292, 733–737 (2001). This paper was the first to show the mechanical unfolding of RNA structures, and it laid the groundwork for subsequent measurements of enzymatic unfolding of nucleic acid structures.

    CAS  PubMed  Google Scholar 

  66. Dumont, S. et al. RNA translocation and unwinding mechanism of HCV NS3 helicase and its coordination by ATP. Nature 439, 105–108 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  67. Friese, M.E.J., Nieminen, T.A., Heckenberg, N.R. & Rubinsztein-Dunlop, H. Optical alignment and spinning of laser-trapped microscopic particles. Nature 394, 348–350 (1998).

    CAS  Google Scholar 

  68. Deufel, C., Forth, S., Simmons, C.R., Dejgosha, S. & Wang, M.D. Nanofabricated quartz cylinders for angular trapping: DNA supercoiling torque detection. Nat. Methods 4, 223–225 (2007).

    CAS  PubMed  Google Scholar 

  69. La Porta, A. & Wang, M.D. Optical torque wrench: angular trapping, rotation, and torque detection of quartz microparticles. Phys. Rev. Lett. 92, 190801 (2004).

    PubMed  Google Scholar 

  70. Gross, S.P. Application of optical traps in vivo. Methods Enzymol. 361, 162–174 (2003).

    CAS  PubMed  Google Scholar 

  71. Peterman, E.J.G., Gittes, F. & Schmidt, C.F. Laser-induced heating in optical traps. Biophys. J. 84, 1308–1316 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  72. Abbondanzieri, E.A., Shaevitz, J.W. & Block, S.M. Picocalorimetry of transcription by RNA polymerase. Biophys. J. 89, L61–L63 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  73. Seol, Y., Carpenter, A.E. & Perkins, T.T. Gold nanoparticles: enhanced optical trapping and sensitivity coupled with significant heating. Opt. Lett. 31, 2429–2431 (2006).

    CAS  PubMed  Google Scholar 

  74. Liang, H. et al. Wavelength dependence of cell cloning efficiency after optical trapping. Biophys. J. 70, 1529–1533 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  75. Strick, T., Allemand, J., Croquette, V. & Bensimon, D. Twisting and stretching single DNA molecules. Prog. Biophys. Mol. Biol. 74, 115–140 (2000).

    CAS  PubMed  Google Scholar 

  76. Charvin, G., Strick, T.R., Bensimon, D. & Croquette, V. Tracking topoisomerase activity at the single-molecule level. Annu. Rev. Biophys. Biomol. Struct. 34, 201–219 (2005).

    CAS  PubMed  Google Scholar 

  77. Strick, T.R., Croquette, V. & Bensimon, D. Single-molecule analysis of DNA uncoiling by a type II topoisomerase. Nature 404, 901–904 (2000). This was the first single-molecule measurement of topoisomerase activity using magnetic tweezers.

    CAS  PubMed  Google Scholar 

  78. Gore, J. et al. Mechanochemical analysis of DNA gyrase using rotor bead tracking. Nature 439, 100–104 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  79. Itoh, H. et al. Mechanically driven ATP synthesis by F-1-ATPase. Nature 427, 465–468 (2004).

    CAS  PubMed  Google Scholar 

  80. Fisher, J.K. et al. Thin-foil magnetic force system for high-numerical-aperture microscopy. Rev. Sci. Instrum. 77, 023702 (2006).

    Google Scholar 

  81. Yan, J., Skoko, D. & Marko, J.F. Near-field-magnetic-tweezer manipulation of single DNA molecules. Phys. Rev. E 70, 011905 (2004).

    Google Scholar 

  82. Gosse, C. & Croquette, V. Magnetic tweezers: micromanipulation and force measurement at the molecular level. Biophys. J. 82, 3314–3329 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  83. Lee, S.H. et al. Characterizing and tracking single colloidal particles with video holographic microscopy. Opt. Express 15, 18275–18282 (2007).

    PubMed  Google Scholar 

  84. Lionnet, T., Spiering, M.M., Benkovic, S.J., Bensimon, D. & Croquette, V. Real-time observation of bacteriophage T4 gp41 helicase reveals an unwinding mechanism. Proc. Natl. Acad. Sci. USA 104, 19790–19795 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  85. Strick, T.R., Allemand, J.F., Bensimon, D., Bensimon, A. & Croquette, V. The elasticity of a single supercoiled DNA molecule. Science 271, 1835–1837 (1996).

    CAS  PubMed  Google Scholar 

  86. Strick, T.R., Allemand, J.F., Bensimon, D. & Croquette, V. Behavior of supercoiled DNA. Biophys. J. 74, 2016–2028 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  87. Dekker, N.H. et al. The mechanism of type IA topoisomerases. Proc. Natl. Acad. Sci. USA 99, 12126–12131 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  88. Charvin, G., Bensimon, D. & Croquette, V. Single-molecule study of DNA unlinking by eukaryotic and prokaryotic type-II topoisomerases. Proc. Natl. Acad. Sci. USA 100, 9820–9825 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  89. Conroy, R.S. Force spectroscopy with optical and magnetic tweezers. in Handbook of Molecular Force Spectroscopy (ed. Noy, A.) 23–96 (Springer US, New York, 2008).

    Google Scholar 

  90. Bausch, A.R., Ziemann, F., Boulbitch, A.A., Jacobson, K. & Sackmann, E. Local measurements of viscoelastic parameters of adherent cell surfaces by magnetic bead microrheometry. Biophys. J. 75, 2038–2049 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  91. de Vries, A.H., Krenn, B.E., van Driel, R. & Kanger, J.S. Micro magnetic tweezers for nanomanipulation inside live cells. Biophys. J. 88, 2137–2144 (2005).

    CAS  PubMed  Google Scholar 

  92. Bausch, A.R., Moller, W. & Sackmann, E. Measurement of local viscoelasticity and forces in living cells by magnetic tweezers. Biophys. J. 76, 573–579 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  93. Keller, M., Schilling, J. & Sackmann, E. Oscillatory magnetic bead rheometer for complex fluid microrheometry. Rev. Sci. Instrum. 72, 3626–3634 (2001).

    CAS  Google Scholar 

  94. Danilowicz, C., Greenfield, D. & Prentiss, M. Dissociation of ligand-receptor complexes using magnetic tweezers. Anal. Chem. 77, 3023–3028 (2005).

    CAS  PubMed  Google Scholar 

  95. Koster, D.A., Croquette, V., Dekker, C., Shuman, S. & Dekker, N.H. Friction and torque govern the relaxation of DNA supercoils by eukaryotic topoisomerase IB. Nature 434, 671–674 (2005).

    CAS  PubMed  Google Scholar 

  96. Koster, D.A., Palle, K., Bot, E.S., Bjornsti, M.A. & Dekker, N.H. Antitumour drugs impede DNA uncoiling by topoisomerase I. Nature 448, 213–217 (2007). The mechanism of action of a type I topoisomerase inhibitor measured at the single-molecule level was shown to be directly related to its effects in vivo.

    CAS  PubMed  Google Scholar 

  97. Revyakin, A., Ebright, R.H. & Strick, T.R. Single-molecule DNA nanomanipulation: improved resolution through use of shorter DNA fragments. Nat. Methods 2, 127–138 (2005).

    CAS  PubMed  Google Scholar 

  98. Revyakin, A., Liu, C., Ebright, R.H. & Strick, T.R. Abortive initiation and productive initiation by RNA polymerase involve DNA scrunching. Science 314, 1139–1143 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  99. Binnig, G., Quate, C.F. & Gerber, C. Atomic force microscope. Phys. Rev. Lett. 56, 930–933 (1986).

    CAS  PubMed  Google Scholar 

  100. Lee, G.U., Chrisey, L.A. & Colton, R.J. Direct measurement of the forces between complementary strands of DNA. Science 266, 771–773 (1994).

    CAS  PubMed  Google Scholar 

  101. Dai, P. et al. X-ray-diffraction and scanning-tunneling-microscopy studies of a liquid-crystal film adsorbed on single-crystal graphite. Phys. Rev. B Condens. Matter 47, 7401–7407 (1993).

    CAS  PubMed  Google Scholar 

  102. Binnig, G., Garcia, N. & Rohrer, H. Conductivity sensitivity of inelastic scanning tunneling microscopy. Phys. Rev. B Condens. Matter 32, 1336–1338 (1985).

    CAS  PubMed  Google Scholar 

  103. Marti, O. et al. Scanning probe microscopy of biological samples and other surfaces. J. Microsc. 152, 803–809 (1988).

    CAS  PubMed  Google Scholar 

  104. Engel, A. Biological applications of scanning probe microscopes. Annu. Rev. Biophys. Biophys. Chem. 20, 79–108 (1991).

    CAS  PubMed  Google Scholar 

  105. Lindsay, S.M. Biological scanning probe microscopy comes of age. Biophys. J. 67, 2134–2135 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  106. Bustamante, J.O., Liepins, A., Prendergast, R.A., Hanover, J.A. & Oberleithner, H. Patch clamp and atomic force microscopy demonstrate TATA-binding protein (TBP) interactions with the nuclear pore complex. J. Membr. Biol. 146, 263–272 (1995).

    CAS  PubMed  Google Scholar 

  107. Shao, Z., Yang, J. & Somlyo, A.P. Biological atomic force microscopy: from microns to nanometers and beyond. Annu. Rev. Cell Dev. Biol. 11, 241–265 (1995).

    CAS  PubMed  Google Scholar 

  108. Drake, B. et al. Imaging crystals, polymers, and processes in water with the atomic force microscope. Science 243, 1586–1589 (1989).

    CAS  PubMed  Google Scholar 

  109. Bustamante, C., Rivetti, C. & Keller, D.J. Scanning force microscopy under aqueous solutions. Curr. Opin. Struct. Biol. 7, 709–716 (1997).

    CAS  PubMed  Google Scholar 

  110. Rief, M., Oesterhelt, F., Heymann, B. & Gaub, H.E. Single molecule force spectroscopy on polysaccharides by atomic force microscopy. Science 275, 1295–1297 (1997).

    CAS  PubMed  Google Scholar 

  111. Cumpson, P.J., Zhdan, P. & Hedley, J. Calibration of AFM cantilever stiffness: a microfabricated array of reflective springs. Ultramicroscopy 100, 241–251 (2004).

    CAS  PubMed  Google Scholar 

  112. Sarkar, A., Robertson, R.B. & Fernandez, J.M. Simultaneous atomic force microscope and fluorescence measurements of protein unfolding using a calibrated evanescent wave. Proc. Natl. Acad. Sci. USA 101, 12882–12886 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  113. Rief, M., Gautel, M., Oesterhelt, F., Fernandez, J.M. & Gaub, H.E. Reversible unfolding of individual titin immunoglobulin domains by AFM. Science 276, 1109–1112 (1997).

    CAS  PubMed  Google Scholar 

  114. Leake, M.C., Wilson, D., Gautel, M. & Simmons, R.M. The elasticity of single titin molecules using a two-bead optical tweezers assay. Biophys. J. 87, 1112–1135 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  115. Kellermayer, M.S. Delayed dissociation of in vitro moving actin filaments from heavy meromyosin induced by low concentrations of Triton X-100. Biophys. Chem. 67, 199–210 (1997).

    CAS  PubMed  Google Scholar 

  116. Smith, S.B., Cui, Y. & Bustamante, C. Overstretching B-DNA: the elastic response of individual double-stranded and single-stranded DNA molecules. Science 271, 795–799 (1996).

    CAS  PubMed  Google Scholar 

  117. Florin, E.L., Moy, V.T. & Gaub, H.E. Adhesion forces between individual ligand-receptor pairs. Science 264, 415–417 (1994).

    CAS  PubMed  Google Scholar 

  118. Yasuda, R., Noji, H., Kinosita, K. Jr & Yoshida, M. F1-ATPase is a highly efficient molecular motor that rotates with discrete 120 degree steps. Cell 93, 1117–1124 (1998).

    CAS  PubMed  Google Scholar 

  119. Rief, M., Gautel, M., Schemmel, A. & Gaub, H.E. The mechanical stability of immunoglobulin and fibronectin III domains in the muscle protein titin measured by atomic force microscopy. Biophys. J. 75, 3008–3014 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  120. Baumgartner, W. et al. Cadherin interaction probed by atomic force microscopy. Proc. Natl. Acad. Sci. USA 97, 4005–4010 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  121. Marszalek, P.E. et al. Mechanical unfolding intermediates in titin modules. Nature 402, 100–103 (1999).

    CAS  PubMed  Google Scholar 

  122. Carrion-Vazquez, M. et al. Mechanical design of proteins studied by single-molecule force spectroscopy and protein engineering. Prog. Biophys. Mol. Biol. 74, 63–91 (2000).

    CAS  PubMed  Google Scholar 

  123. Steward, A., Toca-Herrera, J.L. & Clarke, J. Versatile cloning system for construction of multimeric proteins for use in atomic force microscopy. Protein Sci. 11, 2179–2183 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  124. Katz, E. & Willner, I. Biomolecule-functionalized carbon nanotubes: applications in nanobioelectronics. ChemPhysChem 5, 1084–1104 (2004).

    CAS  PubMed  Google Scholar 

  125. Bustamante, C., Marko, J.F., Siggia, E.D. & Smith, S. Entropic elasticity of lambda-phage DNA. Science 265, 1599–1600 (1994). In this work the non-linear elasticity of DNA was measured and fit with an analytical expression. These results underpin the subsequent single-molecule force spectroscopy measurements of DNA and DNA processing enzymes.

    CAS  PubMed  Google Scholar 

  126. Fernandez, J.M. & Li, H.B. Force-clamp spectroscopy monitors the folding trajectory of a single protein. Science 303, 1674–1678 (2004).

    CAS  PubMed  Google Scholar 

  127. Schwaiger, I., Kardinal, A., Schleicher, M., Noegel, A.A. & Rief, M. A mechanical unfolding intermediate in an actin-crosslinking protein. Nat. Struct. Mol. Biol. 11, 81–85 (2004).

    CAS  PubMed  Google Scholar 

  128. Carrion-Vazquez, M. et al. The mechanical stability of ubiquitin is linkage dependent. Nat. Struct. Biol. 10, 738–743 (2003).

    CAS  PubMed  Google Scholar 

  129. Dietz, H., Berkemeier, F., Bertz, M. & Rief, M. Anisotropic deformation response of single protein molecules. Proc. Natl. Acad. Sci. USA 103, 12724–12728 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  130. Wright, C.F., Lindorff-Larsen, K., Randles, L.G. & Clarke, J. Parallel protein-unfolding pathways revealed and mapped. Nat. Struct. Biol. 10, 658–662 (2003).

    CAS  PubMed  Google Scholar 

  131. Wiita, A.P. et al. Probing the chemistry of thioredoxin catalysis with force. Nature 450, 124–127 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  132. Szoszkiewicz, R. et al. Dwell time analysis of a single-molecule mechanochemical reaction. Langmuir 24, 1356–1364 (2008).

    CAS  PubMed  Google Scholar 

  133. Greene, D. et al. Single-molecule force spectroscopy reveals a stepwise unfolding of C. elegans giant protein kinase domains. Biophys. J. (in the press) (2008).

  134. Brown, A.E., Litvinov, R.I., Discher, D.E. & Weisel, J.W. Forced unfolding of coiled-coils in fibrinogen by single-molecule AFM. Biophys. J. 92, L39–L41 (2007).

    CAS  PubMed  Google Scholar 

  135. Kellermayer, M.S. et al. Reversible mechanical unzipping of amyloid beta-fibrils. J. Biol. Chem. 280, 8464–8470 (2005).

    CAS  PubMed  Google Scholar 

  136. Oesterhelt, F. et al. Unfolding pathways of individual bacteriorhodopsins. Science 288, 143–146 (2000).

    CAS  PubMed  Google Scholar 

  137. Ando, T. et al. A high-speed atomic force microscope for studying biological macromolecules. Proc. Natl. Acad. Sci. USA 98, 12468–12472 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  138. Wen, J.D. et al. Following translation by single ribosomes one codon at a time. Nature 452, 598–603 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  139. Tanner, N.A. et al. Single-molecule studies of fork dynamics in Escherichia coli DNA replication. Nat. Struct. Mol. Biol. 15, 170–176 (2008).

    CAS  PubMed  Google Scholar 

  140. Hamdan, S.M. et al. Dynamic DNA helicase-DNA polymerase interactions assure processive replication fork movement. Mol. Cell 27, 539–549 (2007).

    CAS  PubMed  Google Scholar 

  141. van Oijen, A.M. Single-molecule studies of complex systems: the replisome. Mol. Biosyst. 3, 117–125 (2007).

    CAS  PubMed  Google Scholar 

  142. Lee, J.B. et al. DNA primase acts as a molecular brake in DNA replication. Nature 439, 621–624 (2006).

    CAS  PubMed  Google Scholar 

  143. Ishijima, A. et al. Simultaneous observation of individual ATPase and mechanical events by a single myosin molecule during interaction with actin. Cell 92, 161–171 (1998).

    CAS  PubMed  Google Scholar 

  144. Lang, M.J., Fordyce, P.M., Engh, A.M., Neuman, K.C. & Block, S.M. Simultaneous, coincident optical trapping and single-molecule fluorescence. Nat. Methods 1, 133–139 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  145. Li, P.T., Bustamante, C. & Tinoco, I. Jr. Real-time control of the energy landscape by force directs the folding of RNA molecules. Proc. Natl. Acad. Sci. USA 104, 7039–7044 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  146. Chiou, P.Y., Ohta, A.T. & Wu, M.C. Massively parallel manipulation of single cells and microparticles using optical images. Nature 436, 370–372 (2005).

    CAS  PubMed  Google Scholar 

  147. Cohen, A.E. & Moerner, W.E. Suppressing Brownian motion of individual biomolecules in solution. Proc. Natl. Acad. Sci. USA 103, 4362–4365 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  148. Berg-Sorensen, K. & Flyvbjerg, H. Power spectrum analysis for optical tweezers. Rev. Sci. Instrum. 75, 594–612 (2004). This is an invaluable reference for the treatment and fitting of Brownian noise and power spectra in single-molecule force spectroscopy measurements.

    CAS  Google Scholar 

Download references

Acknowledgements

K.C.N. and A.N. are supported by the Intramural Program of the National Heart, Lung, and Blood Institute, National Institutes of Health. We thank G. Liou, R. Neuman and Y. Takagi for critical reading of the manuscript. K.C.N. acknowledges D. Bensimon, V. Croquette and S. Block, in addition to members of their labs.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Keir C Neuman.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Neuman, K., Nagy, A. Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy. Nat Methods 5, 491–505 (2008). https://doi.org/10.1038/nmeth.1218

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.1218

This article is cited by

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