Single-Molecule Manipulation Using Optical Traps

  • Michael T. Woodside
  • Megan T. Valentine


One of the most sensitive tools for manipulating single molecules and measuring their properties is the optical trap, also known as optical tweezers. Consisting essentially of a strongly focused light beam, optical traps were first developed and demonstrated in the 1970s and 1980s by Arthur Ashkin and colleagues (Ashkin et al. 1986). These early pioneers showed that micron-sized dielectric particles could be held and manipulated in solution by using optical forces to create a stable, three-dimensional potential well. Since then, optical trapping instrumentation has been refined and developed such that piconewton forces are now routinely applied, while at the same time measuring the resultant displacements to nanometre or even angström resolution. As a result of these advances, optical traps have been applied widely, from cytometry to the study of mesoscopic colloids and polymers and of course the properties of single biological macromolecules. This chapter begins with a description of the theory and design of optical traps, followed by an illustrative discussion of applications to the study of structure formation and molecular motors, a description of typical “tricks of the trade” for using optical traps, and a brief look at techniques for extending the capabilities of traps.


Persistence Length Optical Trap Optical Trapping Trap Centre Force Spectroscopy 



We thank Cuauhtémoc García-García for critical reading of the manuscript and Charles Asbury, Joshua Shaevitz, and Kristina Herbert for providing the raw data used in Figures 12.10 and 12.11. MTV is supported by a Career Award at the Scientific Interface from the Burroughs Wellcome Fund.


  1. Abbondanzieri EA, Greenleaf WJ, Shaevitz JW, et al. (2005). Direct observation of base-pair stepping by RNA polymerase. Nature 438:460–465.ADSCrossRefGoogle Scholar
  2. Ashkin A, Dziedzic JM, Bjorkholm JE, Chu S (1986). Observation of a single-beam gradient force optical trap for dielectric particles. Optics Lett 11:288–290.ADSCrossRefGoogle Scholar
  3. Bell GI (1978). Models for specific adhesion of cells to cells. Science 200:618–627.ADSCrossRefGoogle Scholar
  4. Berg-Sørensen K, Flyvbjerg H (2004). Power spectrum analysis for optical tweezers. Rev Sci Instrum 75:594–612.ADSCrossRefGoogle Scholar
  5. Best RB, Paci E, Hummer G, Dudko O (2008). Pulling direction as a reaction coordinate for the mechanical unfolding of single molecules. J Phys Chem B 112: 5968–5976.CrossRefGoogle Scholar
  6. Block SM (1998). Constructing optical tweezers. In: Spector DL, Goldman RD, Leinwand LA (eds.), Cells: A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Google Scholar
  7. Block SM, Asbury CL, Shaevitz JW, Lang MJ (2003). Probing the kinesin reaction cycle with a 2D optical force clamp. Proc Natl Acad Sci USA 100:2351–2356.ADSCrossRefGoogle Scholar
  8. Borgia A, Williams PM, Clarke J (2008). Single-molecule studies of protein folding. Annu Rev Biochem 77:6.1–6.25.CrossRefGoogle Scholar
  9. Brower-Towland BD, Smith CL, et al. (2002). Mechanical disruption of individual nucleosomes reveals a reversible multistage release of DNA. Proc Natl Acad Sci USA 99:1960–1965.ADSCrossRefGoogle Scholar
  10. Bustamante C, Chemla YR, Forde NR, Izhaky D (2004). Mechanical processes in biochemistry. Annu Rev Biochem 73:705–748.CrossRefGoogle Scholar
  11. Carter AR, King GM, Ulrich TA, et al. (2007). Stabilization of an optical microscope to 0.1 nm in three dimensions. Appl Opt 46:421–427.ADSCrossRefGoogle Scholar
  12. Carter BC, Vershinin M, Gross SP (2008). A comparison of step-detection methods: How well can you do? Biophys J 94:306–319.CrossRefGoogle Scholar
  13. Cecconi C, Shank EA, Bustamante C, Marqusee S (2005). Direct observation of the three-state folding of a single protein molecule. Science 309:2057–2059.ADSCrossRefGoogle Scholar
  14. Collin D, Ritort F, Jarzynski C, et al. (2005). Verification of the Crooks fluctuation theorem and recovery of RNA folding free energies. Nature 437:231–234.ADSCrossRefGoogle Scholar
  15. Crooks GE (1999). Entropy production fluctuation theorem and the nonequilibrium work relation for free energy differences. Phys Rev E 60:2721–2726.ADSCrossRefGoogle Scholar
  16. Dalal RV, Larson MH, Neuman KC, et al. (2006). Pulling on the nascent RNA during transcription does not alter kinetics of elongation or ubiquitous pausing. Mol Cell 23:231–239.CrossRefGoogle Scholar
  17. Dame RT, Noom MC, Wuite GJ (2006). Bacterial chromatin organization by H-NS protein unravelled using dual DNA manipulation. Nature 444:387–390.ADSCrossRefGoogle Scholar
  18. deCastro MJ, Fondecave RM, Clarke LA, et al. (2000). Working strokes by single molecules of the kinesin-related microtubule motor ncd. Nat Cell Biol 2:724–729.CrossRefGoogle Scholar
  19. Dessinges MN, Maier B, Zhang Y, et al. (2002). Stretching single stranded DNA, a model polyelectrolyte. Phys Rev Lett 89:248102.ADSCrossRefGoogle Scholar
  20. Dietz H, Berkemeier F, Bertz M, Reif M (2006). Anisotropic deformation response of single protein molecules. Proc Natl Acad Sci USA 103:12724–12728.ADSCrossRefGoogle Scholar
  21. Dudko O, Hummer G, Szabo A (2006). Intrinsic rates and activation free energies from single-molecule pulling experiments. Phys Rev Lett 96:108101.ADSCrossRefGoogle Scholar
  22. Dumont S, Cheng W, Serebov V, et al. (2006). RNA translocation and unwinding mechanism of HCV NS3 helicase and its coordination by ATP. Nature 439:105–108.ADSCrossRefGoogle Scholar
  23. Enger J, Goksor M, Ramser K, et al. (2004). Optical tweezers applied to a microfluidic system. Lab Chip 4:196–200.CrossRefGoogle Scholar
  24. Evans E, Ritchie K (1997). Dynamic strength of molecular adhesion bonds. Biophys J 72:1541–1555.CrossRefGoogle Scholar
  25. Finer JT, Simmons RM, Spudich JA (1994). Single myosin mechanics: picoNewton forces and nanometre steps. Nature 368: 113–119.ADSCrossRefGoogle Scholar
  26. Fordyce PM, Valentine MT, Block SM (2007). Advances in surface-based assays for single molecules. In: Selvin P, Ha T (eds.), Single-molecule techniques: A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 431--460.Google Scholar
  27. Gilbert SP and Mackey AT (2000). Kinetics: A tool to study molecular motors. Methods 22:337–354.CrossRefGoogle Scholar
  28. Gore J, Ritort F, Bustamante C (2003). Bias and error in estimates of equilibrium free-energy differences from nonequilibrium measurements. Proc Natl Acad Sci USA 100:12564–12569.MathSciNetADSMATHCrossRefGoogle Scholar
  29. Greenleaf WJ, Woodside MT, Abbondanzieri EA, Block SM (2005). Passive all-optical force clamp for high-resolution laser trapping. Phys Rev Lett 95:208102.ADSCrossRefGoogle Scholar
  30. Greenleaf WJ, Woodside MT, Block SM (2007). High-resolution, single-molecule measurements of biomolecular motion. Annu Rev Biophys Biomol Struct 36:171–190.CrossRefGoogle Scholar
  31. Greenleaf WJ, Frieda KL, Foster DNA, et al. (2008). Direct observation of hierarchical folding in single riboswitch aptamers. Science 319:630–633.CrossRefGoogle Scholar
  32. Grier DG, Roichman Y (2006). Holographic optical trapping. Appl Opt 45:880–887.ADSCrossRefGoogle Scholar
  33. Harada Y, Asakura T (1996). Radiation forces on a dielectric sphere in the Rayleigh scattering regime. Opt Commun 124:529–541.ADSCrossRefGoogle Scholar
  34. Herbert KM, La Porta A, Wong BJ, et al. (2006). Sequence-resolved detection of pausing by single RNA polymerase molecules. Cell 125:1084–1094.CrossRefGoogle Scholar
  35. Herbert KM, Greenleaf WJ, Block SM (2008). Single-molecule studies of RNA polymerase: motoring along. Annu Rev Biochem 77:149–176.CrossRefGoogle Scholar
  36. Hermanson GT (1996). Bioconjugate techniques. Academic Press, San Diego, CA.Google Scholar
  37. Hohng S, Zhou R, Nahas MK, et al. (2007). Fluorescence-force spectroscopy maps two-dimensional reaction landscape of the Holliday junction. Science 318:279–283.ADSCrossRefGoogle Scholar
  38. Hummer G, Szabo A (2005). Free energy surfaces from single-molecule force spectroscopy. Acc Chem Res 38: 504–513.CrossRefGoogle Scholar
  39. Hyeon C, Thirumalai D (2008). Multiple probes are required to explore and control the rugged energy landscape of RNA hairpins. J Am Chem Soc 130:1538–1539.CrossRefGoogle Scholar
  40. Ishijima A, Kojima H, Funatsu T, et al. (1998). Simultaneous observation of individual ATPase and mechanical events by a single myosin molecule during interaction with actin. Cell 92:161–171.CrossRefGoogle Scholar
  41. Jarzynski C (1997). Non-equilibrium equality for free energy differences. Phys Rev Lett 78:2690–2693.ADSCrossRefGoogle Scholar
  42. Kawaguchi K, Ishiwata S (2001). Nucleotide-dependent single- to double-headed binding of kinesin. Science 291:667–669.ADSCrossRefGoogle Scholar
  43. Kimura Y, Bianco PR (2006). Single molecule studies of DNA binding proteins using optical tweezers. Analyst 131:868–874.ADSCrossRefGoogle Scholar
  44. Koch SJ, Shundrovsky A, Jantzen BC, Wang MD (2002). Probing protein–DNA interactions by unzipping a single DNA double helix. Biophys J 83:1098–1105.CrossRefGoogle Scholar
  45. Lang MJ, Fordyce PM, Engh AM, et al. (2004). Simultaneous, coincident optical trapping and single molecule fluorescence. Nat Methods 1:133–139.CrossRefGoogle Scholar
  46. Lang MJ, Asbury CL, Shaevitz, JW, Block SM (2002). An automated two-dimensional optical force clamp for single molecule studies. Biophys J 83:491–501.CrossRefGoogle Scholar
  47. La Porta A, Wang MD (2004). Optical torque wrench: Angular trapping, rotation and torque detection using quartz microparticles. Phys Rev Lett 92: 190801.CrossRefGoogle Scholar
  48. Larson MH, Greenleaf WJ, Landick R, Block SM (2008). Applied force reveals mechanistic and energetic details of transcription termination. Cell 132:971–982.CrossRefGoogle Scholar
  49. Li PTX, Collin D, Smith SB, et al. (2006). Probing the mechanical folding kinetics of TAR RNA by hopping, force-jump, and force-ramp methods. Biophys J 90:250–260.CrossRefGoogle Scholar
  50. Lister I, Schmitz S, Walker M, et al. (2004). A monomeric myosin VI with a large working stroke. EMBO J 23: 1729–1738.CrossRefGoogle Scholar
  51. Mañosas M, Collin D, Ritort F (2006). Force-dependent fragility in RNA hairpins. Phys Rev Lett 96:218301.ADSCrossRefGoogle Scholar
  52. Mañosas M, Wen JD, Li PTX, et al. (2007). Force unfolding kinetics of RNA using optical tweezers. II. Modeling experiments. Biophys J 92:3010–3021.CrossRefGoogle Scholar
  53. Marko JF, Siggia ED (1995). Stretching DNA. Macromolecules 28:8759–8770.ADSCrossRefGoogle Scholar
  54. Moffitt JR, Chemla YR, Izhaky D, Bustamante C (2006). Differential detection of dual traps improves the spatial resolution of optical tweezers. Proc Natl Acad Sci USA 103:9006–9011.ADSCrossRefGoogle Scholar
  55. Moffitt JR, Chemla YR, Smith SB, Bustamante C (2008). Recent advances in optical tweezers. Annu Rev Biochem. 77:19.1–19.24.CrossRefGoogle Scholar
  56. Molloy JE, Burns JE, Kendrick-Jones J, et al. (1995). Movement and force produced by a single myosin head. Nature 378:209–212.ADSCrossRefGoogle Scholar
  57. Nambiar R, Gajraj A, Meiner JC (2004). All-optical constant-force laser tweezers. Biophys J 87:1972–1980.CrossRefGoogle Scholar
  58. Neuman KC, Chadd EH, Liou GF, et al. (1999). Characterization of photodamage to Escherichia coli in optical traps. Biophys J 77:2656–2863.CrossRefGoogle Scholar
  59. Neuman KC, Block SM (2004). Optical trapping. Rev Sci Instrum 75:2787–2809.ADSCrossRefGoogle Scholar
  60. Neuman KC, Abbondanzieri EA, Block SM (2005). Measurement of the effective focal shift in an optical trap. Opt Lett 30:1318–1320.ADSCrossRefGoogle Scholar
  61. Neuman KC, Nagy A (2008). Single-molecule force spectroscopy: Optical tweezers, magnetic tweezers and atomic force microscopy. Nat Methods 5:491–505.CrossRefGoogle Scholar
  62. Nishizaka T, Miyata H, Yoshikawa H, et al. (1995). Unbinding force of a single motor molecule of muscle using optical tweezers. Nature 337:251–254.ADSCrossRefGoogle Scholar
  63. Nugent-Glandorf L, Perkins TT (2004). Measuring 0.1-nm motion in 1 ms in an optical microscope with differential back-focal-plane detection. Opt Lett 29:2611–2613.ADSCrossRefGoogle Scholar
  64. Rasnik I, McKinney SA, Ha T (2006). Nonblinking and long-lasting single-molecule fluorescence imaging. Nat Methods 3: 891–893.CrossRefGoogle Scholar
  65. Rock RS, Rief M, Mehta AD, Spudich JA (2000). In vitro assays of processive myosin motors. Methods 22:373–381.CrossRefGoogle Scholar
  66. Rohrbach A, Stelzer EHK (2001). Optical trapping of dielectric particles in arbitrary fields. J Opt Soc Am A 18: 839–853.ADSCrossRefGoogle Scholar
  67. Schnitzer MJ, Visscher K, Block SM (2000). Mechanism of force production by single kinesin motors. Nat Cell Biol 2:718–723.CrossRefGoogle Scholar
  68. Seol Y, Li J, Nelson PC, et al. (2007). Elasticity of short DNA molecules: Theory and experiment for contour lengths of 0.6–7 micron. Biophys J 93:4360–4373.CrossRefGoogle Scholar
  69. Shaevitz JW, Block SM, Schnitzer MJ (2005). Statistical kinetics of macromolecular dynamics. Biophys J. 89: 2275–2285.CrossRefGoogle Scholar
  70. Shaevitz JW, Abbondanzieri EA, Landick R, Block SM (2003). Backtracking by single RNA polymerase molecules observed at near-base-pair resolution. Nature 426:684–687.ADSCrossRefGoogle Scholar
  71. Smith SB, Cui Y, Bustamante C (1996). Overstretching B-DNA: The elastic response of individual double-stranded and single-stranded DNA molecules. Science 271: 795–799.ADSCrossRefGoogle Scholar
  72. Smith SB, Cui Y, Bustamante C (2003). Optical-trap force transducer that operates by direct measurement of light momentum. Methods Enzymol 361:134–162.CrossRefGoogle Scholar
  73. Svoboda K, Block SM (1994a). Biological applications of optical forces. Annu Rev Biophys Biomol Struct 23: 247–285.CrossRefGoogle Scholar
  74. Svoboda K, Block SM (1994b). Force and velocity measured for single kinesin molecules. Cell 77: 773–784.CrossRefGoogle Scholar
  75. Tolić-Nørrelykke SF, Schäffer E, Howard J, et al. (2006). Calibration of optical tweezers with positional detection in the back focal plane. Rev Sci Instrum 77:103101.ADSCrossRefGoogle Scholar
  76. Trapagnier EH, Radenovic A, Sivak D, et al. (2007). Controlling DNA capture and propagation through artificial nanopores. Nano Lett 7:2824–2830.ADSCrossRefGoogle Scholar
  77. Valentine MT, Fordyce PM, Krzysiak TC, et al. (2006). Individual dimers of the mitotic kinesin motor Eg5 step processively and support substantial loads in vitro. Nat Cell Biol 8:470–476.CrossRefGoogle Scholar
  78. Veigel C, Schmitz S, Wang F, Sellers JR (2005). Load-dependent kinetics of myosin-V can explain its high processivity. Nat Cell Biol 7:861–869.CrossRefGoogle Scholar
  79. Vilar JMG, Rubi JM (2008). Failure of the work-Hamiltonian connection for free-energy calculations. Phys Rev Lett 100:020601.ADSCrossRefGoogle Scholar
  80. Visscher K, Gross SP, Block SM (1996). Construction of multiple-beam optical traps with nanometer-resolution position sensing. IEEE J Sel Top Quant Electr 2:1066–1076.CrossRefGoogle Scholar
  81. Visscher K, Block SM (1998). Versatile optical traps with feedback control. Methods Enzymol 298: 460–489.CrossRefGoogle Scholar
  82. Visscher K, Schnitzer MJ, Block SM (1999). Single kinesin molecules studied with a molecular force clamp. Nature 400:184–189.ADSCrossRefGoogle Scholar
  83. Wang MD, Yin H, Landick R, et al. (1997). Stretching DNA with optical tweezers. Biophys J 72:1335–1346.CrossRefGoogle Scholar
  84. Williams PM, Fowler SB, Best RB, et al. (2003). Hidden complexity on the mechanical properties of titin. Nature 422:446–449.ADSCrossRefGoogle Scholar
  85. Woodside MT, Behnke-Parks WM, Larizadeh K, et al. (2006a). Nanomechanical measurements of the sequence-dependent folding landscapes of single nucleic acid hairpins. Proc Natl Acad Sci USA 103:6190–6195.ADSCrossRefGoogle Scholar
  86. Woodside MT, Anthony PC, Behnke-Parks WM, et al. (2006b). Direct measurement of the full, sequence-dependent folding landscape of a nucleic acid. Science 314:1001–1004.ADSCrossRefGoogle Scholar
  87. Woodside MT, García-García C, Block SM (2008). Folding and unfolding single RNA molecules under tension. Curr Opin Chem Biol, vol. 12, pp. 640--646.Google Scholar
  88. Wuite GJL, Smith SB, Young M, et al. (2000). Single-molecule studies of the effect of template tension on T7 DNA polymerase activity. Nature 404:103–106.ADSCrossRefGoogle Scholar
  89. Xu F, Ren K, Gouesbet G, et al. (2007). Generalized Lorenz-Mie theory for an arbitrarily oriented, located, and shaped beam scatted by a homogeneous spheroid. J Opt Soc Am A 24:119–131.ADSMATHCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Michael T. Woodside
    • 1
  • Megan T. Valentine
    • 2
  1. 1.National Institute for NanotechnologyNational Research Council of Canada, and Department of Physics, University of AlbertaAlbertaCanada
  2. 2.Department of Mechanical EngineeringUniversity of CaliforniaSanta BarbaraUSA

Personalised recommendations