High-Resolution “Fleezers”: Dual-Trap Optical Tweezers Combined with Single-Molecule Fluorescence Detection

  • Kevin D. Whitley
  • Matthew J. Comstock
  • Yann R. Chemla
Part of the Methods in Molecular Biology book series (MIMB, volume 1486)


Recent advances in optical tweezers have greatly expanded their measurement capabilities. A new generation of hybrid instrument that combines nanomechanical manipulation with fluorescence detection—fluorescence optical tweezers, or “fleezers”—is providing a powerful approach to study complex macromolecular dynamics. Here, we describe a combined high-resolution optical trap/confocal fluorescence microscope that can simultaneously detect sub-nanometer displacements, sub-piconewton forces, and single-molecule fluorescence signals. The primary technical challenge to these hybrid instruments is how to combine both measurement modalities without sacrificing the sensitivity of either one. We present general design principles to overcome this challenge and provide detailed, step-by-step instructions to implement them in the construction and alignment of the instrument. Lastly, we present a set of protocols to perform a simple, proof-of-principle experiment that highlights the instrument capabilities.

Key words

Optical tweezers Optical trapping Single-molecule fluorescence Förster resonance energy transfer FRET Confocal microscopy Fleezers 



We thank members of the Chemla, Ha, and Comstock laboratories for scientific discussion. Funding was provided by NSF grants MCB-0952442 (CAREER to Y.R.C.), PHY-1430124 (Center for the Physics of Living Cells to Y.R.C.), MCB-1514706 (to M.J.C.), and NIH grant R21 RR025341 (to Y.R.C.).


  1. 1.
    Ashkin A (1986) Observation of a single-beam gradient force optical trap for dielectric particles. Opt Lett 11:288–290CrossRefGoogle Scholar
  2. 2.
    Woodside MT, Block SM (2014) Reconstructing folding energy landscapes by single-molecule force spectroscopy. Annu Rev Biophys 43:19–39CrossRefGoogle Scholar
  3. 3.
    Ritchie DB, Woodside MT (2015) Probing the structural dynamics of proteins and nucleic acids with optical tweezers. Curr Opin Struct Biol 34:43–51CrossRefGoogle Scholar
  4. 4.
    Hilario J, Kowalczykowski SC (2010) Visualizing protein–DNA interactions at the single-molecule level. Curr Opin Chem Biol 14:15–22CrossRefGoogle Scholar
  5. 5.
    Heller I, Hoekstra TP, King GA et al (2014) Optical Tweezers Analysis of DNA−Protein Complexes. Chem Rev 1:3087–3119CrossRefGoogle Scholar
  6. 6.
    Mehta AD, Rief M, Spudich JA et al (1999) Single-molecule biomechanics with optical methods. Science 283:1689–1695CrossRefGoogle Scholar
  7. 7.
    Bustamante C, Cheng W, Mejia YX (2011) Revisiting the central dogma one molecule at a time. Cell 144:480–497CrossRefGoogle Scholar
  8. 8.
    Abbondanzieri EA, Greenleaf WJ, Shaevitz JW et al (2005) Direct observation of base-pair stepping by RNA polymerase. Nature 438:460–465CrossRefGoogle Scholar
  9. 9.
    Moffitt JR, Chemla YR, Izhaky D et al (2006) Differential detection of dual traps improves the spatial resolution of optical tweezers. Proc Natl Acad Sci U S A 103:9006–9011CrossRefGoogle Scholar
  10. 10.
    Chemla YR (2010) Revealing the base pair stepping dynamics of nucleic acid motor proteins with optical traps. Phys Chem Chem Phys 12:3080–3095CrossRefGoogle Scholar
  11. 11.
    Larson MH, Landick R, Block SM (2011) Single-molecule studies of RNA polymerase: one singular sensation, every little step it takes. Mol Cell 41:249–262CrossRefGoogle Scholar
  12. 12.
    Wen J, Lancaster L, Hodges C et al (2008) Following translation by single ribosomes one codon at a time. Nature 452:598–603CrossRefGoogle Scholar
  13. 13.
    Cheng W, Arunajadai SG, Moffitt JR et al (2011) Single-base pair unwinding and asynchronous RNA release by the hepatitis C virus NS3 helicase. Science 333:1746–1749CrossRefGoogle Scholar
  14. 14.
    Qi Z, Pugh RA, Spies M et al (2013) Sequence-dependent base pair stepping dynamics in XPD helicase unwinding. Elife 2:1–23CrossRefGoogle Scholar
  15. 15.
    Moffitt JR, Chemla YR, Aathavan K et al (2009) Intersubunit coordination in a homomeric ring ATPase. Nature 457:446–450Google Scholar
  16. 16.
    Comstock MJ, Ha T, Chemla YR (2011) Ultrahigh-resolution optical trap with single-fluorophore sensitivity. Nat Methods 8:335–340CrossRefGoogle Scholar
  17. 17.
    La Porta A, Wang MD (2004) Optical torque wrench: angular trapping, rotation, and torque detection of quartz microparticles. Phys Rev Lett 92(190801):190801–190804CrossRefGoogle Scholar
  18. 18.
    Lang MJ, Fordyce PM, Engh AM et al (2004) Simultaneous, coincident optical trapping and single-molecule fluorescence. Nat Methods 1:1–7CrossRefGoogle Scholar
  19. 19.
    Hohng S, Zhou R, Nahas MK et al (2007) Fluorescence-force spectroscopy maps two-dimensional reaction landscape of the holliday junction. Science 318:279–283CrossRefGoogle Scholar
  20. 20.
    Van mameren J, Peterman EJ, Wuite GJ (2008) See me, feel me: methods to concurrently visualize and manipulate single DNA molecules and associated proteins. Nucleic Acids Res 36:4381–4389CrossRefGoogle Scholar
  21. 21.
    Lee KS, Balci H, Jia H et al (2013) Direct imaging of single UvrD helicase dynamics on long single-stranded DNA. Nat Commun 4:1–9Google Scholar
  22. 22.
    Suksombat S, Khafizov R, Kozlov AG et al (2015) Structural dynamics of E. coli single-stranded DNA binding protein reveal DNA wrapping and unwrapping pathways. Elife 4:1–23CrossRefGoogle Scholar
  23. 23.
    Comstock MJ, Whitley KD, Jia H et al (2015) Direct observation of structure-function relationship in a nucleic acid – processing enzyme. Science 348:352–354CrossRefGoogle Scholar
  24. 24.
    van Dijk MA, Kapitein LC, Mameren J et al (2004) Combining optical trapping and single-molecule fluorescence spectroscopy: enhanced photobleaching of fluorophores. J Phys Chem B 108:6479–6484CrossRefGoogle Scholar
  25. 25.
    Brau RR, Tarsa PB, Ferrer JM et al (2006) Interlaced optical force-fluorescence measurements for single molecule biophysics. Biophys J 91:1069–1077CrossRefGoogle Scholar
  26. 26.
    Bustamante C, Chemla YR, Moffitt JR (2008) In: Selvin P, Ha TJ (eds) Single-molecule techniques: a laboratory manual. Cold Spring Harbor Laboratory Press, Woodbury, New YorkGoogle Scholar
  27. 27.
    Block SM (1998) In: Spector D, Goldman R, Leinward L (eds) Cells: a laboratory manual. Cold Spring Harbor Press, New YorkGoogle Scholar
  28. 28.
    Van mameren J, Wuite GJ, Heller I (2011) Introduction to optical tweezers: background, system designs, and commercial solutions. Methods Mol Biol 783:1–20CrossRefGoogle Scholar
  29. 29.
    Neuman KC, Block SM (2004) Optical trapping. Rev Sci Instrum 75:2787–2809CrossRefGoogle Scholar
  30. 30.
    Visscher K, Brakenhoff GJ, Krol JJ (1993) Micromanipulation by multiple optical traps created by a single fast scanning trap integrated with the bilateral confocal scanning laser microscope. Cytometry 14:105–114CrossRefGoogle Scholar
  31. 31.
    Visscher K, Gross SP, Block SM (1996) Construction of multiple-beam optical traps with nanometer-resolution position sensing. IEEE JSTQE 2:1066–1076Google Scholar
  32. 32.
    Wuite GJ, Davenport RJ, Rappaport A et al (2000) An integrated laser trap/flow control video microscope for the study of single biomolecules. Biophys J 79:1155–1167CrossRefGoogle Scholar
  33. 33.
    Gittes F, Schmidt CF (1998) Interference model for back-focal-plane displacement detection in optical tweezers. Opt Lett 23:7–9CrossRefGoogle Scholar
  34. 34.
    Pralle A, Prummer M, Florin E et al (1999) Three-dimensional high-resolution particle tracking for optical tweezers by forward scattered light. Microsc Res Tech 44:378–386CrossRefGoogle Scholar
  35. 35.
    Huisstede JHG, van Rooijen BD, van der Werf KO et al (2006) Dependence of silicon position-detector bandwidth on wavelength, power, and bias. Opt Lett 31:610–612CrossRefGoogle Scholar
  36. 36.
    Analog Devices (2007) User’s Manual for CMOS 300 MSPS Complete DDS: AD9852, Rev. E. p. 1–52Google Scholar
  37. 37.
    Ha T (2001) Single-molecule fluorescence resonance energy transfer. Methods 25:78–86CrossRefGoogle Scholar
  38. 38.
    Landry MP, McCall PM, Qi Z et al (2008) Characterization of photoactivated singlet oxygen damage in single-molecule optical trap experiments. Biophys J 97:2128–2136CrossRefGoogle Scholar
  39. 39.
    Rasnik I, McKinney SA, Ha T (2006) Nonblinking and long-lasting single-molecule fluorescence imaging. Nat Methods 3:891–893CrossRefGoogle Scholar
  40. 40.
    Joo C, Ha T (2008) In: Selvin PR, Ha T (eds) Single-molecule techniques: a laboratory manual. Cold Spring Harbor Laboratory Press, Woodbury, New YorkGoogle Scholar
  41. 41.
    Swoboda M, Cheng H, Brugger D et al (2012) Enzymatic oxygen scavenging for photostability without pH drop in single-molecule experiments. ACS Nano 6:6364–6369CrossRefGoogle Scholar
  42. 42.
    Brewer LR, Bianco PR (2008) Laminar flow cells for single-molecule studies of DNA-protein interactions. Nat Methods 5:517–525CrossRefGoogle Scholar
  43. 43.
    Min TL, Mears PJ, Golding I et al (2012) Chemotactic adaptation kinetics of individual Escherichia coli cells. Proc Natl Acad Sci U S A 109:9869–9874CrossRefGoogle Scholar
  44. 44.
    Landry MP, Zou X, Wang L et al (2013) DNA target sequence identification mechanism for dimer-active protein complexes. Nucleic Acids Res 41:2416–2427CrossRefGoogle Scholar
  45. 45.
    Ha T, Rasnik I, Cheng W et al (2002) Initiation and re-initiation of DNA unwinding by the Escherichia coli Rep helicase. Nature 419:638–641CrossRefGoogle Scholar
  46. 46.
    Berg-Sørensen K, Flyvbjerg H (2004) Power spectrum analysis for optical tweezers. Rev Sci Instrum 75:594–612CrossRefGoogle Scholar
  47. 47.
    Nicholas MP, Rao L, Gennerich A (2014) An improved optical tweezers assay for measuring the force generation of single kinesin molecules. Methods Mol Biol 1136:171–246CrossRefGoogle Scholar
  48. 48.
    Humphrey W, Dalke A, Schulten K (1996) VMD: visual molecular dynamics. J Mol Graph 14(33–38):27–28Google Scholar
  49. 49.
    Marko JF, Siggia ED (1995) Stretching DNA. Macromolecules 28:8759–8770CrossRefGoogle Scholar
  50. 50.
    Odijk T (1995) Stiff chains and filaments under tension. Macromolecules 28:7016–7018CrossRefGoogle Scholar
  51. 51.
    Bustamante C, Marko JF, Siggia E et al (1994) Entropic elasticity of lambda-phage DNA. Science 265:1599–1600CrossRefGoogle Scholar
  52. 52.
    Wang MD, Yin H, Landick R et al (1997) Stretching DNA with optical tweezers. Biophys J 72:1335–1346CrossRefGoogle Scholar
  53. 53.
    Woodside MT, Behnke-Parks WM, Larizadeh K et al (2006) Nanomechanical measurements of the sequence-dependent folding landscapes of single nucleic acid hairpins. Proc Natl Acad Sci U S A 103:6190–6195CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Kevin D. Whitley
    • 1
  • Matthew J. Comstock
    • 2
  • Yann R. Chemla
    • 1
  1. 1.Center for the Physics of Living Cells & Center for Biophysics and Quantitative BiologyUniversity of IllinoisUrbanaUSA
  2. 2.Department of Physics and AstronomyMichigan State UniversityEast LansingUSA

Personalised recommendations