Skip to main content
SpringerLink
Log in

High-Resolution Optical Tweezers Combined with Multicolor Single-Molecule Microscopy

  • Protocol
  • First Online:
Optical Tweezers

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2478))

Abstract

We present an instrument that combines high-resolution optical tweezers and multicolor confocal fluorescence spectroscopy. Biological macromolecules exhibit complex conformation and stoichiometry changes in coordination with their motion and activity. To further our understanding of the complex machinery of life, we need methods that can simultaneously probe more than one degree of freedom of single molecules and complexes. Fluorescence optical tweezers, or “fleezers,” combine the capabilities of optical tweezers and single-molecule fluorescence microscopy into a single instrument. Here we present the latest generation of a high-resolution fleezers instrument integrated with multicolor fluorescence spectroscopy. The tweezers portion of the instrument can manipulate biological macromolecules with pN scale forces while measuring subnanometer distances. Simultaneous with tweezers measurements, the multicolor fluorescence capability allows the direct observation of multiple molecules or multiple degrees of freedom which allows, for example, the observation of multiple proteins simultaneously within a complex. The instrument incorporates three fluorescence excitation lasers, all sourced from a single-mode optical fiber allowing a reliable alignment scheme, that allows, for example, three independent fluorescent probes or fluorescence resonance energy transfer (FRET) measurements and also increases flexibility in the choice of fluorescent probes. To avoid photobleaching and improve tweezers stability, the instrument implements a timesharing (using a single trap laser to produce a pair of traps via rapid switching between two locations) and interlacing (turning the trapping beam off when the fluorescence excitation beams are on and vice versa) scheme using acousto-optic modulators (AOM) to rapidly and precisely modulate lasers. Our latest “random phase” trap AOM control method obliterates previous residual trap positioning and bead position measurement errors. Here we present the general design principles and detailed construction and testing protocols for the instrument.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Mehta AD, Rief M, Spudich JA, Smith DA, Simmons RM (1999) Single-molecule biomechanics with optical methods. Science 283(5408):1689–1695

    Article  ADS  Google Scholar 

  2. Hilario J, Kowalczykowski SC (2010) Visualizing protein–DNA interactions at the single-molecule level. Curr Opin Chem Biol 14(1):15–22

    Article  Google Scholar 

  3. Bustamante C, Cheng W, Mejia YX (2011) Revisiting the central dogma one molecule at a time. Cell 144(4):480–497

    Article  Google Scholar 

  4. Larson MH, Landick R, Block SM (2011) Single-molecule studies of RNA polymerase: one singular sensation, every little step it takes. Mol Cell 41(3):249–262

    Article  Google Scholar 

  5. Heller I, Hoekstra TP, King GA, Peterman EJG, Wuite GJL (2014) Optical tweezers analysis of DNA – protein complexes. Chem Rev 1(14):3087–3119

    Article  Google Scholar 

  6. Woodside MT, Block SM (2014) Reconstructing folding energy landscapes by single-molecule force spectroscopy. Annu Rev Biophys 43:19–39

    Article  Google Scholar 

  7. Ashkin A (1986) Observation of a single-beam gradient force optical trap for dielectric particles. Opt Lett 11(5):288–290

    Article  ADS  Google Scholar 

  8. Neuman KC, Nagy A (2008) Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy. Nat Methods 5(6):491–505. https://doi.org/10.1038/nmeth.1218

    Article  Google Scholar 

  9. Ritchie DB, Woodside MT (2015) Probing the structural dynamics of proteins and nucleic acids with optical tweezers. Curr Opin Struct Biol 34:43–51. https://doi.org/10.1016/j.sbi.2015.06.006

    Article  Google Scholar 

  10. 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 U S A 103(24):9006–9011

    Article  ADS  Google Scholar 

  11. Chuang C-Y, Zammit M, Whitmore ML, Comstock MJ (2019) Combined high-resolution optical tweezers and multicolor single-molecule fluorescence with an automated single-molecule assembly line. J Phys Chem A 123(44):9612–9620. https://doi.org/10.1021/acs.jpca.9b08282

    Article  Google Scholar 

  12. Patrick EM, Slivka JD, Payne B, Comstock MJ, Schmidt JC (2020) Observation of processive telomerase catalysis using high-resolution optical tweezers. Nat Chem Biol 16(7):801–809. https://doi.org/10.1038/s41589-020-0478-0

    Article  Google Scholar 

  13. Comstock MJ, Whitley KD, Jia H, Sokoloski J, Lohman TM, Ha T, Chemla YR (2015) Direct observation of structure-function relationship in a nucleic acid – processing enzyme. Science 348(6232):352–354

    Article  ADS  Google Scholar 

  14. Humphrey W, Dalke A, Schulten K (1996) VMD: visual molecular dynamics. J Mol Graph 14(1):33–38. 27–28

    Article  Google Scholar 

  15. Abbondanzieri EA, Greenleaf WJ, Shaevitz JW, Landick R, Block SM (2005) Direct observation of base-pair stepping by RNA polymerase. Nature 438(7067):460–465

    Article  ADS  Google Scholar 

  16. Righini M, Lee A, Cañari-Chumpitaz C, Lionberger T, Gabizon R, Coello Y, Tinoco I, Bustamante C (2018) Full molecular trajectories of RNA polymerase at single base-pair resolution. Proc Natl Acad Sci 115(6):1286–1291. https://doi.org/10.1073/pnas.1719906115

    Article  Google Scholar 

  17. Moffitt JR, Chemla YR, Aathavan K, Grimes S, Jardine PJ, Anderson DL, Bustamante C (2009) Intersubunit coordination in a homomeric ring ATPase. Nature 457:446. https://doi.org/10.1038/nature07637. nature07637 [pii]

    Article  ADS  Google Scholar 

  18. Cheng W, Arunajadai SG, Moffitt JR, Tinoco I, Bustamante C (2011) Single-base pair unwinding and asynchronous RNA release by the hepatitis C virus NS3 helicase. Science 333(6050):1746–1749

    Article  ADS  Google Scholar 

  19. Qi Z, Pugh RA, Spies M, Chemla YR (2013) Sequence-dependent base pair stepping dynamics in XPD helicase unwinding. elife 2:1–23

    Article  Google Scholar 

  20. Patrick EM, Srinivasan S, Jankowsky E, Comstock MJ (2017) The RNA helicase Mtr4p is a duplex-sensing translocase. Nat Chem Biol 13(1):99–104. https://doi.org/10.1038/nchembio.2234

    Article  Google Scholar 

  21. Landry MP, Zou X, Wang L, Huang WM, Schulten K, Chemla YR (2013) DNA target sequence identification mechanism for dimer-active protein complexes. Nucleic Acids Res 41(4):2416–2427

    Article  Google Scholar 

  22. Suksombat S, Khafizov R, Kozlov AG, Lohman TM, Chemla YR (2015) Structural dynamics of E. coli single-stranded DNA binding protein reveal DNA wrapping and unwrapping pathways. elife 4:1–23

    Article  Google Scholar 

  23. Yu H, Liu X, Neupane K, Gupta AN, Brigley AM, Solanki A, Sosova I, Woodside MT (2012) Direct observation of multiple misfolding pathways in a single prion protein molecule. Proc Natl Acad Sci 109(14):5283–5288. https://doi.org/10.1073/pnas.1107736109

    Article  ADS  Google Scholar 

  24. Yu H, Dee DR, Liu X, Brigley AM, Sosova I, Woodside MT (2015) Protein misfolding occurs by slow diffusion across multiple barriers in a rough energy landscape. Proc Natl Acad Sci 112(27):8308–8313. https://doi.org/10.1073/pnas.1419197112

    Article  ADS  Google Scholar 

  25. Izadi D, Chen Y, Whitmore ML, Slivka JD, Ching K, Lapidus LJ, Comstock MJ (2018) Combined force ramp and equilibrium high-resolution investigations reveal multipath heterogeneous unfolding of protein G. J Phys Chem B 122(49):11155–11165. https://doi.org/10.1021/acs.jpcb.8b06199

    Article  Google Scholar 

  26. Hohng S, Zhou R, Nahas MK, Yu J, Schulten K, Lilley DM, Ha T (2007) Fluorescence-force spectroscopy maps two-dimensional reaction landscape of the Holliday Junction. Science 318(5848):279–283

    Article  ADS  Google Scholar 

  27. Lee KS, Balci H, Jia H, Lohman TM, Ha T (2013) Direct imaging of single UvrD helicase dynamics on long single-stranded DNA. Nat Commun 4(1878):1–9

    Google Scholar 

  28. Comstock MJ, Ha T, Chemla YR (2011) Ultrahigh-resolution optical trap with single-fluorophore sensitivity. Nat Methods 8(4):335–340

    Article  Google Scholar 

  29. Whitley KD, Comstock MJ, Chemla YR (2016) Elasticity of the transition state for oligonucleotide hybridization. Nucleic Acids Res 45(2):547–555. https://doi.org/10.1093/nar/gkw1173

    Article  Google Scholar 

  30. Whitley KD, Comstock MJ, Chemla YR (2018) Ultrashort nucleic acid duplexes exhibit long wormlike chain behavior with force-dependent edge effects. Phys Rev Lett 120(6):068102. https://doi.org/10.1103/PhysRevLett.120.068102

    Article  ADS  Google Scholar 

  31. Duesterberg VK, Fischer-Hwang IT, Perez CF, Hogan DW, Block SM (2015) Observation of long-range tertiary interactions during ligand binding by the TPP riboswitch aptamer. elife 4:e12362. https://doi.org/10.7554/eLife.12362

    Article  Google Scholar 

  32. Mitra J, Makurath MA, Ngo TTM, Troitskaia A, Chemla YR, Ha T (2019) Extreme mechanical diversity of human telomeric DNA revealed by fluorescence-force spectroscopy. Proc Natl Acad Sci 116(17):8350–8359. https://doi.org/10.1073/pnas.1815162116

    Article  Google Scholar 

  33. Desai VP, Frank F, Lee A, Righini M, Lancaster L, Noller HF, Tinoco I, Bustamante C (2019) Co-temporal force and fluorescence measurements reveal a ribosomal gear shift mechanism of translation regulation by structured mRNAs. Mol Cell 75(5):1007–1019.e1005. https://doi.org/10.1016/j.molcel.2019.07.024

    Article  Google Scholar 

  34. Whitley KD, Comstock MJ, Chemla YR (2017) High-resolution “fleezers”: dual-trap optical tweezers combined with single-molecule fluorescence detection. In: Gennerich A (ed) Optical tweezers: methods and protocols. Springer, New York, NY, pp 183–256. https://doi.org/10.1007/978-1-4939-6421-5_8

    Chapter  Google Scholar 

  35. Baker AG, Chuang CY, Whitmore M, Comstock MJ (2018) Randomizing phase to remove acousto-optic device wiggle errors for high-resolution optical tweezers. Appl Opt 57(8):1752–1756. https://doi.org/10.1364/AO.57.001752

    Article  ADS  Google Scholar 

  36. van Dijk MA, Kapitein LC, Mameren J, Schmidt CF, Peterman EJ (2004) Combining optical trapping and single-molecule fluorescence spectroscopy: enhanced photobleaching of fluorophores. J Phys Chem B 108:6479–6484

    Article  Google Scholar 

  37. Brau RR, Tarsa PB, Ferrer JM, Lee P, Lang MJ (2006) Interlaced optical force-fluorescence measurements for single molecule biophysics. Biophys J 91(3):1069–1077

    Article  Google Scholar 

  38. Bustamante C, Chemla YR, Moffitt JR (2008) High resolution dual trap optical tweezers with differential detection. In: Selvin P, Taekjip H (eds) Single-molecule techniques: a laboratory manual. Cold Spring Harbor Laboratory Press, Woodbury, NY

    Google Scholar 

  39. Neuman KC, Block SM (2004) Optical trapping. Rev Sci Instrum 75(9):2787–2809

    Article  ADS  Google Scholar 

  40. Van Mameren J, Wuite GJL, Heller I (2011) Introduction to optical tweezers: background, system designs, and commercial solutions. Methods Mol Biol 783:1–20

    Article  Google Scholar 

  41. Block SM (1998) Constructing optical tweezers. In: Spector D, Goldman R, Leinward L (eds) Cells: a laboratory manual. Cold Spring Harbor Press, New York, NY

    Google Scholar 

  42. 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 14:105–114

    Article  Google Scholar 

  43. Visscher K, Gross SP, Block SM (1996) Construction of multiple-beam optical traps with nanometer-resolution position sensing. IEEE J Sel Top Quantum Electron 2(4):1066–1076

    Article  ADS  Google Scholar 

  44. Valentine MT, Guydosh NR, Gutierrez-Medina B, Fehr AN, Andreasson JO, Block SM (2008) Precision steering of an optical trap by electro-optic deflection. Opt Lett 33(6):599–601. https://doi.org/10.1364/ol.33.000599

    Article  ADS  Google Scholar 

  45. Gittes F, Schmidt CF (1998) Interference model for back-focal-plane displacement detection in optical tweezers. Opt Lett 23(1):7–9

    Article  ADS  Google Scholar 

  46. Pralle A, Prummer M, Florin E, Stelzer EHK, Horber JK (1999) Three-dimensional high-resolution particle tracking for optical tweezers by forward scattered light. Microsc Res Tech 44(5):378–386

    Article  Google Scholar 

  47. Huisstede JHG, van Rooijen BD, van der Werf KO, Bennink ML, Subramaniam V (2006) Dependence of silicon position-detector bandwidth on wavelength, power, and bias. Opt Lett 31(5):610–612

    Article  ADS  Google Scholar 

  48. Analog Devices (2007) User’s manual for CMOS 300 MSPS complete DDS: AD9852, Rev. E

    Google Scholar 

  49. Landry MP, McCall PM, Qi Z, Chemla YR (2008) Characterization of photoactivated singlet oxygen damage in single-molecule optical trap experiments. Biophys J 97(8):2128–2136

    Article  Google Scholar 

  50. Rasnik I, McKinney SA, Ha T (2006) Nonblinking and long-lasting single-molecule fluorescence imaging. Nat Methods 3(11):891–893

    Article  Google Scholar 

  51. Ha T (2001) Single-molecule fluorescence resonance energy transfer. Methods 25(1):78–86

    Article  MathSciNet  Google Scholar 

  52. Swoboda M, Cheng H, Brugger D, Haltrich D, Plumere N, Schlierf M (2012) Enzymatic oxygen scavenging for photostability without pH drop in single-molecule experiments. ACS Nano 6(7):6364–6369

    Article  Google Scholar 

  53. Aitken CE, Marshall RA, Puglisi JD (2008) An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments. Biophys J 94(5):1826–1835. https://doi.org/10.1529/biophysj.107.117689

    Article  Google Scholar 

  54. Ha T, Rasnik I, Cheng W, Babcock HP, Gauss GH, Lohman TM, Chu S (2002) Initiation and re-initiation of DNA unwinding by the Escherichia coli Rep helicase. Nature 419(6907):638–641

    Article  ADS  Google Scholar 

  55. Berg-Sørensen K, Flyvbjerg H (2004) Power spectrum analysis for optical tweezers. Rev Sci Instrum 75(3):594–612

    Article  ADS  Google Scholar 

Download references

Acknowledgments

We thank members of the Chemla and TJ Ha laboratories for scientific discussion. We thank previous members of the Comstock lab for instrument design and building, and scientific discussions, especially Cho-Ying Chuang, Miles Whitmore, Andrew Baker, and Matthew Zammit. Funding was provided by NSF grant MCB-1919439 (to M.J.C.)

Author information

Authors and Affiliations

  1. Department of Physics and Astronomy, Michigan State University, East Lansing, MI, USA

    Rajeev Yadav, Kasun B. Senanayake & Matthew J. Comstock

Authors
  1. Rajeev Yadav
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kasun B. Senanayake
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Matthew J. Comstock
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Matthew J. Comstock .

Editor information

Editors and Affiliations

  1. Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY, USA

    Arne Gennerich

Rights and permissions

Reprints and permissions

Copyright information

© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Yadav, R., Senanayake, K.B., Comstock, M.J. (2022). High-Resolution Optical Tweezers Combined with Multicolor Single-Molecule Microscopy. In: Gennerich, A. (eds) Optical Tweezers. Methods in Molecular Biology, vol 2478. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2229-2_8

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-2229-2_8

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2228-5

  • Online ISBN: 978-1-0716-2229-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation