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A practical guide to single-molecule FRET

Abstract

Single-molecule fluorescence resonance energy transfer (smFRET) is one of the most general and adaptable single-molecule techniques. Despite the explosive growth in the application of smFRET to answer biological questions in the last decade, the technique has been practiced mostly by biophysicists. We provide a practical guide to using smFRET, focusing on the study of immobilized molecules that allow measurements of single-molecule reaction trajectories from 1 ms to many minutes. We discuss issues a biologist must consider to conduct successful smFRET experiments, including experimental design, sample preparation, single-molecule detection and data analysis. We also describe how a smFRET-capable instrument can be built at a reasonable cost with off-the-shelf components and operated reliably using well-established protocols and freely available software.

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Figure 1: smFRET description.
Figure 2
Figure 3: Schematic for smFRET spectroscopy.
Figure 4: Surface immobilization strategies for smFRET experiments.
Figure 5: Sample chamber.

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References

  1. Feynman, R.P. There's plenty of room at the bottom. J. Microelectromech. Syst. 1, 60–66 (1992).

    Google Scholar 

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

    PubMed  Google Scholar 

  3. Moerner, W.E. & Fromm, D.P. Methods of single-molecule fluorescence spectroscopy and microscopy. Rev. Sci. Instrum. 74, 3597–3619 (2003).An extensive review of single-molecule fluorescence methods.

    CAS  Google Scholar 

  4. Förster, T. Experimental and theoretical investigation of the intermolecular transfer of electronic excitation energy. Z. Naturforsch. A 4, 321–327 (1949).

    Google Scholar 

  5. Ha, T. Single-molecule fluorescence resonance energy transfer. Methods 25, 78–86 (2001).

    CAS  PubMed  Google Scholar 

  6. Weiss, S. Fluorescence spectroscopy of single biomolecules. Science 283, 1676–1683 (1999).

    CAS  PubMed  Google Scholar 

  7. Joo, C. & Ha, T. Single-molecule FRET with total internal reflection microscopy. in Single Molecule Techniques: a Laboratory Manual. (eds. P. Selvin & T. Ha) 3–36 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2007).A step-by-step 'how-to' manual for single-molecule FRET with TIR microscopy.

    Google Scholar 

  8. Ha, T. et al. Probing the interaction between two single molecules: fluorescence resonance energy transfer between a single donor and a single acceptor. Proc. Natl. Acad. Sci. USA 93, 6264–6268 (1996).First detection of single-molecule FRET.

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Kapanidis, A.N. et al. Alternating-laser excitation of single molecules. Acc. Chem. Res. 38, 523–533 (2005).A review of the alternating laser excitation (ALEX) methods for probing FRET in diffusing single-molecules in solution.

    CAS  PubMed  Google Scholar 

  10. Michalet, X., Weiss, S. & Jager, M. Single-molecule fluorescence studies of protein folding and conformational dynamics. Chem. Rev. 106, 1785–1813 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Seidel, R. & Dekker, C. Single-molecule studies of nucleic acid motors. Curr. Opin. Struct. Biol. 17, 80–86 (2007).

    CAS  PubMed  Google Scholar 

  12. Smiley, R.D. & Hammes, G.G. Single molecule studies of enzyme mechanisms. Chem. Rev. 106, 3080–3094 (2006).

    CAS  PubMed  Google Scholar 

  13. Zhuang, X. Single-molecule RNA science. Annu. Rev. Biophys. Biomol. Struct. 34, 399–414 (2005).

    CAS  PubMed  Google Scholar 

  14. Förster, T. Delocalized excitation and excitation transfer. in Modern Quantum Chemistry (ed., O. Shinanoglu) 93–137 (Academic Press, New York, 1967).

    Google Scholar 

  15. Stryer, L. & Haugland, R.P. Energy transfer: a spectroscopic ruler. Proc. Natl. Acad. Sci. USA 58, 719–726 (1967).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Deniz, A.A. et al. Single-pair fluorescence resonance energy transfer on freely diffusing molecules: observation of Förster distance dependence and subpopulations. Proc. Natl. Acad. Sci. USA 96, 3670–3675 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Best, R.B. et al. Effect of flexibility and cis residues in single-molecule FRET studies of polyproline. Proc. Natl. Acad. Sci. USA 104, 18964–18969 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Merchant, K.A., Best, R.B., Louis, J.M., Gopich, I.V. & Eaton, W.A. Characterizing the unfolded states of proteins using single-molecule FRET spectroscopy and molecular simulations. Proc. Natl. Acad. Sci. USA 104, 1528–1533 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Schuler, B. & Eaton, W.A. Protein folding studied by single-molecule FRET. Curr. Opin. Struct. Biol. 18, 16–26 (2008).A review of single-molecule FRET studies applied to extract quantitative distance information during protein folding.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Ha, T., Chemla, D.S., Enderle, T. & Weiss, S. Single molecule spectroscopy with automated positioning. Appl. Phys. Lett. 70, 782–784 (1997).

    CAS  Google Scholar 

  21. Sabanayagam, C.R., Eid, J.S. & Meller, A. High-throughput scanning confocal microscope for single molecule analysis. Appl. Phys. Lett. 84, 1216–1218 (2004).

    CAS  Google Scholar 

  22. Zhuang, X. et al. A single-molecule study of RNA catalysis and folding. Science 288, 2048–2051 (2000).

    CAS  PubMed  Google Scholar 

  23. Brasselet, S., Peterman, E.J.G., Miyawaki, A. & Moerner, W.E. Single-molecule fluorescence resonant energy transfer in calcium concentration dependent cameleon. J. Phys. Chem. B 104, 3676–3682 (2000).

    CAS  Google Scholar 

  24. Hohng, S. & Ha, T. Single-molecule quantum-dot fluorescence resonance energy transfer. ChemPhysChem 6, 956–960 (2005).

    CAS  PubMed  Google Scholar 

  25. Hohng, S. & Ha, T. Near-complete suppression of quantum dot blinking in ambient conditions. J. Am. Chem. Soc. 126, 1324–1325 (2004).

    CAS  PubMed  Google Scholar 

  26. Kapanidis, A.N. & Weiss, S. Fluorescent probes and bioconjugation chemistries for single-molecule fluorescence analysis of biomolecules. J. Chem. Phys. 117, 10953–10964 (2002).A review of fluorescent dyes and conjugation chemistries for single-molecule fluorescence experiments.

    CAS  Google Scholar 

  27. Hubner, C.G., Renn, A., Renge, I. & Wild, U.P. Direct observation of the triplet lifetime quenching of single dye molecules by molecular oxygen. J. Chem. Phys. 115, 9619–9622 (2001).

    CAS  Google Scholar 

  28. Rasnik, I., McKinney, S.A. & Ha, T. Nonblinking and long-lasting single-molecule fluorescence imaging. Nat. Methods 3, 891–893 (2006).

    CAS  PubMed  Google Scholar 

  29. Rust, M.J., Bates, M. & Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 3, 793–795 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Widengren, J., Chmyrov, A., Eggeling, C., Lofdahl, P.A. & Seidel, C. Strategies to improve photostabilities in ultrasensitive fluorescence spectroscopy. J. Phys. Chem. A 111, 429–440 (2007).

    CAS  PubMed  Google Scholar 

  31. Benesch, R.E. & Benesch, R. Enzymatic removal of oxygen for polarography and related methods. Science 118, 447–448 (1953).

    CAS  PubMed  Google Scholar 

  32. Aitken, C.E., Marshall, R.A. & Puglisi, J. An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments. Biophys. J. 94, 1826–1835 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Wu, P.G. & Brand, L. Resonance energy transfer: methods and applications. Anal. Biochem. 218, 1–13 (1994).

    CAS  PubMed  Google Scholar 

  34. Clegg, R.M. Fluorescence resonance energy transfer and nucleic acids. Methods Enzymol. 211, 353–388 (1992).

    CAS  PubMed  Google Scholar 

  35. Murphy, M.C., Rasnik, I., Cheng, W., Lohman, T.M. & Ha, T. Probing single-stranded DNA conformational flexibility using fluorescence spectroscopy. Biophys. J. 86, 2530–2537 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Ryu, Y.H. & Schultz, P.G. Efficient incorporation of unnatural amino acids into proteins in Escherichia coli. Nat. Methods 3, 263–265 (2006).

    CAS  PubMed  Google Scholar 

  37. Higuchi, R., Krummel, B. & Saiki, R.K. A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res. 16, 7351–7367 (1988).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Braman, J. In vitro mutagenesis protocols, vol. 182,. 2nd edn. (Humana Press, Totowa, New Jersey, 2001).

    Google Scholar 

  39. Pennington, M.W. Site-specific chemical modification procedures. Methods Mol. Biol. 35, 171–185 (1994).

    CAS  PubMed  Google Scholar 

  40. Axelrod, D. Total internal reflection fluorescence at biological surfaces. in Noninvasive Techniques in Cell Biology. (eds. J.K. Foskett & S. Grinstein) 93–127 (Wiley-Liss, New York, 1990).

    Google Scholar 

  41. Axelrod, D. Total internal reflection fluorescence microscopy in cell biology. Methods Enzymol. 361, 1–33 (2003).

    CAS  PubMed  Google Scholar 

  42. Michalet, X. et al. Detectors for single-molecule fluorescence imaging and spectroscopy. J. Mod. Opt. 54, 239–281 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Hohng, S., Joo, C. & Ha, T. Single-molecule three-color FRET. Biophys. J. 87, 1328–1337 (2004).Design and validation of three-color FRET at the single-molecule level.

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Ha, T. et al. Initiation and re-initiation of DNA unwinding by the Escherichia coli Rep helicase. Nature 419, 638–641 (2002).

    Article  CAS  PubMed  Google Scholar 

  45. Sofia, S.J., Premnath, V.V. & Merrill, E.W. Poly(ethylene oxide) grafted to silicon surfaces: grafting density and protein adsorption. Macromolecules 31, 5059–5070 (1998).

    CAS  PubMed  Google Scholar 

  46. Schroeder, C.M., Blainey, P.C., Kim, S. & Xie, X.S. Hydrodynamic flow-stretching assay for single-molecule studies of nucleic acid–protein interactions. in Single Molecule Techniques: A Laboratory Manual. (eds. P. Selvin & T. Ha) 461–492 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; 2007).

    Google Scholar 

  47. Heyes, C.D., Kobitski, A.Y., Amirgoulova, E.V. & Nienhaus, G.U. Biocompatible surfaces for specific tethering of individual protein molecules. J. Phys. Chem. B 108, 13387–13394 (2004).

    CAS  Google Scholar 

  48. Heyes, C.D., Groll, J., Moller, M. & Nienhaus, G.U. Synthesis, patterning and applications of star-shaped poly(ethylene glycol) biofunctionalized surfaces. Mol. Biosyst. 3, 419–430 (2007).

    CAS  PubMed  Google Scholar 

  49. Cha, T., Guo, A. & Zhu, X.Y. Enzymatic activity on a chip: the critical role of protein orientation. Proteomics 5, 416–419 (2005).

    CAS  PubMed  Google Scholar 

  50. Rhoades, E., Gussakovsky, E. & Haran, G. Watching proteins fold one molecule at a time. Proc. Natl. Acad. Sci. USA 100, 3197–3202 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Okumus, B., Wilson, T.J., Lilley, D.M. & Ha, T. Vesicle encapsulation studies reveal that single molecule ribozyme heterogeneities are intrinsic. Biophys. J. 87, 2798–2806 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Benitez, J.J. et al. Probing transient copper chaperone-Wilson disease protein interactions at the single-molecule level with nanovesicle trapping. J. Am. Chem. Soc. 130, 2446–2447 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Cisse, I., Okumus, B., Joo, C. & Ha, T. Fueling protein-DNA interactions inside porous nanocontainers. Proc. Natl. Acad. Sci. USA 104, 12646–12650 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Myong, S., Rasnik, I., Joo, C., Lohman, T.M. & Ha, T. Repetitive shuttling of a motor protein on DNA. Nature 437, 1321–1325 (2005).

    CAS  PubMed  Google Scholar 

  55. Van der Meer, B.W. Resonance energy transfer. (Wiley, Chichester, UK, 1999).

    Google Scholar 

  56. Ha, T. et al. Single-molecule fluorescence spectroscopy of enzyme conformational dynamics and cleavage mechanism. Proc. Natl. Acad. Sci. USA 96, 893–898 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. Joo, C. et al. Real-time observation of RecA filament dynamics with single monomer resolution. Cell 126, 515–527 (2006).

    CAS  PubMed  Google Scholar 

  58. Luo, G., Wang, M., Konigsberg, W.H. & Xie, X.S. Single-molecule and ensemble fluorescence assays for a functionally important conformational change in T7 DNA polymerase. Proc. Natl. Acad. Sci. USA 104, 12610–12615 (2007).Use of intensity fluctuations of a single fluorophore to report on the biochemical reactions of a DNA-enzyme complex.

    CAS  PubMed  PubMed Central  Google Scholar 

  59. Clegg, R.M., Murchie, A.I., Zechel, A. & Lilley, D.M. Observing the helical geometry of double-stranded DNA in solution by fluorescence resonance energy transfer. Proc. Natl. Acad. Sci. USA 90, 2994–2998 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  60. Cooper, J.P. & Hagerman, P.J. Analysis of fluorescence energy transfer in duplex and branched DNA molecules. Biochemistry 29, 9261–9268 (1990).

    CAS  PubMed  Google Scholar 

  61. Lee, S.P., Porter, D., Chirikjian, J.G., Knutson, J.R. & Han, M.K. A fluorometric assay for DNA cleavage reactions characterized with BamHI restriction endonuclease. Anal. Biochem. 220, 377–383 (1994).

    CAS  PubMed  Google Scholar 

  62. Ha, T.J. et al. Temporal fluctuations of fluorescence resonance energy transfer between two dyes conjugated to a single protein. Chem. Phys. 247, 107–118 (1999).

    CAS  Google Scholar 

  63. Colquhoun, D. & Hawkes, A.G. The principles of the stochastic interpretation of ion-channel mechanism. in Single Channel Recording. (eds. B. Sakmann & E. Neher) 397–482 (Plenum Press, New York, 1995).This chapter explains determination of kinetic parameters from stochastic fluctuations of single ion-channel time trajectories. The same concepts are equally applicable to single-molecule FRET trajectories, and the chapter is highly recommended to the beginners in the field.

    Google Scholar 

  64. Kim, H.D. et al. Mg2+-dependent conformational change of RNA studied by fluorescence correlation and FRET on immobilized single molecules. Proc. Natl. Acad. Sci. USA 99, 4284–4289 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  65. McKinney, S.A., Joo, C. & Ha, T. Analysis of single-molecule FRET trajectories using hidden Markov modeling. Biophys. J. 91, 1941–1951 (2006).Hidden Markov model–based analysis of single-molecule FRET time trajectories.

    CAS  PubMed  PubMed Central  Google Scholar 

  66. Munro, J.B., Altman, R.B., O'Connor, N. & Blanchard, S.C. Identification of two distinct hybrid state intermediates on the ribosome. Mol. Cell 25, 505–517 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  67. Myong, S., Bruno, M.M., Pyle, A.M. & Ha, T. Spring-loaded mechanism of DNA unwinding by hepatitis C virus NS3 helicase. Science 317, 513–516 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  68. Yang, H. & Xie, X.S. Probing single-molecule dynamics photon by photon. J. Chem. Phys. 117, 10965–10979 (2002).

    CAS  Google Scholar 

  69. Andrec, M., Levy, R.M. & Talaga, D.S. Direct determination of kinetic rates from single-molecule photon arrival trajectories using hidden Markov models. J. Phys. Chem. A 107, 7454–7464 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  70. Schroder, G.F. & Grubmuller, H. Maximum likelihood trajectories from single molecule fluorescence resonance energy transfer experiments. J. Chem. Phys. 119, 9920–9924 (2003).

    Google Scholar 

  71. Blanchard, S.C., Gonzalez, R.L., Kim, H.D., Chu, S. & Puglisi, J.D. tRNA selection and kinetic proofreading in translation. Nat. Struct. Mol. Biol. 11, 1008–1014 (2004).

    CAS  PubMed  Google Scholar 

  72. Smith, G.J., Sosnick, T.R., Scherer, N.F. & Pan, T. Efficient fluorescence labeling of a large RNA through oligonucleotide hybridization. RNA 11, 234–239 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  73. Dorywalska, M. et al. Site-specific labeling of the ribosome for single-molecule spectroscopy. Nucleic Acids Res. 33, 182–189 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  74. Deniz, A.A. et al. Single-molecule protein folding: diffusion fluorescence resonance energy transfer studies of the denaturation of chymotrypsin inhibitor 2. Proc. Natl. Acad. Sci. USA 97, 5179–5184 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  75. Jager, M., Nir, E. & Weiss, S. Site-specific labeling of proteins for single-molecule FRET by combining chemical and enzymatic modification. Protein Sci. 15, 640–646 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  76. Dale, R.E., Eisinger, J. & Blumberg, W.E. Orientational freedom of molecular probes: orientation factor in intra-molecular energy transfer. Biophys. J. 26, 161–193 (1979).

    CAS  PubMed  PubMed Central  Google Scholar 

  77. Schuler, B., Lipman, E.A., Steinbach, P.J., Kumke, M. & Eaton, W.A. Polyproline and the “spectroscopic ruler” revisited with single-molecule fluorescence. Proc. Natl. Acad. Sci. USA 102, 2754–2759 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  78. Rothwell, P.J. et al. Multiparameter single-molecule fluorescence spectroscopy reveals heterogeneity of HIV-1 reverse transcriptase:primer/template complexes. Proc. Natl. Acad. Sci. USA 100, 1655–1660 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  79. Rasnik, I., Myong, S., Cheng, W., Lohman, T.M. & Ha, T. DNA-binding orientation and domain conformation of the E. coli rep helicase monomer bound to a partial duplex junction: single-molecule studies of fluorescently labeled enzymes. J. Mol. Biol. 336, 395–408 (2004).

    CAS  PubMed  Google Scholar 

  80. Andrecka, J. et al. Single-molecule tracking of mRNA exiting from RNA polymerase II. Proc. Natl. Acad. Sci. USA 105, 135–140 (2008).

    CAS  PubMed  Google Scholar 

  81. Clamme, J.P. & Deniz, A.A. Three-color single-molecule fluorescence resonance energy transfer. ChemPhysChem 6, 74–77 (2005).

    CAS  PubMed  Google Scholar 

  82. Heinze, K.G., Jahnz, M. & Schwille, P. Triple-color coincidence analysis: one step further in following higher order molecular complex formation. Biophys. J. 86, 506–516 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  83. Kapanidis, A.N. et al. Fluorescence-aided molecule sorting: analysis of structure and interactions by alternating-laser excitation of single molecules. Proc. Natl. Acad. Sci. USA 101, 8936–8941 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  84. Muller, B.K., Zaychikov, E., Brauchle, C. & Lamb, D.C. Pulsed interleaved excitation. Biophys. J. 89, 3508–3522 (2005).

    PubMed  PubMed Central  Google Scholar 

  85. Lee, N.K. et al. Three-color alternating-laser excitation of single molecules: monitoring multiple interactions and distances. Biophys. J. 92, 303–312 (2007).

    CAS  PubMed  Google Scholar 

  86. Heilemann, M. et al. Multistep energy transfer in single molecular photonic wires. J. Am. Chem. Soc. 126, 6514–6515 (2004).

    CAS  PubMed  Google Scholar 

  87. Lang, M., Fordyce, P. & Block, S. Combined optical trapping and single-molecule fluorescence. J. Biol. 2, 6 (2003).

    PubMed  PubMed Central  Google Scholar 

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

    CAS  Google Scholar 

  89. Shroff, H. et al. Biocompatible force sensor with optical readout and dimensions of 6 nm(3). Nano Lett. 5, 1509–1514 (2005).

    CAS  PubMed  Google Scholar 

  90. 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 

  91. Borisenko, V. et al. Simultaneous optical and electrical recording of single gramicidin channels. Biophys. J. 84, 612–622 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  92. Harms, G.S. et al. Probing conformational changes of gramicidin ion channels by single-molecule patch-clamp fluorescence microscopy. Biophys. J. 85, 1826–1838 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  93. Tan, E. et al. A four-way junction accelerates hairpin ribozyme folding via a discrete intermediate. Proc. Natl. Acad. Sci. USA 100, 9308–9313 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  94. Jager, M., Michalet, X. & Weiss, S. Protein-protein interactions as a tool for site-specific labeling of proteins. Protein Sci. 14, 2059–2068 (2005).

    PubMed  PubMed Central  Google Scholar 

  95. Ratner, V., Kahana, E., Eichler, M. & Haas, E. A general strategy for site-specific double labeling of globular proteins for kinetic FRET studies. Bioconjug. Chem. 13, 1163–1170 (2002).

    CAS  PubMed  Google Scholar 

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Acknowledgements

We acknowledge I. Rasnik, S. Mckinney, C. Joo, R. Clegg, S. Myong, members of Ha group and K. Drexhage for expert advice and discussion; S. Syed (University of Illinois) for procurement of the dyes and reagents; and P. Cornish, M. Brenner and L. Supriya for carefully reading the manuscript. C. Joo prepared the video instruction on PEG slide preparation. Authors' work on single-molecule FRET was funded by the US National Institutes of Health, National Science Foundation career award and Howard Hughes Medical Institute. S.H. was also supported by Research Settlement Fund for the new faculty at Seoul National University (Korea), Ministry of Science and Technology grant (RH0-2005-000-01003-0, 2007) and Basic Science Research Grant from the Korea Research Foundation.

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Correspondence to Taekjip Ha.

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Roy, R., Hohng, S. & Ha, T. A practical guide to single-molecule FRET. Nat Methods 5, 507–516 (2008). https://doi.org/10.1038/nmeth.1208

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