Abstract

Single-molecule fluorescence imaging has greatly contributed to our understanding of many bio-molecular systems. While reactions occurring in the range of several minutes can be readily studied using conventional single-molecule fluorescence microscopes, data acquisition for longer time scales is hindered by the focal drift of high numerical aperture objectives, which should be corrected in real time. Here, we developed a robust autofocusing system based on optical astigmatism analysis of single-molecule images. Compared to the previously developed methods, our approach has a merit of simplicity in that neither fiducial makers nor an additional laser-detector system is required. As a demonstration, we observed B-Z transition dynamics occurring for several hours.

© 2012 OSA

Full Article  |  PDF Article

References

  • View by:
  • |
  • |
  • |

  1. C. Joo, H. Balci, Y. Ishitsuka, C. Buranachai, and T. Ha, “Advances in single-molecule fluorescence methods for molecular biology,” Annu. Rev. Biochem. 77(1), 51–76 (2008).
    [CrossRef] [PubMed]
  2. B. Schuler and W. A. Eaton, “Protein folding studied by single-molecule FRET,” Curr. Opin. Struct. Biol. 18(1), 16–26 (2008).
    [CrossRef] [PubMed]
  3. J. Hohlbein, K. Gryte, M. Heilemann, and A. N. Kapanidis, “Surfing on a new wave of single-molecule fluorescence methods,” Phys. Biol. 7(3), 031001 (2010).
    [CrossRef] [PubMed]
  4. D. Klostermeier, “Single-molecule FRET reveals nucleotide-driven conformational changes in molecular machines and their link to RNA unwinding and DNA supercoiling,” Biochem. Soc. Trans. 39(2), 611–616 (2011).
    [CrossRef] [PubMed]
  5. M. Guizar-Sicairos, S. T. Thurman, and J. R. Fienup, “Efficient subpixel image registration algorithms,” Opt. Lett. 33(2), 156–158 (2008).
    [CrossRef] [PubMed]
  6. M. J. Mlodzianoski, J. M. Schreiner, S. P. Callahan, K. Smolková, A. Dlasková, J. Santorová, P. Ježek, and J. Bewersdorf, “Sample drift correction in 3D fluorescence photoactivation localization microscopy,” Opt. Express 19(16), 15009–15019 (2011).
    [CrossRef] [PubMed]
  7. S. H. Lee, M. Baday, M. Tjioe, P. D. Simonson, R. Zhang, E. Cai, and P. R. Selvin, “Using fixed fiduciary markers for stage drift correction,” Opt. Express 20(11), 12177–12183 (2012).
    [CrossRef] [PubMed]
  8. M. P. Elenko, J. W. Szostak, and A. M. van Oijen, “Single-molecule binding experiments on long time scales,” Rev. Sci. Instrum. 81(8), 083705 (2010).
    [CrossRef] [PubMed]
  9. A. Pertsinidis, Y. Zhang, and S. Chu, “Subnanometre single-molecule localization, registration and distance measurements,” Nature 466(7306), 647–651 (2010).
    [CrossRef] [PubMed]
  10. J. Peters, “Nikon Instruments TiE-PFS Dynamic Focusing System,” Nat. Methods | Application Notes (2008).
  11. S. Lee, J. Lee, and S. Hohng, “Single-molecule three-color FRET with both negligible spectral overlap and long observation time,” PLoS ONE 5(8), e12270 (2010).
    [CrossRef] [PubMed]
  12. J. Lee, S. Lee, K. Ragunathan, C. Joo, T. Ha, and S. Hohng, “Single-molecule four-color FRET,” Angew. Chem. Int. Ed. Engl. 49(51), 9922–9925 (2010).
    [CrossRef] [PubMed]
  13. W. Hwang, V. Arluison, and S. Hohng, “Dynamic competition of DsrA and rpoS fragments for the proximal binding site of Hfq as a means for efficient annealing,” Nucleic Acids Res. 39(12), 5131–5139 (2011).
    [CrossRef] [PubMed]
  14. D. K. Cohen, W. H. Gee, M. Ludeke, and J. Lewkowicz, “Automatic focus control: the astigmatic lens approach,” Appl. Opt. 23(4), 565–570 (1984).
    [CrossRef] [PubMed]
  15. B. Huang, W. Wang, M. Bates, and X. Zhuang, “Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy,” Science 319(5864), 810–813 (2008).
    [CrossRef] [PubMed]
  16. R. Roy, S. Hohng, and T. Ha, “A practical guide to single-molecule FRET,” Nat. Methods 5(6), 507–516 (2008).
    [CrossRef] [PubMed]
  17. J. F. Brenner, B. S. Dew, J. B. Horton, T. King, P. W. Neurath, and W. D. Selles, “An automated microscope for cytologic research a preliminary evaluation,” J. Histochem. Cytochem. 24(1), 100–111 (1976).
    [CrossRef] [PubMed]
  18. Y. Sun, S. Duthaler, and B. J. Nelson, “Autofocusing in computer microscopy: selecting the optimal focus algorithm,” Microsc. Res. Tech. 65(3), 139–149 (2004).
    [CrossRef] [PubMed]
  19. S. Yazdanfar, K. B. Kenny, K. Tasimi, A. D. Corwin, E. L. Dixon, and R. J. Filkins, “Simple and robust image-based autofocusing for digital microscopy,” Opt. Express 16(12), 8670–8677 (2008).
    [CrossRef] [PubMed]
  20. A. Rich, “DNA comes in many forms,” Gene 135(1-2), 99–109 (1993).
    [CrossRef] [PubMed]
  21. J. Choi and T. Majima, “Conformational changes of non-B DNA,” Chem. Soc. Rev. 40(12), 5893–5909 (2011).
    [CrossRef] [PubMed]
  22. S. Bae, H. Son, Y.-G. Kim, and S. Hohng, “Z-DNA is stabilized by the Hofmeister effect of salts,” (manuscript in preparation).
  23. J. E. Bronson, J. Fei, J. M. Hofman, R. L. Gonzalez, and C. H. Wiggins, “Learning Rates and States from Biophysical Time Series: A Bayesian approach to model selection and single-molecule FRET data,” Biophys. J. 97(12), 3196–3205 (2009).
    [CrossRef] [PubMed]
  24. S. A. McKinney, C. Joo, and T. Ha, “Analysis of single-molecule FRET trajectories using hidden markov modeling,” Biophys. J. 91(5), 1941–1951 (2006).
    [CrossRef] [PubMed]
  25. S. Hohng, C. Joo, and T. Ha, “Single-molecule three-color FRET,” Biophys. J. 87(2), 1328–1337 (2004).
    [CrossRef] [PubMed]
  26. V. DeRocco, T. Anderson, J. Piehler, D. A. Erie, and K. Weninger, “Four-color single-molecule fluorescence with noncovalent dye labeling to monitor dynamic multimolecular complexes,” Biotechniques 49(5), 807–816 (2010).
    [CrossRef] [PubMed]

2012

2011

M. J. Mlodzianoski, J. M. Schreiner, S. P. Callahan, K. Smolková, A. Dlasková, J. Santorová, P. Ježek, and J. Bewersdorf, “Sample drift correction in 3D fluorescence photoactivation localization microscopy,” Opt. Express 19(16), 15009–15019 (2011).
[CrossRef] [PubMed]

D. Klostermeier, “Single-molecule FRET reveals nucleotide-driven conformational changes in molecular machines and their link to RNA unwinding and DNA supercoiling,” Biochem. Soc. Trans. 39(2), 611–616 (2011).
[CrossRef] [PubMed]

W. Hwang, V. Arluison, and S. Hohng, “Dynamic competition of DsrA and rpoS fragments for the proximal binding site of Hfq as a means for efficient annealing,” Nucleic Acids Res. 39(12), 5131–5139 (2011).
[CrossRef] [PubMed]

J. Choi and T. Majima, “Conformational changes of non-B DNA,” Chem. Soc. Rev. 40(12), 5893–5909 (2011).
[CrossRef] [PubMed]

2010

M. P. Elenko, J. W. Szostak, and A. M. van Oijen, “Single-molecule binding experiments on long time scales,” Rev. Sci. Instrum. 81(8), 083705 (2010).
[CrossRef] [PubMed]

A. Pertsinidis, Y. Zhang, and S. Chu, “Subnanometre single-molecule localization, registration and distance measurements,” Nature 466(7306), 647–651 (2010).
[CrossRef] [PubMed]

S. Lee, J. Lee, and S. Hohng, “Single-molecule three-color FRET with both negligible spectral overlap and long observation time,” PLoS ONE 5(8), e12270 (2010).
[CrossRef] [PubMed]

J. Lee, S. Lee, K. Ragunathan, C. Joo, T. Ha, and S. Hohng, “Single-molecule four-color FRET,” Angew. Chem. Int. Ed. Engl. 49(51), 9922–9925 (2010).
[CrossRef] [PubMed]

J. Hohlbein, K. Gryte, M. Heilemann, and A. N. Kapanidis, “Surfing on a new wave of single-molecule fluorescence methods,” Phys. Biol. 7(3), 031001 (2010).
[CrossRef] [PubMed]

V. DeRocco, T. Anderson, J. Piehler, D. A. Erie, and K. Weninger, “Four-color single-molecule fluorescence with noncovalent dye labeling to monitor dynamic multimolecular complexes,” Biotechniques 49(5), 807–816 (2010).
[CrossRef] [PubMed]

2009

J. E. Bronson, J. Fei, J. M. Hofman, R. L. Gonzalez, and C. H. Wiggins, “Learning Rates and States from Biophysical Time Series: A Bayesian approach to model selection and single-molecule FRET data,” Biophys. J. 97(12), 3196–3205 (2009).
[CrossRef] [PubMed]

2008

C. Joo, H. Balci, Y. Ishitsuka, C. Buranachai, and T. Ha, “Advances in single-molecule fluorescence methods for molecular biology,” Annu. Rev. Biochem. 77(1), 51–76 (2008).
[CrossRef] [PubMed]

B. Schuler and W. A. Eaton, “Protein folding studied by single-molecule FRET,” Curr. Opin. Struct. Biol. 18(1), 16–26 (2008).
[CrossRef] [PubMed]

B. Huang, W. Wang, M. Bates, and X. Zhuang, “Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy,” Science 319(5864), 810–813 (2008).
[CrossRef] [PubMed]

R. Roy, S. Hohng, and T. Ha, “A practical guide to single-molecule FRET,” Nat. Methods 5(6), 507–516 (2008).
[CrossRef] [PubMed]

M. Guizar-Sicairos, S. T. Thurman, and J. R. Fienup, “Efficient subpixel image registration algorithms,” Opt. Lett. 33(2), 156–158 (2008).
[CrossRef] [PubMed]

S. Yazdanfar, K. B. Kenny, K. Tasimi, A. D. Corwin, E. L. Dixon, and R. J. Filkins, “Simple and robust image-based autofocusing for digital microscopy,” Opt. Express 16(12), 8670–8677 (2008).
[CrossRef] [PubMed]

2006

S. A. McKinney, C. Joo, and T. Ha, “Analysis of single-molecule FRET trajectories using hidden markov modeling,” Biophys. J. 91(5), 1941–1951 (2006).
[CrossRef] [PubMed]

2004

S. Hohng, C. Joo, and T. Ha, “Single-molecule three-color FRET,” Biophys. J. 87(2), 1328–1337 (2004).
[CrossRef] [PubMed]

Y. Sun, S. Duthaler, and B. J. Nelson, “Autofocusing in computer microscopy: selecting the optimal focus algorithm,” Microsc. Res. Tech. 65(3), 139–149 (2004).
[CrossRef] [PubMed]

1993

A. Rich, “DNA comes in many forms,” Gene 135(1-2), 99–109 (1993).
[CrossRef] [PubMed]

1984

1976

J. F. Brenner, B. S. Dew, J. B. Horton, T. King, P. W. Neurath, and W. D. Selles, “An automated microscope for cytologic research a preliminary evaluation,” J. Histochem. Cytochem. 24(1), 100–111 (1976).
[CrossRef] [PubMed]

Anderson, T.

V. DeRocco, T. Anderson, J. Piehler, D. A. Erie, and K. Weninger, “Four-color single-molecule fluorescence with noncovalent dye labeling to monitor dynamic multimolecular complexes,” Biotechniques 49(5), 807–816 (2010).
[CrossRef] [PubMed]

Arluison, V.

W. Hwang, V. Arluison, and S. Hohng, “Dynamic competition of DsrA and rpoS fragments for the proximal binding site of Hfq as a means for efficient annealing,” Nucleic Acids Res. 39(12), 5131–5139 (2011).
[CrossRef] [PubMed]

Baday, M.

Balci, H.

C. Joo, H. Balci, Y. Ishitsuka, C. Buranachai, and T. Ha, “Advances in single-molecule fluorescence methods for molecular biology,” Annu. Rev. Biochem. 77(1), 51–76 (2008).
[CrossRef] [PubMed]

Bates, M.

B. Huang, W. Wang, M. Bates, and X. Zhuang, “Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy,” Science 319(5864), 810–813 (2008).
[CrossRef] [PubMed]

Bewersdorf, J.

Brenner, J. F.

J. F. Brenner, B. S. Dew, J. B. Horton, T. King, P. W. Neurath, and W. D. Selles, “An automated microscope for cytologic research a preliminary evaluation,” J. Histochem. Cytochem. 24(1), 100–111 (1976).
[CrossRef] [PubMed]

Bronson, J. E.

J. E. Bronson, J. Fei, J. M. Hofman, R. L. Gonzalez, and C. H. Wiggins, “Learning Rates and States from Biophysical Time Series: A Bayesian approach to model selection and single-molecule FRET data,” Biophys. J. 97(12), 3196–3205 (2009).
[CrossRef] [PubMed]

Buranachai, C.

C. Joo, H. Balci, Y. Ishitsuka, C. Buranachai, and T. Ha, “Advances in single-molecule fluorescence methods for molecular biology,” Annu. Rev. Biochem. 77(1), 51–76 (2008).
[CrossRef] [PubMed]

Cai, E.

Callahan, S. P.

Choi, J.

J. Choi and T. Majima, “Conformational changes of non-B DNA,” Chem. Soc. Rev. 40(12), 5893–5909 (2011).
[CrossRef] [PubMed]

Chu, S.

A. Pertsinidis, Y. Zhang, and S. Chu, “Subnanometre single-molecule localization, registration and distance measurements,” Nature 466(7306), 647–651 (2010).
[CrossRef] [PubMed]

Cohen, D. K.

Corwin, A. D.

DeRocco, V.

V. DeRocco, T. Anderson, J. Piehler, D. A. Erie, and K. Weninger, “Four-color single-molecule fluorescence with noncovalent dye labeling to monitor dynamic multimolecular complexes,” Biotechniques 49(5), 807–816 (2010).
[CrossRef] [PubMed]

Dew, B. S.

J. F. Brenner, B. S. Dew, J. B. Horton, T. King, P. W. Neurath, and W. D. Selles, “An automated microscope for cytologic research a preliminary evaluation,” J. Histochem. Cytochem. 24(1), 100–111 (1976).
[CrossRef] [PubMed]

Dixon, E. L.

Dlasková, A.

Duthaler, S.

Y. Sun, S. Duthaler, and B. J. Nelson, “Autofocusing in computer microscopy: selecting the optimal focus algorithm,” Microsc. Res. Tech. 65(3), 139–149 (2004).
[CrossRef] [PubMed]

Eaton, W. A.

B. Schuler and W. A. Eaton, “Protein folding studied by single-molecule FRET,” Curr. Opin. Struct. Biol. 18(1), 16–26 (2008).
[CrossRef] [PubMed]

Elenko, M. P.

M. P. Elenko, J. W. Szostak, and A. M. van Oijen, “Single-molecule binding experiments on long time scales,” Rev. Sci. Instrum. 81(8), 083705 (2010).
[CrossRef] [PubMed]

Erie, D. A.

V. DeRocco, T. Anderson, J. Piehler, D. A. Erie, and K. Weninger, “Four-color single-molecule fluorescence with noncovalent dye labeling to monitor dynamic multimolecular complexes,” Biotechniques 49(5), 807–816 (2010).
[CrossRef] [PubMed]

Fei, J.

J. E. Bronson, J. Fei, J. M. Hofman, R. L. Gonzalez, and C. H. Wiggins, “Learning Rates and States from Biophysical Time Series: A Bayesian approach to model selection and single-molecule FRET data,” Biophys. J. 97(12), 3196–3205 (2009).
[CrossRef] [PubMed]

Fienup, J. R.

Filkins, R. J.

Gee, W. H.

Gonzalez, R. L.

J. E. Bronson, J. Fei, J. M. Hofman, R. L. Gonzalez, and C. H. Wiggins, “Learning Rates and States from Biophysical Time Series: A Bayesian approach to model selection and single-molecule FRET data,” Biophys. J. 97(12), 3196–3205 (2009).
[CrossRef] [PubMed]

Gryte, K.

J. Hohlbein, K. Gryte, M. Heilemann, and A. N. Kapanidis, “Surfing on a new wave of single-molecule fluorescence methods,” Phys. Biol. 7(3), 031001 (2010).
[CrossRef] [PubMed]

Guizar-Sicairos, M.

Ha, T.

J. Lee, S. Lee, K. Ragunathan, C. Joo, T. Ha, and S. Hohng, “Single-molecule four-color FRET,” Angew. Chem. Int. Ed. Engl. 49(51), 9922–9925 (2010).
[CrossRef] [PubMed]

R. Roy, S. Hohng, and T. Ha, “A practical guide to single-molecule FRET,” Nat. Methods 5(6), 507–516 (2008).
[CrossRef] [PubMed]

C. Joo, H. Balci, Y. Ishitsuka, C. Buranachai, and T. Ha, “Advances in single-molecule fluorescence methods for molecular biology,” Annu. Rev. Biochem. 77(1), 51–76 (2008).
[CrossRef] [PubMed]

S. A. McKinney, C. Joo, and T. Ha, “Analysis of single-molecule FRET trajectories using hidden markov modeling,” Biophys. J. 91(5), 1941–1951 (2006).
[CrossRef] [PubMed]

S. Hohng, C. Joo, and T. Ha, “Single-molecule three-color FRET,” Biophys. J. 87(2), 1328–1337 (2004).
[CrossRef] [PubMed]

Heilemann, M.

J. Hohlbein, K. Gryte, M. Heilemann, and A. N. Kapanidis, “Surfing on a new wave of single-molecule fluorescence methods,” Phys. Biol. 7(3), 031001 (2010).
[CrossRef] [PubMed]

Hofman, J. M.

J. E. Bronson, J. Fei, J. M. Hofman, R. L. Gonzalez, and C. H. Wiggins, “Learning Rates and States from Biophysical Time Series: A Bayesian approach to model selection and single-molecule FRET data,” Biophys. J. 97(12), 3196–3205 (2009).
[CrossRef] [PubMed]

Hohlbein, J.

J. Hohlbein, K. Gryte, M. Heilemann, and A. N. Kapanidis, “Surfing on a new wave of single-molecule fluorescence methods,” Phys. Biol. 7(3), 031001 (2010).
[CrossRef] [PubMed]

Hohng, S.

W. Hwang, V. Arluison, and S. Hohng, “Dynamic competition of DsrA and rpoS fragments for the proximal binding site of Hfq as a means for efficient annealing,” Nucleic Acids Res. 39(12), 5131–5139 (2011).
[CrossRef] [PubMed]

J. Lee, S. Lee, K. Ragunathan, C. Joo, T. Ha, and S. Hohng, “Single-molecule four-color FRET,” Angew. Chem. Int. Ed. Engl. 49(51), 9922–9925 (2010).
[CrossRef] [PubMed]

S. Lee, J. Lee, and S. Hohng, “Single-molecule three-color FRET with both negligible spectral overlap and long observation time,” PLoS ONE 5(8), e12270 (2010).
[CrossRef] [PubMed]

R. Roy, S. Hohng, and T. Ha, “A practical guide to single-molecule FRET,” Nat. Methods 5(6), 507–516 (2008).
[CrossRef] [PubMed]

S. Hohng, C. Joo, and T. Ha, “Single-molecule three-color FRET,” Biophys. J. 87(2), 1328–1337 (2004).
[CrossRef] [PubMed]

Horton, J. B.

J. F. Brenner, B. S. Dew, J. B. Horton, T. King, P. W. Neurath, and W. D. Selles, “An automated microscope for cytologic research a preliminary evaluation,” J. Histochem. Cytochem. 24(1), 100–111 (1976).
[CrossRef] [PubMed]

Huang, B.

B. Huang, W. Wang, M. Bates, and X. Zhuang, “Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy,” Science 319(5864), 810–813 (2008).
[CrossRef] [PubMed]

Hwang, W.

W. Hwang, V. Arluison, and S. Hohng, “Dynamic competition of DsrA and rpoS fragments for the proximal binding site of Hfq as a means for efficient annealing,” Nucleic Acids Res. 39(12), 5131–5139 (2011).
[CrossRef] [PubMed]

Ishitsuka, Y.

C. Joo, H. Balci, Y. Ishitsuka, C. Buranachai, and T. Ha, “Advances in single-molecule fluorescence methods for molecular biology,” Annu. Rev. Biochem. 77(1), 51–76 (2008).
[CrossRef] [PubMed]

Ježek, P.

Joo, C.

J. Lee, S. Lee, K. Ragunathan, C. Joo, T. Ha, and S. Hohng, “Single-molecule four-color FRET,” Angew. Chem. Int. Ed. Engl. 49(51), 9922–9925 (2010).
[CrossRef] [PubMed]

C. Joo, H. Balci, Y. Ishitsuka, C. Buranachai, and T. Ha, “Advances in single-molecule fluorescence methods for molecular biology,” Annu. Rev. Biochem. 77(1), 51–76 (2008).
[CrossRef] [PubMed]

S. A. McKinney, C. Joo, and T. Ha, “Analysis of single-molecule FRET trajectories using hidden markov modeling,” Biophys. J. 91(5), 1941–1951 (2006).
[CrossRef] [PubMed]

S. Hohng, C. Joo, and T. Ha, “Single-molecule three-color FRET,” Biophys. J. 87(2), 1328–1337 (2004).
[CrossRef] [PubMed]

Kapanidis, A. N.

J. Hohlbein, K. Gryte, M. Heilemann, and A. N. Kapanidis, “Surfing on a new wave of single-molecule fluorescence methods,” Phys. Biol. 7(3), 031001 (2010).
[CrossRef] [PubMed]

Kenny, K. B.

King, T.

J. F. Brenner, B. S. Dew, J. B. Horton, T. King, P. W. Neurath, and W. D. Selles, “An automated microscope for cytologic research a preliminary evaluation,” J. Histochem. Cytochem. 24(1), 100–111 (1976).
[CrossRef] [PubMed]

Klostermeier, D.

D. Klostermeier, “Single-molecule FRET reveals nucleotide-driven conformational changes in molecular machines and their link to RNA unwinding and DNA supercoiling,” Biochem. Soc. Trans. 39(2), 611–616 (2011).
[CrossRef] [PubMed]

Lee, J.

J. Lee, S. Lee, K. Ragunathan, C. Joo, T. Ha, and S. Hohng, “Single-molecule four-color FRET,” Angew. Chem. Int. Ed. Engl. 49(51), 9922–9925 (2010).
[CrossRef] [PubMed]

S. Lee, J. Lee, and S. Hohng, “Single-molecule three-color FRET with both negligible spectral overlap and long observation time,” PLoS ONE 5(8), e12270 (2010).
[CrossRef] [PubMed]

Lee, S.

S. Lee, J. Lee, and S. Hohng, “Single-molecule three-color FRET with both negligible spectral overlap and long observation time,” PLoS ONE 5(8), e12270 (2010).
[CrossRef] [PubMed]

J. Lee, S. Lee, K. Ragunathan, C. Joo, T. Ha, and S. Hohng, “Single-molecule four-color FRET,” Angew. Chem. Int. Ed. Engl. 49(51), 9922–9925 (2010).
[CrossRef] [PubMed]

Lee, S. H.

Lewkowicz, J.

Ludeke, M.

Majima, T.

J. Choi and T. Majima, “Conformational changes of non-B DNA,” Chem. Soc. Rev. 40(12), 5893–5909 (2011).
[CrossRef] [PubMed]

McKinney, S. A.

S. A. McKinney, C. Joo, and T. Ha, “Analysis of single-molecule FRET trajectories using hidden markov modeling,” Biophys. J. 91(5), 1941–1951 (2006).
[CrossRef] [PubMed]

Mlodzianoski, M. J.

Nelson, B. J.

Y. Sun, S. Duthaler, and B. J. Nelson, “Autofocusing in computer microscopy: selecting the optimal focus algorithm,” Microsc. Res. Tech. 65(3), 139–149 (2004).
[CrossRef] [PubMed]

Neurath, P. W.

J. F. Brenner, B. S. Dew, J. B. Horton, T. King, P. W. Neurath, and W. D. Selles, “An automated microscope for cytologic research a preliminary evaluation,” J. Histochem. Cytochem. 24(1), 100–111 (1976).
[CrossRef] [PubMed]

Pertsinidis, A.

A. Pertsinidis, Y. Zhang, and S. Chu, “Subnanometre single-molecule localization, registration and distance measurements,” Nature 466(7306), 647–651 (2010).
[CrossRef] [PubMed]

Piehler, J.

V. DeRocco, T. Anderson, J. Piehler, D. A. Erie, and K. Weninger, “Four-color single-molecule fluorescence with noncovalent dye labeling to monitor dynamic multimolecular complexes,” Biotechniques 49(5), 807–816 (2010).
[CrossRef] [PubMed]

Ragunathan, K.

J. Lee, S. Lee, K. Ragunathan, C. Joo, T. Ha, and S. Hohng, “Single-molecule four-color FRET,” Angew. Chem. Int. Ed. Engl. 49(51), 9922–9925 (2010).
[CrossRef] [PubMed]

Rich, A.

A. Rich, “DNA comes in many forms,” Gene 135(1-2), 99–109 (1993).
[CrossRef] [PubMed]

Roy, R.

R. Roy, S. Hohng, and T. Ha, “A practical guide to single-molecule FRET,” Nat. Methods 5(6), 507–516 (2008).
[CrossRef] [PubMed]

Santorová, J.

Schreiner, J. M.

Schuler, B.

B. Schuler and W. A. Eaton, “Protein folding studied by single-molecule FRET,” Curr. Opin. Struct. Biol. 18(1), 16–26 (2008).
[CrossRef] [PubMed]

Selles, W. D.

J. F. Brenner, B. S. Dew, J. B. Horton, T. King, P. W. Neurath, and W. D. Selles, “An automated microscope for cytologic research a preliminary evaluation,” J. Histochem. Cytochem. 24(1), 100–111 (1976).
[CrossRef] [PubMed]

Selvin, P. R.

Simonson, P. D.

Smolková, K.

Sun, Y.

Y. Sun, S. Duthaler, and B. J. Nelson, “Autofocusing in computer microscopy: selecting the optimal focus algorithm,” Microsc. Res. Tech. 65(3), 139–149 (2004).
[CrossRef] [PubMed]

Szostak, J. W.

M. P. Elenko, J. W. Szostak, and A. M. van Oijen, “Single-molecule binding experiments on long time scales,” Rev. Sci. Instrum. 81(8), 083705 (2010).
[CrossRef] [PubMed]

Tasimi, K.

Thurman, S. T.

Tjioe, M.

van Oijen, A. M.

M. P. Elenko, J. W. Szostak, and A. M. van Oijen, “Single-molecule binding experiments on long time scales,” Rev. Sci. Instrum. 81(8), 083705 (2010).
[CrossRef] [PubMed]

Wang, W.

B. Huang, W. Wang, M. Bates, and X. Zhuang, “Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy,” Science 319(5864), 810–813 (2008).
[CrossRef] [PubMed]

Weninger, K.

V. DeRocco, T. Anderson, J. Piehler, D. A. Erie, and K. Weninger, “Four-color single-molecule fluorescence with noncovalent dye labeling to monitor dynamic multimolecular complexes,” Biotechniques 49(5), 807–816 (2010).
[CrossRef] [PubMed]

Wiggins, C. H.

J. E. Bronson, J. Fei, J. M. Hofman, R. L. Gonzalez, and C. H. Wiggins, “Learning Rates and States from Biophysical Time Series: A Bayesian approach to model selection and single-molecule FRET data,” Biophys. J. 97(12), 3196–3205 (2009).
[CrossRef] [PubMed]

Yazdanfar, S.

Zhang, R.

Zhang, Y.

A. Pertsinidis, Y. Zhang, and S. Chu, “Subnanometre single-molecule localization, registration and distance measurements,” Nature 466(7306), 647–651 (2010).
[CrossRef] [PubMed]

Zhuang, X.

B. Huang, W. Wang, M. Bates, and X. Zhuang, “Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy,” Science 319(5864), 810–813 (2008).
[CrossRef] [PubMed]

Angew. Chem. Int. Ed. Engl.

J. Lee, S. Lee, K. Ragunathan, C. Joo, T. Ha, and S. Hohng, “Single-molecule four-color FRET,” Angew. Chem. Int. Ed. Engl. 49(51), 9922–9925 (2010).
[CrossRef] [PubMed]

Annu. Rev. Biochem.

C. Joo, H. Balci, Y. Ishitsuka, C. Buranachai, and T. Ha, “Advances in single-molecule fluorescence methods for molecular biology,” Annu. Rev. Biochem. 77(1), 51–76 (2008).
[CrossRef] [PubMed]

Appl. Opt.

Biochem. Soc. Trans.

D. Klostermeier, “Single-molecule FRET reveals nucleotide-driven conformational changes in molecular machines and their link to RNA unwinding and DNA supercoiling,” Biochem. Soc. Trans. 39(2), 611–616 (2011).
[CrossRef] [PubMed]

Biophys. J.

J. E. Bronson, J. Fei, J. M. Hofman, R. L. Gonzalez, and C. H. Wiggins, “Learning Rates and States from Biophysical Time Series: A Bayesian approach to model selection and single-molecule FRET data,” Biophys. J. 97(12), 3196–3205 (2009).
[CrossRef] [PubMed]

S. A. McKinney, C. Joo, and T. Ha, “Analysis of single-molecule FRET trajectories using hidden markov modeling,” Biophys. J. 91(5), 1941–1951 (2006).
[CrossRef] [PubMed]

S. Hohng, C. Joo, and T. Ha, “Single-molecule three-color FRET,” Biophys. J. 87(2), 1328–1337 (2004).
[CrossRef] [PubMed]

Biotechniques

V. DeRocco, T. Anderson, J. Piehler, D. A. Erie, and K. Weninger, “Four-color single-molecule fluorescence with noncovalent dye labeling to monitor dynamic multimolecular complexes,” Biotechniques 49(5), 807–816 (2010).
[CrossRef] [PubMed]

Chem. Soc. Rev.

J. Choi and T. Majima, “Conformational changes of non-B DNA,” Chem. Soc. Rev. 40(12), 5893–5909 (2011).
[CrossRef] [PubMed]

Curr. Opin. Struct. Biol.

B. Schuler and W. A. Eaton, “Protein folding studied by single-molecule FRET,” Curr. Opin. Struct. Biol. 18(1), 16–26 (2008).
[CrossRef] [PubMed]

Gene

A. Rich, “DNA comes in many forms,” Gene 135(1-2), 99–109 (1993).
[CrossRef] [PubMed]

J. Histochem. Cytochem.

J. F. Brenner, B. S. Dew, J. B. Horton, T. King, P. W. Neurath, and W. D. Selles, “An automated microscope for cytologic research a preliminary evaluation,” J. Histochem. Cytochem. 24(1), 100–111 (1976).
[CrossRef] [PubMed]

Microsc. Res. Tech.

Y. Sun, S. Duthaler, and B. J. Nelson, “Autofocusing in computer microscopy: selecting the optimal focus algorithm,” Microsc. Res. Tech. 65(3), 139–149 (2004).
[CrossRef] [PubMed]

Nat. Methods

R. Roy, S. Hohng, and T. Ha, “A practical guide to single-molecule FRET,” Nat. Methods 5(6), 507–516 (2008).
[CrossRef] [PubMed]

Nature

A. Pertsinidis, Y. Zhang, and S. Chu, “Subnanometre single-molecule localization, registration and distance measurements,” Nature 466(7306), 647–651 (2010).
[CrossRef] [PubMed]

Nucleic Acids Res.

W. Hwang, V. Arluison, and S. Hohng, “Dynamic competition of DsrA and rpoS fragments for the proximal binding site of Hfq as a means for efficient annealing,” Nucleic Acids Res. 39(12), 5131–5139 (2011).
[CrossRef] [PubMed]

Opt. Express

Opt. Lett.

Phys. Biol.

J. Hohlbein, K. Gryte, M. Heilemann, and A. N. Kapanidis, “Surfing on a new wave of single-molecule fluorescence methods,” Phys. Biol. 7(3), 031001 (2010).
[CrossRef] [PubMed]

PLoS ONE

S. Lee, J. Lee, and S. Hohng, “Single-molecule three-color FRET with both negligible spectral overlap and long observation time,” PLoS ONE 5(8), e12270 (2010).
[CrossRef] [PubMed]

Rev. Sci. Instrum.

M. P. Elenko, J. W. Szostak, and A. M. van Oijen, “Single-molecule binding experiments on long time scales,” Rev. Sci. Instrum. 81(8), 083705 (2010).
[CrossRef] [PubMed]

Science

B. Huang, W. Wang, M. Bates, and X. Zhuang, “Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy,” Science 319(5864), 810–813 (2008).
[CrossRef] [PubMed]

Other

J. Peters, “Nikon Instruments TiE-PFS Dynamic Focusing System,” Nat. Methods | Application Notes (2008).

S. Bae, H. Son, Y.-G. Kim, and S. Hohng, “Z-DNA is stabilized by the Hofmeister effect of salts,” (manuscript in preparation).

Cited By

OSA participates in CrossRef's Cited-By Linking service. Citing articles from OSA journals and other participating publishers are listed here.

Alert me when this article is cited.


Figures (4)

Fig. 1
Fig. 1

A schematic of the autofocusing system. (a) The optical setup. To add the autofocusing capability to a conventional single-molecule FRET microscope [16], we installed a piezoelectric stage (PZT, Physick Instrument, P-721) and a cylindrical lens (CL, Thorlabs, LJ1516L1-A, f = 1000 mm), which was positioned 75 mm from the camera (EM-CCD). The other key elements are as follows. OBJ: an objective lens (Olympus, UPlanSApo 60X), F: a longpass filter (Chroma, LP03-532RU), L2: a plano-convex lens (Melles Griot, 01 LAO 538, f = 120 mm), D: a dichroic mirror (Chroma, 640dcxr), L3: a plano-convex lens (Melles Griot, 01 LAO 638, f = 260.1 mm), M: a dielectric mirror (Thorlabs, BB2-E02), and EM-CCD: an electron multiplying charge coupled device (Andor, iXon DV897). (b) Cy3 images of immobilized DNA1 at different axial positions. The single-molecule images, which are circular in focus (top), become elongated in the x-direction for negative defocus (z = −0.5 µm, bottom left) and in the y-direction for positive defocus (z = + 0.5 µm, bottom right). (c) FOM at varying axial positions. (d) The FOMs in the range −0.2–0.2 μm (solid squares) and their linear fit (red line). The error bars in (c) and (d) were generated from 30 independent measurements.

Fig. 2
Fig. 2

Autofocusing from large defocuses. (a) Representative time traces of the fluorescence intensities (top; Cy3: green line, Cy5: red line), the position of the objective lens (middle), and the displacement of the objective lens for each step of autofocusing (bottom). The orange and black bars on top of the graphs indicate the on and off states of the autofocusing system, respectively. While the autofocusing system was off, the microscope was intentionally defocused downward or upward. (b) The autofocusing time for varying defocusing distances. Four different SNR conditions of Cy3 signal (2.0, 2.9, 4.3 and 6.1) were tested. The error bars were generated from five measurements. (c) The position stability of the objective lens while the image focus was maintained. Four different SNR conditions of Cy3 signal (2.0, 2.9, 4.3 and 6.1) were tested. The error bars indicate the standard error of the mean generated from 4 independent movies. The experiments were performed with a 0.1-s exposure time.

Fig. 3
Fig. 3

Focus maintenance. (a) Representative time traces of single molecule fluorescence intensities (top) and the SNR of the Cy3 signal (bottom). The experiment was performed with the autofocusing system turned off. (b) Representative time traces of the single-molecule fluorescence intensities (top panel), the SNR of the Cy3 signal (2nd panel), the FOM (3rd panel), the number of remaining Cy3 molecules (4th panel), and the position of the objective lens (bottom). The experiment was performed with the autofocusing system activated. The SNR was obtained by analyzing the nearest 50 frames of the Cy3 images (n = 10). The experiments were performed with a 3-s exposure time.

Fig. 4
Fig. 4

The real-time observation of B-Z transition kinetics at high-salt conditions. (a) The sequences of the DNA construct (B(CG)6B). (b) The experimental scheme. The interdye distance increases in the Z-form, resulting in decreased FRET. (c) Representative time traces of the fluorescence intensities (top) and the corresponding FRET (gray, bottom). The most probable FRET time trace generated via hidden Markov modeling (HMM) [23] is overlaid (blue, bottom). (d) FRET histograms showing Z-DNA formation at 4.0 M NaClO4. The histogram is fitted to two Gaussian functions. The low-FRET state (red) corresponds to Z-DNA, and the high-FRET state (green) corresponds to B-DNA. (e) The transition density plot at 4.0 M NaClO4 obtained from hidden Markov modeling [24]. (f) The salt dependence of the B-Z transition rates. The experiments were performed with a 3-s exposure time.

Equations (1)

Equations on this page are rendered with MathJax. Learn more.

FOM= i,j [I(i,j)I(i+2,j)] 2 i,j I(i,j) 2   i,j [I(i,j)I(i,j+2)] 2 i,j I(i,j) 2  

Metrics