Abstract

This paper discusses the application of objective-type total internal reflection fluorescence correlation spectroscopy (TIR-FCS) to the study of the kinetics of immobilized horseradish peroxidase on a single molecule level. Objective-type TIR-FCS combines the advantages of FCS with TIRF microscopy in a way that allows for simultaneous ultra-sensitive spectroscopic measurements using a single-point detector and convenient localization of single molecules on a surface by means of parallel imaging.

© 2007 Optical Society of America

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  1. E. B. Shera, N. K. Seitzinger, L. M. Davis, R. A. Keller, and S. A. Soper "Detection of single fluorescent molecules," Chem. Phys. Lett. 174,553-557 (1990).
    [CrossRef]
  2. R. Rigler and J. Widengren, "Ultrasensitive detection of single molecules by fluorescence correlation spectroscopy," in BioScience, B. Klinge and C. Owman, eds., (Lund University Press, Lund, Sweden, 1990), pp. 180- 183.
  3. R. Rigler, U. Mets, J. Widengren, and P. Kask, "Fluorescence Correlation Spectroscopy with High Count Rate and Low Background - Analysis of Translational Diffusion," Eur. Biophys. J. Biophys. Lett. 22,169-175 (1993).
    [CrossRef]
  4. M. Eigen and R. Rigler, "Sorting single molecules - application to diagnostics and evolutionary biotechnology," Proc. Natl. Acad. Sci. USA 91,5740-5747 (1994).
    [CrossRef] [PubMed]
  5. S. Weiss, "Fluorescence spectroscopy of single biomolecules," Science 283,1676-1683 (1999).
    [CrossRef] [PubMed]
  6. W. P. Ambrose, P. M. Goodwin, J. H. Jett, A. Van Orden, J. H. Werner, and R. A. Keller, "Single molecule fluorescence spectroscopy at ambient temperature," Chem. Rev. 99,2929-2956 (1999).
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  7. J. Enderlein, R. A. Keller, and C. Zander, "Single Molecule Detection in Liquids and on Surfaces under Ambient Conditions: Introduction and Historical Overview," in Single Molecule Detection in Solution, C. Zander, J. Enderlein, and R. A. Keller, eds., (Wiley-VCH, Berlin, 2002), pp 1-19.
  8. T. Funatsu, Y. Harada, M. Tokunaga, K. Saito, and T. Yanagida, "Imaging of Single Fluorescent Molecules and Individual Atp Turnovers by Single Myosin Molecules in Aqueous-Solution," Nature 374,555-559 (1995).
    [CrossRef]
  9. R. D. Vale, T. Funatsu, D. W. Pierce, L. Romberg, Y. Harada, and T. Yanagida, "Direct observation of single kinesin molecules moving along microtubules," Nature 380,451-453 (1996).
    [CrossRef] [PubMed]
  10. H. P. Lu, L. Y. Xun, and X. S. Xie, "Single-molecule enzymatic dynamics," Science 282,1877-1882 (1998).
    [CrossRef]
  11. L. Edman, Z. Földes-Papp, S. Wennmalm, and R. Rigler, "The fluctuating enzyme: a single molecule approach," Chem. Phys. 247,11-22 (1999).
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  13. H. Frauenfelder and B. H. McMahon, "Energy landscape and fluctuations in proteins," Ann. Phys. 9,655-667 (2000).
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  14. K. Velonia, O. Flomenbom, D. Loos, S. Masuo, M. Cotlet, Y. Engelborghs, J. Hofkens, A. E. Rowan, J. Klafter, R. J. M. Nolte, and F. C. de Schryver, "Single-enzyme kinetics of CALB-catalyzed hydrolysis," Angew. Chem. Int. Ed. 44,560-564 (2005).
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  15. O. Flomenbom, K. Velonia, D. Loos, S. Masuo, M. Cotlet, Y. Engelborghs, J. Hofkens, A. E. Rowan, R. J. M. Nolte, M. Van der Auweraer, F. C. de Schryver, and J. Klafter, "Stretched exponential decay and correlations in the catalytic activity of fluctuating single lipase molecules," Proc. Natl. Acad. Sci. USA 102,2368-2372 (2005).
    [CrossRef] [PubMed]
  16. B. P. English,W. Min, A. M. van Oijen, K. T. Lee, G. B. Luo, H. Y. Sun, B. J. Cherayil, S. C. Kou, and X. S. Xie, "Ever-fluctuating single enzyme molecules: Michaelis-Menten equation revisited," Nat. Chem. Biol. 2,87-94 (2006).
    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
  18. D. Magde, W. W. Webb, and E. Elson, "Thermodynamic fluctuations in a reacting system - Measurement by Fluorescence Correlation Spectroscopy," Phys. Rev. Lett. 29,705-708 (1972).
    [CrossRef]
  19. R. Rigler and E. L. Elson, Fluorescence Correlation Spectroscopy: Theory and Applications (Springer, Berlin, 2001).
    [CrossRef]
  20. K. Hassler, T. Anhut, R. Rigler, M. Gösch, and T. Lasser, "High count rates with total internal reflection fluorescence correlation spectroscopy," Biophys. J. 88,L1-L3 (2005).
    [CrossRef]
  21. K. Hassler, M. Leutenegger, P. Rigler, R. Rao, R. Rigler, M. G¨osch and T. Lasser, "Total internal reflection fluorescence correlation spectroscopy (TIR-FCS) with low background and high count-rate per molecule," Opt. Express 13,7415-7423 (2005).
    [CrossRef] [PubMed]
  22. D. Axelrod, T. P. Burghardt, and N. L. Thompson, "Total Internal-Reflection Fluorescence," Ann. Rev. Biophys. Bioeng. 13,247-268 (1984).
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  24. D. Axelrod, "Total internal reflection fluorescence microscopy in cell biology," Traffic 2,764-774 (2001).
    [CrossRef] [PubMed]
  25. L. Banci, "Structural properties of peroxidases," J. Biotechnol. 53,253-263 (1997).
    [CrossRef] [PubMed]
  26. M. J. Swann, L. L. Peel, S. Carrington, and N. J. Freeman, "Dual-polarization interferometry: an analytical technique to measure changes in protein structure in real time, to determine the stoichiometry of binding events, and to differentiate between specific and nonspecific interactions," Anal. Biochem. 329,190-198 (2004).
    [CrossRef] [PubMed]
  27. N. P. Huang, J. Voros, S. M. De Paul, M. Textor, and N. D. Spencer, "Biotin-derivatized poly(L-lysine)-gpoly (ethylene glycol): A novel polymeric interface for bioaffinity sensing," Langmuir 18,220-230 (2002).
    [CrossRef]
  28. L. A. Ruiz-Taylor, T. L. Martin, and P. Wagner, "X-ray photoelectron spectroscopy and radiometry studies of biotin-derivatized poly(L-lysine)-grafted-poly(ethylene glycol) monolayers on metal oxides," Langmuir 17,7313-7322 (2001).
    [CrossRef]
  29. J. Widengren, Ü. Mets, and R. Rigler, "Fluorescence Correlation Spectroscopy of Triplet States in Solution: A Theoretical and Experimental Study," J. Phys. Chem. 99,13368-13379 (1995).
    [CrossRef]
  30. T. E. Starr and N. L. Thompson, "Total internal reflection with fluorescence correlation spectroscopy: Combined surface reaction and solution diffusion," Biophys. J. 80,1575-1584 (2001).
    [CrossRef] [PubMed]
  31. K. Hassler, "Single molecule detection and fluorescence correlation spectroscopy on surfaces," Ph.D. Dissertation, École Polytechnique Fèdèrale de Lausanne, Lausanne, Switzerland (2005).

2006

B. P. English,W. Min, A. M. van Oijen, K. T. Lee, G. B. Luo, H. Y. Sun, B. J. Cherayil, S. C. Kou, and X. S. Xie, "Ever-fluctuating single enzyme molecules: Michaelis-Menten equation revisited," Nat. Chem. Biol. 2,87-94 (2006).
[CrossRef] [PubMed]

2005

K. Velonia, O. Flomenbom, D. Loos, S. Masuo, M. Cotlet, Y. Engelborghs, J. Hofkens, A. E. Rowan, J. Klafter, R. J. M. Nolte, and F. C. de Schryver, "Single-enzyme kinetics of CALB-catalyzed hydrolysis," Angew. Chem. Int. Ed. 44,560-564 (2005).
[CrossRef]

O. Flomenbom, K. Velonia, D. Loos, S. Masuo, M. Cotlet, Y. Engelborghs, J. Hofkens, A. E. Rowan, R. J. M. Nolte, M. Van der Auweraer, F. C. de Schryver, and J. Klafter, "Stretched exponential decay and correlations in the catalytic activity of fluctuating single lipase molecules," Proc. Natl. Acad. Sci. USA 102,2368-2372 (2005).
[CrossRef] [PubMed]

K. Hassler, T. Anhut, R. Rigler, M. Gösch, and T. Lasser, "High count rates with total internal reflection fluorescence correlation spectroscopy," Biophys. J. 88,L1-L3 (2005).
[CrossRef]

K. Hassler, M. Leutenegger, P. Rigler, R. Rao, R. Rigler, M. G¨osch and T. Lasser, "Total internal reflection fluorescence correlation spectroscopy (TIR-FCS) with low background and high count-rate per molecule," Opt. Express 13,7415-7423 (2005).
[CrossRef] [PubMed]

2004

M. J. Swann, L. L. Peel, S. Carrington, and N. J. Freeman, "Dual-polarization interferometry: an analytical technique to measure changes in protein structure in real time, to determine the stoichiometry of binding events, and to differentiate between specific and nonspecific interactions," Anal. Biochem. 329,190-198 (2004).
[CrossRef] [PubMed]

2002

N. P. Huang, J. Voros, S. M. De Paul, M. Textor, and N. D. Spencer, "Biotin-derivatized poly(L-lysine)-gpoly (ethylene glycol): A novel polymeric interface for bioaffinity sensing," Langmuir 18,220-230 (2002).
[CrossRef]

2001

L. A. Ruiz-Taylor, T. L. Martin, and P. Wagner, "X-ray photoelectron spectroscopy and radiometry studies of biotin-derivatized poly(L-lysine)-grafted-poly(ethylene glycol) monolayers on metal oxides," Langmuir 17,7313-7322 (2001).
[CrossRef]

T. E. Starr and N. L. Thompson, "Total internal reflection with fluorescence correlation spectroscopy: Combined surface reaction and solution diffusion," Biophys. J. 80,1575-1584 (2001).
[CrossRef] [PubMed]

D. Axelrod, "Total internal reflection fluorescence microscopy in cell biology," Traffic 2,764-774 (2001).
[CrossRef] [PubMed]

2000

H. Frauenfelder and B. H. McMahon, "Energy landscape and fluctuations in proteins," Ann. Phys. 9,655-667 (2000).
[CrossRef]

1999

S. Weiss, "Fluorescence spectroscopy of single biomolecules," Science 283,1676-1683 (1999).
[CrossRef] [PubMed]

W. P. Ambrose, P. M. Goodwin, J. H. Jett, A. Van Orden, J. H. Werner, and R. A. Keller, "Single molecule fluorescence spectroscopy at ambient temperature," Chem. Rev. 99,2929-2956 (1999).
[CrossRef]

L. Edman, Z. Földes-Papp, S. Wennmalm, and R. Rigler, "The fluctuating enzyme: a single molecule approach," Chem. Phys. 247,11-22 (1999).
[CrossRef]

1998

H. P. Lu, L. Y. Xun, and X. S. Xie, "Single-molecule enzymatic dynamics," Science 282,1877-1882 (1998).
[CrossRef]

1997

L. Banci, "Structural properties of peroxidases," J. Biotechnol. 53,253-263 (1997).
[CrossRef] [PubMed]

1996

R. D. Vale, T. Funatsu, D. W. Pierce, L. Romberg, Y. Harada, and T. Yanagida, "Direct observation of single kinesin molecules moving along microtubules," Nature 380,451-453 (1996).
[CrossRef] [PubMed]

1995

T. Funatsu, Y. Harada, M. Tokunaga, K. Saito, and T. Yanagida, "Imaging of Single Fluorescent Molecules and Individual Atp Turnovers by Single Myosin Molecules in Aqueous-Solution," Nature 374,555-559 (1995).
[CrossRef]

J. Widengren, Ü. Mets, and R. Rigler, "Fluorescence Correlation Spectroscopy of Triplet States in Solution: A Theoretical and Experimental Study," J. Phys. Chem. 99,13368-13379 (1995).
[CrossRef]

1994

M. Eigen and R. Rigler, "Sorting single molecules - application to diagnostics and evolutionary biotechnology," Proc. Natl. Acad. Sci. USA 91,5740-5747 (1994).
[CrossRef] [PubMed]

1993

R. Rigler, U. Mets, J. Widengren, and P. Kask, "Fluorescence Correlation Spectroscopy with High Count Rate and Low Background - Analysis of Translational Diffusion," Eur. Biophys. J. Biophys. Lett. 22,169-175 (1993).
[CrossRef]

1991

H. Frauenfelder, S. G. Sligar, and P. G. Wolynes, "The Energy Landscapes and Motions of Proteins," Science 254,1598-1603 (1991).
[CrossRef] [PubMed]

1990

E. B. Shera, N. K. Seitzinger, L. M. Davis, R. A. Keller, and S. A. Soper "Detection of single fluorescent molecules," Chem. Phys. Lett. 174,553-557 (1990).
[CrossRef]

1984

D. Axelrod, T. P. Burghardt, and N. L. Thompson, "Total Internal-Reflection Fluorescence," Ann. Rev. Biophys. Bioeng. 13,247-268 (1984).
[CrossRef]

1972

D. Magde, W. W. Webb, and E. Elson, "Thermodynamic fluctuations in a reacting system - Measurement by Fluorescence Correlation Spectroscopy," Phys. Rev. Lett. 29,705-708 (1972).
[CrossRef]

Ambrose, W. P.

W. P. Ambrose, P. M. Goodwin, J. H. Jett, A. Van Orden, J. H. Werner, and R. A. Keller, "Single molecule fluorescence spectroscopy at ambient temperature," Chem. Rev. 99,2929-2956 (1999).
[CrossRef]

Anhut, T.

K. Hassler, T. Anhut, R. Rigler, M. Gösch, and T. Lasser, "High count rates with total internal reflection fluorescence correlation spectroscopy," Biophys. J. 88,L1-L3 (2005).
[CrossRef]

Axelrod, D.

D. Axelrod, "Total internal reflection fluorescence microscopy in cell biology," Traffic 2,764-774 (2001).
[CrossRef] [PubMed]

D. Axelrod, T. P. Burghardt, and N. L. Thompson, "Total Internal-Reflection Fluorescence," Ann. Rev. Biophys. Bioeng. 13,247-268 (1984).
[CrossRef]

Banci, L.

L. Banci, "Structural properties of peroxidases," J. Biotechnol. 53,253-263 (1997).
[CrossRef] [PubMed]

Burghardt, T. P.

D. Axelrod, T. P. Burghardt, and N. L. Thompson, "Total Internal-Reflection Fluorescence," Ann. Rev. Biophys. Bioeng. 13,247-268 (1984).
[CrossRef]

Carrington, S.

M. J. Swann, L. L. Peel, S. Carrington, and N. J. Freeman, "Dual-polarization interferometry: an analytical technique to measure changes in protein structure in real time, to determine the stoichiometry of binding events, and to differentiate between specific and nonspecific interactions," Anal. Biochem. 329,190-198 (2004).
[CrossRef] [PubMed]

Cherayil, B. J.

B. P. English,W. Min, A. M. van Oijen, K. T. Lee, G. B. Luo, H. Y. Sun, B. J. Cherayil, S. C. Kou, and X. S. Xie, "Ever-fluctuating single enzyme molecules: Michaelis-Menten equation revisited," Nat. Chem. Biol. 2,87-94 (2006).
[CrossRef] [PubMed]

Cotlet, M.

K. Velonia, O. Flomenbom, D. Loos, S. Masuo, M. Cotlet, Y. Engelborghs, J. Hofkens, A. E. Rowan, J. Klafter, R. J. M. Nolte, and F. C. de Schryver, "Single-enzyme kinetics of CALB-catalyzed hydrolysis," Angew. Chem. Int. Ed. 44,560-564 (2005).
[CrossRef]

O. Flomenbom, K. Velonia, D. Loos, S. Masuo, M. Cotlet, Y. Engelborghs, J. Hofkens, A. E. Rowan, R. J. M. Nolte, M. Van der Auweraer, F. C. de Schryver, and J. Klafter, "Stretched exponential decay and correlations in the catalytic activity of fluctuating single lipase molecules," Proc. Natl. Acad. Sci. USA 102,2368-2372 (2005).
[CrossRef] [PubMed]

Davis, L. M.

E. B. Shera, N. K. Seitzinger, L. M. Davis, R. A. Keller, and S. A. Soper "Detection of single fluorescent molecules," Chem. Phys. Lett. 174,553-557 (1990).
[CrossRef]

De Paul, S. M.

N. P. Huang, J. Voros, S. M. De Paul, M. Textor, and N. D. Spencer, "Biotin-derivatized poly(L-lysine)-gpoly (ethylene glycol): A novel polymeric interface for bioaffinity sensing," Langmuir 18,220-230 (2002).
[CrossRef]

de Schryver, F. C.

O. Flomenbom, K. Velonia, D. Loos, S. Masuo, M. Cotlet, Y. Engelborghs, J. Hofkens, A. E. Rowan, R. J. M. Nolte, M. Van der Auweraer, F. C. de Schryver, and J. Klafter, "Stretched exponential decay and correlations in the catalytic activity of fluctuating single lipase molecules," Proc. Natl. Acad. Sci. USA 102,2368-2372 (2005).
[CrossRef] [PubMed]

K. Velonia, O. Flomenbom, D. Loos, S. Masuo, M. Cotlet, Y. Engelborghs, J. Hofkens, A. E. Rowan, J. Klafter, R. J. M. Nolte, and F. C. de Schryver, "Single-enzyme kinetics of CALB-catalyzed hydrolysis," Angew. Chem. Int. Ed. 44,560-564 (2005).
[CrossRef]

Edman, L.

L. Edman, Z. Földes-Papp, S. Wennmalm, and R. Rigler, "The fluctuating enzyme: a single molecule approach," Chem. Phys. 247,11-22 (1999).
[CrossRef]

Eigen, M.

M. Eigen and R. Rigler, "Sorting single molecules - application to diagnostics and evolutionary biotechnology," Proc. Natl. Acad. Sci. USA 91,5740-5747 (1994).
[CrossRef] [PubMed]

Elson, E.

D. Magde, W. W. Webb, and E. Elson, "Thermodynamic fluctuations in a reacting system - Measurement by Fluorescence Correlation Spectroscopy," Phys. Rev. Lett. 29,705-708 (1972).
[CrossRef]

Engelborghs, Y.

O. Flomenbom, K. Velonia, D. Loos, S. Masuo, M. Cotlet, Y. Engelborghs, J. Hofkens, A. E. Rowan, R. J. M. Nolte, M. Van der Auweraer, F. C. de Schryver, and J. Klafter, "Stretched exponential decay and correlations in the catalytic activity of fluctuating single lipase molecules," Proc. Natl. Acad. Sci. USA 102,2368-2372 (2005).
[CrossRef] [PubMed]

K. Velonia, O. Flomenbom, D. Loos, S. Masuo, M. Cotlet, Y. Engelborghs, J. Hofkens, A. E. Rowan, J. Klafter, R. J. M. Nolte, and F. C. de Schryver, "Single-enzyme kinetics of CALB-catalyzed hydrolysis," Angew. Chem. Int. Ed. 44,560-564 (2005).
[CrossRef]

English, B. P.

B. P. English,W. Min, A. M. van Oijen, K. T. Lee, G. B. Luo, H. Y. Sun, B. J. Cherayil, S. C. Kou, and X. S. Xie, "Ever-fluctuating single enzyme molecules: Michaelis-Menten equation revisited," Nat. Chem. Biol. 2,87-94 (2006).
[CrossRef] [PubMed]

Flomenbom, O.

K. Velonia, O. Flomenbom, D. Loos, S. Masuo, M. Cotlet, Y. Engelborghs, J. Hofkens, A. E. Rowan, J. Klafter, R. J. M. Nolte, and F. C. de Schryver, "Single-enzyme kinetics of CALB-catalyzed hydrolysis," Angew. Chem. Int. Ed. 44,560-564 (2005).
[CrossRef]

O. Flomenbom, K. Velonia, D. Loos, S. Masuo, M. Cotlet, Y. Engelborghs, J. Hofkens, A. E. Rowan, R. J. M. Nolte, M. Van der Auweraer, F. C. de Schryver, and J. Klafter, "Stretched exponential decay and correlations in the catalytic activity of fluctuating single lipase molecules," Proc. Natl. Acad. Sci. USA 102,2368-2372 (2005).
[CrossRef] [PubMed]

Földes-Papp, Z.

L. Edman, Z. Földes-Papp, S. Wennmalm, and R. Rigler, "The fluctuating enzyme: a single molecule approach," Chem. Phys. 247,11-22 (1999).
[CrossRef]

Frauenfelder, H.

H. Frauenfelder and B. H. McMahon, "Energy landscape and fluctuations in proteins," Ann. Phys. 9,655-667 (2000).
[CrossRef]

H. Frauenfelder, S. G. Sligar, and P. G. Wolynes, "The Energy Landscapes and Motions of Proteins," Science 254,1598-1603 (1991).
[CrossRef] [PubMed]

Freeman, N. J.

M. J. Swann, L. L. Peel, S. Carrington, and N. J. Freeman, "Dual-polarization interferometry: an analytical technique to measure changes in protein structure in real time, to determine the stoichiometry of binding events, and to differentiate between specific and nonspecific interactions," Anal. Biochem. 329,190-198 (2004).
[CrossRef] [PubMed]

Funatsu, T.

R. D. Vale, T. Funatsu, D. W. Pierce, L. Romberg, Y. Harada, and T. Yanagida, "Direct observation of single kinesin molecules moving along microtubules," Nature 380,451-453 (1996).
[CrossRef] [PubMed]

T. Funatsu, Y. Harada, M. Tokunaga, K. Saito, and T. Yanagida, "Imaging of Single Fluorescent Molecules and Individual Atp Turnovers by Single Myosin Molecules in Aqueous-Solution," Nature 374,555-559 (1995).
[CrossRef]

Goodwin, P. M.

W. P. Ambrose, P. M. Goodwin, J. H. Jett, A. Van Orden, J. H. Werner, and R. A. Keller, "Single molecule fluorescence spectroscopy at ambient temperature," Chem. Rev. 99,2929-2956 (1999).
[CrossRef]

Gösch, M.

K. Hassler, T. Anhut, R. Rigler, M. Gösch, and T. Lasser, "High count rates with total internal reflection fluorescence correlation spectroscopy," Biophys. J. 88,L1-L3 (2005).
[CrossRef]

Harada, Y.

R. D. Vale, T. Funatsu, D. W. Pierce, L. Romberg, Y. Harada, and T. Yanagida, "Direct observation of single kinesin molecules moving along microtubules," Nature 380,451-453 (1996).
[CrossRef] [PubMed]

T. Funatsu, Y. Harada, M. Tokunaga, K. Saito, and T. Yanagida, "Imaging of Single Fluorescent Molecules and Individual Atp Turnovers by Single Myosin Molecules in Aqueous-Solution," Nature 374,555-559 (1995).
[CrossRef]

Hassler, K.

Hofkens, J.

K. Velonia, O. Flomenbom, D. Loos, S. Masuo, M. Cotlet, Y. Engelborghs, J. Hofkens, A. E. Rowan, J. Klafter, R. J. M. Nolte, and F. C. de Schryver, "Single-enzyme kinetics of CALB-catalyzed hydrolysis," Angew. Chem. Int. Ed. 44,560-564 (2005).
[CrossRef]

O. Flomenbom, K. Velonia, D. Loos, S. Masuo, M. Cotlet, Y. Engelborghs, J. Hofkens, A. E. Rowan, R. J. M. Nolte, M. Van der Auweraer, F. C. de Schryver, and J. Klafter, "Stretched exponential decay and correlations in the catalytic activity of fluctuating single lipase molecules," Proc. Natl. Acad. Sci. USA 102,2368-2372 (2005).
[CrossRef] [PubMed]

Huang, N. P.

N. P. Huang, J. Voros, S. M. De Paul, M. Textor, and N. D. Spencer, "Biotin-derivatized poly(L-lysine)-gpoly (ethylene glycol): A novel polymeric interface for bioaffinity sensing," Langmuir 18,220-230 (2002).
[CrossRef]

Jett, J. H.

W. P. Ambrose, P. M. Goodwin, J. H. Jett, A. Van Orden, J. H. Werner, and R. A. Keller, "Single molecule fluorescence spectroscopy at ambient temperature," Chem. Rev. 99,2929-2956 (1999).
[CrossRef]

Kask, P.

R. Rigler, U. Mets, J. Widengren, and P. Kask, "Fluorescence Correlation Spectroscopy with High Count Rate and Low Background - Analysis of Translational Diffusion," Eur. Biophys. J. Biophys. Lett. 22,169-175 (1993).
[CrossRef]

Keller, R. A.

W. P. Ambrose, P. M. Goodwin, J. H. Jett, A. Van Orden, J. H. Werner, and R. A. Keller, "Single molecule fluorescence spectroscopy at ambient temperature," Chem. Rev. 99,2929-2956 (1999).
[CrossRef]

E. B. Shera, N. K. Seitzinger, L. M. Davis, R. A. Keller, and S. A. Soper "Detection of single fluorescent molecules," Chem. Phys. Lett. 174,553-557 (1990).
[CrossRef]

Klafter, J.

O. Flomenbom, K. Velonia, D. Loos, S. Masuo, M. Cotlet, Y. Engelborghs, J. Hofkens, A. E. Rowan, R. J. M. Nolte, M. Van der Auweraer, F. C. de Schryver, and J. Klafter, "Stretched exponential decay and correlations in the catalytic activity of fluctuating single lipase molecules," Proc. Natl. Acad. Sci. USA 102,2368-2372 (2005).
[CrossRef] [PubMed]

K. Velonia, O. Flomenbom, D. Loos, S. Masuo, M. Cotlet, Y. Engelborghs, J. Hofkens, A. E. Rowan, J. Klafter, R. J. M. Nolte, and F. C. de Schryver, "Single-enzyme kinetics of CALB-catalyzed hydrolysis," Angew. Chem. Int. Ed. 44,560-564 (2005).
[CrossRef]

Kou, S. C.

B. P. English,W. Min, A. M. van Oijen, K. T. Lee, G. B. Luo, H. Y. Sun, B. J. Cherayil, S. C. Kou, and X. S. Xie, "Ever-fluctuating single enzyme molecules: Michaelis-Menten equation revisited," Nat. Chem. Biol. 2,87-94 (2006).
[CrossRef] [PubMed]

Lasser, T.

K. Hassler, T. Anhut, R. Rigler, M. Gösch, and T. Lasser, "High count rates with total internal reflection fluorescence correlation spectroscopy," Biophys. J. 88,L1-L3 (2005).
[CrossRef]

Lee, K. T.

B. P. English,W. Min, A. M. van Oijen, K. T. Lee, G. B. Luo, H. Y. Sun, B. J. Cherayil, S. C. Kou, and X. S. Xie, "Ever-fluctuating single enzyme molecules: Michaelis-Menten equation revisited," Nat. Chem. Biol. 2,87-94 (2006).
[CrossRef] [PubMed]

Leutenegger, M.

Loos, D.

K. Velonia, O. Flomenbom, D. Loos, S. Masuo, M. Cotlet, Y. Engelborghs, J. Hofkens, A. E. Rowan, J. Klafter, R. J. M. Nolte, and F. C. de Schryver, "Single-enzyme kinetics of CALB-catalyzed hydrolysis," Angew. Chem. Int. Ed. 44,560-564 (2005).
[CrossRef]

O. Flomenbom, K. Velonia, D. Loos, S. Masuo, M. Cotlet, Y. Engelborghs, J. Hofkens, A. E. Rowan, R. J. M. Nolte, M. Van der Auweraer, F. C. de Schryver, and J. Klafter, "Stretched exponential decay and correlations in the catalytic activity of fluctuating single lipase molecules," Proc. Natl. Acad. Sci. USA 102,2368-2372 (2005).
[CrossRef] [PubMed]

Lu, H. P.

H. P. Lu, L. Y. Xun, and X. S. Xie, "Single-molecule enzymatic dynamics," Science 282,1877-1882 (1998).
[CrossRef]

Luo, G. B.

B. P. English,W. Min, A. M. van Oijen, K. T. Lee, G. B. Luo, H. Y. Sun, B. J. Cherayil, S. C. Kou, and X. S. Xie, "Ever-fluctuating single enzyme molecules: Michaelis-Menten equation revisited," Nat. Chem. Biol. 2,87-94 (2006).
[CrossRef] [PubMed]

Magde, D.

D. Magde, W. W. Webb, and E. Elson, "Thermodynamic fluctuations in a reacting system - Measurement by Fluorescence Correlation Spectroscopy," Phys. Rev. Lett. 29,705-708 (1972).
[CrossRef]

Martin, T. L.

L. A. Ruiz-Taylor, T. L. Martin, and P. Wagner, "X-ray photoelectron spectroscopy and radiometry studies of biotin-derivatized poly(L-lysine)-grafted-poly(ethylene glycol) monolayers on metal oxides," Langmuir 17,7313-7322 (2001).
[CrossRef]

Masuo, S.

K. Velonia, O. Flomenbom, D. Loos, S. Masuo, M. Cotlet, Y. Engelborghs, J. Hofkens, A. E. Rowan, J. Klafter, R. J. M. Nolte, and F. C. de Schryver, "Single-enzyme kinetics of CALB-catalyzed hydrolysis," Angew. Chem. Int. Ed. 44,560-564 (2005).
[CrossRef]

O. Flomenbom, K. Velonia, D. Loos, S. Masuo, M. Cotlet, Y. Engelborghs, J. Hofkens, A. E. Rowan, R. J. M. Nolte, M. Van der Auweraer, F. C. de Schryver, and J. Klafter, "Stretched exponential decay and correlations in the catalytic activity of fluctuating single lipase molecules," Proc. Natl. Acad. Sci. USA 102,2368-2372 (2005).
[CrossRef] [PubMed]

McMahon, B. H.

H. Frauenfelder and B. H. McMahon, "Energy landscape and fluctuations in proteins," Ann. Phys. 9,655-667 (2000).
[CrossRef]

Mets, U.

R. Rigler, U. Mets, J. Widengren, and P. Kask, "Fluorescence Correlation Spectroscopy with High Count Rate and Low Background - Analysis of Translational Diffusion," Eur. Biophys. J. Biophys. Lett. 22,169-175 (1993).
[CrossRef]

Mets, Ü.

J. Widengren, Ü. Mets, and R. Rigler, "Fluorescence Correlation Spectroscopy of Triplet States in Solution: A Theoretical and Experimental Study," J. Phys. Chem. 99,13368-13379 (1995).
[CrossRef]

Min, W.

B. P. English,W. Min, A. M. van Oijen, K. T. Lee, G. B. Luo, H. Y. Sun, B. J. Cherayil, S. C. Kou, and X. S. Xie, "Ever-fluctuating single enzyme molecules: Michaelis-Menten equation revisited," Nat. Chem. Biol. 2,87-94 (2006).
[CrossRef] [PubMed]

Nolte, R. J. M.

K. Velonia, O. Flomenbom, D. Loos, S. Masuo, M. Cotlet, Y. Engelborghs, J. Hofkens, A. E. Rowan, J. Klafter, R. J. M. Nolte, and F. C. de Schryver, "Single-enzyme kinetics of CALB-catalyzed hydrolysis," Angew. Chem. Int. Ed. 44,560-564 (2005).
[CrossRef]

O. Flomenbom, K. Velonia, D. Loos, S. Masuo, M. Cotlet, Y. Engelborghs, J. Hofkens, A. E. Rowan, R. J. M. Nolte, M. Van der Auweraer, F. C. de Schryver, and J. Klafter, "Stretched exponential decay and correlations in the catalytic activity of fluctuating single lipase molecules," Proc. Natl. Acad. Sci. USA 102,2368-2372 (2005).
[CrossRef] [PubMed]

Peel, L. L.

M. J. Swann, L. L. Peel, S. Carrington, and N. J. Freeman, "Dual-polarization interferometry: an analytical technique to measure changes in protein structure in real time, to determine the stoichiometry of binding events, and to differentiate between specific and nonspecific interactions," Anal. Biochem. 329,190-198 (2004).
[CrossRef] [PubMed]

Pierce, D. W.

R. D. Vale, T. Funatsu, D. W. Pierce, L. Romberg, Y. Harada, and T. Yanagida, "Direct observation of single kinesin molecules moving along microtubules," Nature 380,451-453 (1996).
[CrossRef] [PubMed]

Rao, R.

Rigler, P.

Rigler, R.

K. Hassler, M. Leutenegger, P. Rigler, R. Rao, R. Rigler, M. G¨osch and T. Lasser, "Total internal reflection fluorescence correlation spectroscopy (TIR-FCS) with low background and high count-rate per molecule," Opt. Express 13,7415-7423 (2005).
[CrossRef] [PubMed]

K. Hassler, T. Anhut, R. Rigler, M. Gösch, and T. Lasser, "High count rates with total internal reflection fluorescence correlation spectroscopy," Biophys. J. 88,L1-L3 (2005).
[CrossRef]

L. Edman, Z. Földes-Papp, S. Wennmalm, and R. Rigler, "The fluctuating enzyme: a single molecule approach," Chem. Phys. 247,11-22 (1999).
[CrossRef]

J. Widengren, Ü. Mets, and R. Rigler, "Fluorescence Correlation Spectroscopy of Triplet States in Solution: A Theoretical and Experimental Study," J. Phys. Chem. 99,13368-13379 (1995).
[CrossRef]

M. Eigen and R. Rigler, "Sorting single molecules - application to diagnostics and evolutionary biotechnology," Proc. Natl. Acad. Sci. USA 91,5740-5747 (1994).
[CrossRef] [PubMed]

R. Rigler, U. Mets, J. Widengren, and P. Kask, "Fluorescence Correlation Spectroscopy with High Count Rate and Low Background - Analysis of Translational Diffusion," Eur. Biophys. J. Biophys. Lett. 22,169-175 (1993).
[CrossRef]

Romberg, L.

R. D. Vale, T. Funatsu, D. W. Pierce, L. Romberg, Y. Harada, and T. Yanagida, "Direct observation of single kinesin molecules moving along microtubules," Nature 380,451-453 (1996).
[CrossRef] [PubMed]

Rowan, A. E.

K. Velonia, O. Flomenbom, D. Loos, S. Masuo, M. Cotlet, Y. Engelborghs, J. Hofkens, A. E. Rowan, J. Klafter, R. J. M. Nolte, and F. C. de Schryver, "Single-enzyme kinetics of CALB-catalyzed hydrolysis," Angew. Chem. Int. Ed. 44,560-564 (2005).
[CrossRef]

O. Flomenbom, K. Velonia, D. Loos, S. Masuo, M. Cotlet, Y. Engelborghs, J. Hofkens, A. E. Rowan, R. J. M. Nolte, M. Van der Auweraer, F. C. de Schryver, and J. Klafter, "Stretched exponential decay and correlations in the catalytic activity of fluctuating single lipase molecules," Proc. Natl. Acad. Sci. USA 102,2368-2372 (2005).
[CrossRef] [PubMed]

Ruiz-Taylor, L. A.

L. A. Ruiz-Taylor, T. L. Martin, and P. Wagner, "X-ray photoelectron spectroscopy and radiometry studies of biotin-derivatized poly(L-lysine)-grafted-poly(ethylene glycol) monolayers on metal oxides," Langmuir 17,7313-7322 (2001).
[CrossRef]

Saito, K.

T. Funatsu, Y. Harada, M. Tokunaga, K. Saito, and T. Yanagida, "Imaging of Single Fluorescent Molecules and Individual Atp Turnovers by Single Myosin Molecules in Aqueous-Solution," Nature 374,555-559 (1995).
[CrossRef]

Seitzinger, N. K.

E. B. Shera, N. K. Seitzinger, L. M. Davis, R. A. Keller, and S. A. Soper "Detection of single fluorescent molecules," Chem. Phys. Lett. 174,553-557 (1990).
[CrossRef]

Shera, E. B.

E. B. Shera, N. K. Seitzinger, L. M. Davis, R. A. Keller, and S. A. Soper "Detection of single fluorescent molecules," Chem. Phys. Lett. 174,553-557 (1990).
[CrossRef]

Sligar, S. G.

H. Frauenfelder, S. G. Sligar, and P. G. Wolynes, "The Energy Landscapes and Motions of Proteins," Science 254,1598-1603 (1991).
[CrossRef] [PubMed]

Soper, S. A.

E. B. Shera, N. K. Seitzinger, L. M. Davis, R. A. Keller, and S. A. Soper "Detection of single fluorescent molecules," Chem. Phys. Lett. 174,553-557 (1990).
[CrossRef]

Spencer, N. D.

N. P. Huang, J. Voros, S. M. De Paul, M. Textor, and N. D. Spencer, "Biotin-derivatized poly(L-lysine)-gpoly (ethylene glycol): A novel polymeric interface for bioaffinity sensing," Langmuir 18,220-230 (2002).
[CrossRef]

Starr, T. E.

T. E. Starr and N. L. Thompson, "Total internal reflection with fluorescence correlation spectroscopy: Combined surface reaction and solution diffusion," Biophys. J. 80,1575-1584 (2001).
[CrossRef] [PubMed]

Sun, H. Y.

B. P. English,W. Min, A. M. van Oijen, K. T. Lee, G. B. Luo, H. Y. Sun, B. J. Cherayil, S. C. Kou, and X. S. Xie, "Ever-fluctuating single enzyme molecules: Michaelis-Menten equation revisited," Nat. Chem. Biol. 2,87-94 (2006).
[CrossRef] [PubMed]

Swann, M. J.

M. J. Swann, L. L. Peel, S. Carrington, and N. J. Freeman, "Dual-polarization interferometry: an analytical technique to measure changes in protein structure in real time, to determine the stoichiometry of binding events, and to differentiate between specific and nonspecific interactions," Anal. Biochem. 329,190-198 (2004).
[CrossRef] [PubMed]

Textor, M.

N. P. Huang, J. Voros, S. M. De Paul, M. Textor, and N. D. Spencer, "Biotin-derivatized poly(L-lysine)-gpoly (ethylene glycol): A novel polymeric interface for bioaffinity sensing," Langmuir 18,220-230 (2002).
[CrossRef]

Thompson, N. L.

T. E. Starr and N. L. Thompson, "Total internal reflection with fluorescence correlation spectroscopy: Combined surface reaction and solution diffusion," Biophys. J. 80,1575-1584 (2001).
[CrossRef] [PubMed]

D. Axelrod, T. P. Burghardt, and N. L. Thompson, "Total Internal-Reflection Fluorescence," Ann. Rev. Biophys. Bioeng. 13,247-268 (1984).
[CrossRef]

Tokunaga, M.

T. Funatsu, Y. Harada, M. Tokunaga, K. Saito, and T. Yanagida, "Imaging of Single Fluorescent Molecules and Individual Atp Turnovers by Single Myosin Molecules in Aqueous-Solution," Nature 374,555-559 (1995).
[CrossRef]

Vale, R. D.

R. D. Vale, T. Funatsu, D. W. Pierce, L. Romberg, Y. Harada, and T. Yanagida, "Direct observation of single kinesin molecules moving along microtubules," Nature 380,451-453 (1996).
[CrossRef] [PubMed]

Van der Auweraer, M.

O. Flomenbom, K. Velonia, D. Loos, S. Masuo, M. Cotlet, Y. Engelborghs, J. Hofkens, A. E. Rowan, R. J. M. Nolte, M. Van der Auweraer, F. C. de Schryver, and J. Klafter, "Stretched exponential decay and correlations in the catalytic activity of fluctuating single lipase molecules," Proc. Natl. Acad. Sci. USA 102,2368-2372 (2005).
[CrossRef] [PubMed]

van Oijen, A. M.

B. P. English,W. Min, A. M. van Oijen, K. T. Lee, G. B. Luo, H. Y. Sun, B. J. Cherayil, S. C. Kou, and X. S. Xie, "Ever-fluctuating single enzyme molecules: Michaelis-Menten equation revisited," Nat. Chem. Biol. 2,87-94 (2006).
[CrossRef] [PubMed]

Van Orden, A.

W. P. Ambrose, P. M. Goodwin, J. H. Jett, A. Van Orden, J. H. Werner, and R. A. Keller, "Single molecule fluorescence spectroscopy at ambient temperature," Chem. Rev. 99,2929-2956 (1999).
[CrossRef]

Velonia, K.

O. Flomenbom, K. Velonia, D. Loos, S. Masuo, M. Cotlet, Y. Engelborghs, J. Hofkens, A. E. Rowan, R. J. M. Nolte, M. Van der Auweraer, F. C. de Schryver, and J. Klafter, "Stretched exponential decay and correlations in the catalytic activity of fluctuating single lipase molecules," Proc. Natl. Acad. Sci. USA 102,2368-2372 (2005).
[CrossRef] [PubMed]

K. Velonia, O. Flomenbom, D. Loos, S. Masuo, M. Cotlet, Y. Engelborghs, J. Hofkens, A. E. Rowan, J. Klafter, R. J. M. Nolte, and F. C. de Schryver, "Single-enzyme kinetics of CALB-catalyzed hydrolysis," Angew. Chem. Int. Ed. 44,560-564 (2005).
[CrossRef]

Voros, J.

N. P. Huang, J. Voros, S. M. De Paul, M. Textor, and N. D. Spencer, "Biotin-derivatized poly(L-lysine)-gpoly (ethylene glycol): A novel polymeric interface for bioaffinity sensing," Langmuir 18,220-230 (2002).
[CrossRef]

Wagner, P.

L. A. Ruiz-Taylor, T. L. Martin, and P. Wagner, "X-ray photoelectron spectroscopy and radiometry studies of biotin-derivatized poly(L-lysine)-grafted-poly(ethylene glycol) monolayers on metal oxides," Langmuir 17,7313-7322 (2001).
[CrossRef]

Webb, W. W.

D. Magde, W. W. Webb, and E. Elson, "Thermodynamic fluctuations in a reacting system - Measurement by Fluorescence Correlation Spectroscopy," Phys. Rev. Lett. 29,705-708 (1972).
[CrossRef]

Weiss, S.

S. Weiss, "Fluorescence spectroscopy of single biomolecules," Science 283,1676-1683 (1999).
[CrossRef] [PubMed]

Wennmalm, S.

L. Edman, Z. Földes-Papp, S. Wennmalm, and R. Rigler, "The fluctuating enzyme: a single molecule approach," Chem. Phys. 247,11-22 (1999).
[CrossRef]

Werner, J. H.

W. P. Ambrose, P. M. Goodwin, J. H. Jett, A. Van Orden, J. H. Werner, and R. A. Keller, "Single molecule fluorescence spectroscopy at ambient temperature," Chem. Rev. 99,2929-2956 (1999).
[CrossRef]

Widengren, J.

J. Widengren, Ü. Mets, and R. Rigler, "Fluorescence Correlation Spectroscopy of Triplet States in Solution: A Theoretical and Experimental Study," J. Phys. Chem. 99,13368-13379 (1995).
[CrossRef]

R. Rigler, U. Mets, J. Widengren, and P. Kask, "Fluorescence Correlation Spectroscopy with High Count Rate and Low Background - Analysis of Translational Diffusion," Eur. Biophys. J. Biophys. Lett. 22,169-175 (1993).
[CrossRef]

Wolynes, P. G.

H. Frauenfelder, S. G. Sligar, and P. G. Wolynes, "The Energy Landscapes and Motions of Proteins," Science 254,1598-1603 (1991).
[CrossRef] [PubMed]

Xie, X. S.

B. P. English,W. Min, A. M. van Oijen, K. T. Lee, G. B. Luo, H. Y. Sun, B. J. Cherayil, S. C. Kou, and X. S. Xie, "Ever-fluctuating single enzyme molecules: Michaelis-Menten equation revisited," Nat. Chem. Biol. 2,87-94 (2006).
[CrossRef] [PubMed]

H. P. Lu, L. Y. Xun, and X. S. Xie, "Single-molecule enzymatic dynamics," Science 282,1877-1882 (1998).
[CrossRef]

Xun, L. Y.

H. P. Lu, L. Y. Xun, and X. S. Xie, "Single-molecule enzymatic dynamics," Science 282,1877-1882 (1998).
[CrossRef]

Yanagida, T.

R. D. Vale, T. Funatsu, D. W. Pierce, L. Romberg, Y. Harada, and T. Yanagida, "Direct observation of single kinesin molecules moving along microtubules," Nature 380,451-453 (1996).
[CrossRef] [PubMed]

T. Funatsu, Y. Harada, M. Tokunaga, K. Saito, and T. Yanagida, "Imaging of Single Fluorescent Molecules and Individual Atp Turnovers by Single Myosin Molecules in Aqueous-Solution," Nature 374,555-559 (1995).
[CrossRef]

Anal. Biochem.

M. J. Swann, L. L. Peel, S. Carrington, and N. J. Freeman, "Dual-polarization interferometry: an analytical technique to measure changes in protein structure in real time, to determine the stoichiometry of binding events, and to differentiate between specific and nonspecific interactions," Anal. Biochem. 329,190-198 (2004).
[CrossRef] [PubMed]

Angew. Chem. Int. Ed.

K. Velonia, O. Flomenbom, D. Loos, S. Masuo, M. Cotlet, Y. Engelborghs, J. Hofkens, A. E. Rowan, J. Klafter, R. J. M. Nolte, and F. C. de Schryver, "Single-enzyme kinetics of CALB-catalyzed hydrolysis," Angew. Chem. Int. Ed. 44,560-564 (2005).
[CrossRef]

Ann. Phys.

H. Frauenfelder and B. H. McMahon, "Energy landscape and fluctuations in proteins," Ann. Phys. 9,655-667 (2000).
[CrossRef]

Ann. Rev. Biophys. Bioeng.

D. Axelrod, T. P. Burghardt, and N. L. Thompson, "Total Internal-Reflection Fluorescence," Ann. Rev. Biophys. Bioeng. 13,247-268 (1984).
[CrossRef]

Biophys. J.

K. Hassler, T. Anhut, R. Rigler, M. Gösch, and T. Lasser, "High count rates with total internal reflection fluorescence correlation spectroscopy," Biophys. J. 88,L1-L3 (2005).
[CrossRef]

T. E. Starr and N. L. Thompson, "Total internal reflection with fluorescence correlation spectroscopy: Combined surface reaction and solution diffusion," Biophys. J. 80,1575-1584 (2001).
[CrossRef] [PubMed]

Chem. Phys.

L. Edman, Z. Földes-Papp, S. Wennmalm, and R. Rigler, "The fluctuating enzyme: a single molecule approach," Chem. Phys. 247,11-22 (1999).
[CrossRef]

Chem. Phys. Lett.

E. B. Shera, N. K. Seitzinger, L. M. Davis, R. A. Keller, and S. A. Soper "Detection of single fluorescent molecules," Chem. Phys. Lett. 174,553-557 (1990).
[CrossRef]

Chem. Rev.

W. P. Ambrose, P. M. Goodwin, J. H. Jett, A. Van Orden, J. H. Werner, and R. A. Keller, "Single molecule fluorescence spectroscopy at ambient temperature," Chem. Rev. 99,2929-2956 (1999).
[CrossRef]

Eur. Biophys. J. Biophys. Lett.

R. Rigler, U. Mets, J. Widengren, and P. Kask, "Fluorescence Correlation Spectroscopy with High Count Rate and Low Background - Analysis of Translational Diffusion," Eur. Biophys. J. Biophys. Lett. 22,169-175 (1993).
[CrossRef]

J. Biotechnol.

L. Banci, "Structural properties of peroxidases," J. Biotechnol. 53,253-263 (1997).
[CrossRef] [PubMed]

J. Phys. Chem.

J. Widengren, Ü. Mets, and R. Rigler, "Fluorescence Correlation Spectroscopy of Triplet States in Solution: A Theoretical and Experimental Study," J. Phys. Chem. 99,13368-13379 (1995).
[CrossRef]

Langmuir

N. P. Huang, J. Voros, S. M. De Paul, M. Textor, and N. D. Spencer, "Biotin-derivatized poly(L-lysine)-gpoly (ethylene glycol): A novel polymeric interface for bioaffinity sensing," Langmuir 18,220-230 (2002).
[CrossRef]

L. A. Ruiz-Taylor, T. L. Martin, and P. Wagner, "X-ray photoelectron spectroscopy and radiometry studies of biotin-derivatized poly(L-lysine)-grafted-poly(ethylene glycol) monolayers on metal oxides," Langmuir 17,7313-7322 (2001).
[CrossRef]

Nat. Chem. Biol.

B. P. English,W. Min, A. M. van Oijen, K. T. Lee, G. B. Luo, H. Y. Sun, B. J. Cherayil, S. C. Kou, and X. S. Xie, "Ever-fluctuating single enzyme molecules: Michaelis-Menten equation revisited," Nat. Chem. Biol. 2,87-94 (2006).
[CrossRef] [PubMed]

Nature

T. Funatsu, Y. Harada, M. Tokunaga, K. Saito, and T. Yanagida, "Imaging of Single Fluorescent Molecules and Individual Atp Turnovers by Single Myosin Molecules in Aqueous-Solution," Nature 374,555-559 (1995).
[CrossRef]

R. D. Vale, T. Funatsu, D. W. Pierce, L. Romberg, Y. Harada, and T. Yanagida, "Direct observation of single kinesin molecules moving along microtubules," Nature 380,451-453 (1996).
[CrossRef] [PubMed]

Opt. Express

Phys. Rev. Lett.

D. Magde, W. W. Webb, and E. Elson, "Thermodynamic fluctuations in a reacting system - Measurement by Fluorescence Correlation Spectroscopy," Phys. Rev. Lett. 29,705-708 (1972).
[CrossRef]

Proc. Natl. Acad. Sci. USA

O. Flomenbom, K. Velonia, D. Loos, S. Masuo, M. Cotlet, Y. Engelborghs, J. Hofkens, A. E. Rowan, R. J. M. Nolte, M. Van der Auweraer, F. C. de Schryver, and J. Klafter, "Stretched exponential decay and correlations in the catalytic activity of fluctuating single lipase molecules," Proc. Natl. Acad. Sci. USA 102,2368-2372 (2005).
[CrossRef] [PubMed]

M. Eigen and R. Rigler, "Sorting single molecules - application to diagnostics and evolutionary biotechnology," Proc. Natl. Acad. Sci. USA 91,5740-5747 (1994).
[CrossRef] [PubMed]

Science

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Figures (5)

Fig. 1:
Fig. 1:

Detailed scheme of the objective-type TIR-FCS setup. EXF: excitation filter; L1 - L4: lenses; GS: tiltable glass slab; BFP: back focal plane of objective; OL: objective lens; DM: dichroic mirror; EMF: emission filter; M: mirror; TL: tube lens; IP1, IP2: image planes; BS: beam splitter; WLS: white-light source.

Fig. 2:
Fig. 2:

Above: simple scheme for the catalytic cycle of horseradish peroxidase. Right: a more complete description proposed by L. Edman et al. [11] taking into account different conformational states of the enzyme and their supposed influence on its catalytic activity. S: substrate; P: product; Ei+ j: enzyme with i = 3, 4, 5, indicating the trivalent, tetravalent and pentavalent redox states, respectively. The subscript j indicates the conformational state.

Fig. 3:
Fig. 3:

A false color TIRF image of immobilized streptavidin, HRP conjugate in the presence of substrate and cosubstrate (H2O2) is shown on the left. The right image shows a surface where streptavidin was immobilized instead of streptavidin, HRP conjugate. Substrate and cosubstrate concentrations were the same in both cases. Intensities are given in arbitrary units.

Fig. 4:
Fig. 4:

Correlogram and part of the intensity time trace for enzyme turnover. A fit to the correlogram is shown in red. The used model is given by Eq. 2.

Fig. 5:
Fig. 5:

Correlogram and part of the intensity time trace measured at a position where no enzyme is located. A fit to the correlogram is shown in red.

Tables (1)

Tables Icon

Table 1: Parameter estimates for data from single enzyme measurements and a control measurement. i: Estimates for the data displayed in Fig. 4. ii, iii: Results from successive measurements for the same enzyme. iv: Results from a control measurement. The data corresponding to the control measurements is displayed in Fig. 5. *fixed parameters.

Equations (3)

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E k a { k b } EP
G ( τ ) = 1 + [ 1 + T 1 T exp ( τ τ T ) ] × { a 1 N γ [ G D ( τ ) 1 ] + a 2 exp ( k a τ ) + a 3 exp [ ( k b τ ) β ] } .
G D = 1 + γ N ( 1 + ω 2 τ τ z ) 1 [ ( 1 τ 2 τ z ) w ( i τ 4 τ z ) + τ π τ z ] ,

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