Abstract

Fluorescence correlation spectroscopy (FCS) has become a powerful and sensitive research tool for the study of molecular dynamics at the single-molecule level. Because photophysical dynamics often dramatically influence FCS measurements, the role of various photophysical processes in FCS measurements must be understood to accurately interpret FCS data. We describe the role of excitation saturation in two-photon fluorescence correlation measurements. We introduce a physical model that characterizes the effects of excitation saturation on the size and shape of the two-photon fluorescence observation volume and derive a new analytical expression for fluorescence correlation functions that includes the influence of saturation. With this model, we can accurately describe both the temporal decay and the amplitude of measured fluorescence correlation functions over a wide range of illumination powers.

© 2003 Optical Society of America

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  1. D. Magde, E. Elson, W. W. Webb, “Thermodynamic fluctuations in a reacting system. Measurement by fluorescence Correlation spectroscopy,” Phys. Rev. Lett. 29, 705–708 (1972).
  2. E. L. Elson, D. Magde, “Fluorescence correlation spectroscopy. I. Conceptual basis and theory,” Biopolymers 13, 1–27 (1974).
  3. D. Magde, E. L. Elson, W. W. Webb, “Fluorescence correlation spectroscopy. II. Experimental realization,” Biopolymers 13, 29–61 (1974).
  4. S. Maiti, U. Haupts, W. W. Webb, “Fluorescence correlation spectroscopy: diagnostics for sparse molecules,” Proc. Natl. Acad. Sci. USA 94, 11753–11757 (1997).
    [PubMed]
  5. N. L. Thompson, “Fluorescence correlation spectroscopy,” in Topics in Fluorescence Spectroscopy, J. R. Lakowicz, ed. (Plenum, New York, 1991), pp. 337–378.
  6. R. Rigler, E. S. Elson, eds., Fluorescence Correlation Spectroscopy Theory and Applications (Springer, New York, 2001), Vol. 45, p. 486.
  7. M. Eigen, R. Rigler, “Sorting single molecules: application to diagnostics and evolutionary biotechnology,” Proc. Natl. Acad. Sci. USA 91, 5740–5747 (1994).
    [CrossRef] [PubMed]
  8. N. L. Thompson, A. M. Leito, N. W. Allo, “Recent advances in fluorescence correlation spectroscopy,” Curr. Opin. Struct. Biol. 12, 634–641 (2002).
    [CrossRef] [PubMed]
  9. S. T. Hess, S. Huang, A. A. Heikal, W. W. Webb, “Biological and chemical applications of fluorescence correlation spectroscopy: a review,” Biochemistry 41, 697–705 (2002).
    [CrossRef] [PubMed]
  10. J. Widengren, R. Rigler, U. Mets, “Triplet-state monitoring by fluorescence correlation spectroscopy,” J. Fluoresc. 4(3), 255–258 (1994).
    [CrossRef]
  11. J. Widengren, R. Rigler, “Mechanisms of photobleaching investigated by fluorescence correlation spectroscopy,” Bioimaging 4(3), 149–157 (1996).
    [CrossRef]
  12. J. Widengren, B. Terry, R. Rigler, “Protonation kinetics of GFP and FITC investigated by FCS—aspects of the use of fluorescent indicators for measuring pH,” Chem. Phys. 249, 259–271 (1999).
    [CrossRef]
  13. J. Widengren, U. Mets, R. Rigler, “Photodynamic properties of green fluorescent proteins investigated by fluorescence correlation spectroscopy,” Chem. Phys. 250, 171–186 (1999).
    [CrossRef]
  14. J. Widengren, U. Mets, R. Rigler, “Fluorescence correlation spectroscopy of triplet states in solution: a theoretical and experimental study,” J. Phys. Chem. 99, 13368–13379 (1995).
    [CrossRef]
  15. P. Schulk, S. Kummer, A. A. Heikel, W. E. Moeiner, W. W. Webb, “Fluorescence Correlation Spectroscopy reveals fast optical excitation-driven intramolecular dynamics of yellow fluorescent proteins,” Proc. Natl. Acad. USA 97, 151–156 (2000).
    [CrossRef]
  16. F. Malvezzi-Campeggi, M. Jahnz, K. G. Heinze, P. Dittrich, P. Schwille, “Light-induced flickering of DsRed provides evidence for distinct and interconvertible fluorescent states,” Biophys. J. 81, 1776–1785 (2001).
    [CrossRef] [PubMed]
  17. U. Haupts, S. Maiti, P. Schwille, W. W. Webb, “Dynamics of fluorescence fluctuations in green fluorescent protein observed by fluorescence correlation spectroscopy,” Proc. Natl. Acad. Sci. USA 95, 13573–13578 (1998).
    [CrossRef] [PubMed]
  18. J. Mertz, “Molecular photodynamics involved in multi-photon excitation microscopy,” Eur. Phys. J. D. 3(24), 53–66 (1998).
    [CrossRef]
  19. W. Denk, J. H. Strickler, W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science, 248, 73–76 (1990).
    [CrossRef] [PubMed]
  20. C. Xu, W. W. Webb, “Multiphoton excitation of molecular fluorophores and nonlinear laser microscopy,” Top. Fluoresc. Spectrosc. 5, 471–540 (1997).
    [CrossRef]
  21. K. M. Berland, P. T. C. So, E. Gratton, “Two-photon fluorescence correlation spectroscopy: method and application to the intracellular environment,” Biophys. J. 68, 694–701 (1995).
    [CrossRef] [PubMed]
  22. P. Schwille, U. Haupts, S. Maiti, W. W. Webb, Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and two-photon excitation, Biophys. J. 77, 2251–2265 (1999).
    [CrossRef] [PubMed]
  23. P. S. Dittrich, P. Schwille, “Photobleaching and stabilization of fluorophores used for single-molecule analysis with one- and two-photon excitation,” Appl. Phys. B 73, 829–873 (2001).
    [CrossRef]
  24. C. Eggeling, J. Widengren, R. Rigler, C. A. M. SeidelG. G. Max-Planck-Institut fuer Biophysikalische Chemie, “Photobleaching of fluorescent dyes under conditions used for single-molecule detection: evidence of two-step photolysis,” Anal. Chem. 70, 2651–2659 (1998).
    [CrossRef] [PubMed]
  25. T. Wohland, R. Rigler, H. Vogel, “The standard deviation in fluorescence correlation spectroscopy,” Biophys. J. 80, 2987–2999 (2001).
    [CrossRef] [PubMed]
  26. Our fitting routines are programmed with conditional statements in the fit function definitions such that the parameter alpha is assigned the value zero whenever the power is less than the saturation threshold parameter. This allows for simultaneous fitting of the entire curve, both above and below the saturation threshold, with asingle fitting function.

2002

N. L. Thompson, A. M. Leito, N. W. Allo, “Recent advances in fluorescence correlation spectroscopy,” Curr. Opin. Struct. Biol. 12, 634–641 (2002).
[CrossRef] [PubMed]

S. T. Hess, S. Huang, A. A. Heikal, W. W. Webb, “Biological and chemical applications of fluorescence correlation spectroscopy: a review,” Biochemistry 41, 697–705 (2002).
[CrossRef] [PubMed]

2001

F. Malvezzi-Campeggi, M. Jahnz, K. G. Heinze, P. Dittrich, P. Schwille, “Light-induced flickering of DsRed provides evidence for distinct and interconvertible fluorescent states,” Biophys. J. 81, 1776–1785 (2001).
[CrossRef] [PubMed]

P. S. Dittrich, P. Schwille, “Photobleaching and stabilization of fluorophores used for single-molecule analysis with one- and two-photon excitation,” Appl. Phys. B 73, 829–873 (2001).
[CrossRef]

T. Wohland, R. Rigler, H. Vogel, “The standard deviation in fluorescence correlation spectroscopy,” Biophys. J. 80, 2987–2999 (2001).
[CrossRef] [PubMed]

2000

P. Schulk, S. Kummer, A. A. Heikel, W. E. Moeiner, W. W. Webb, “Fluorescence Correlation Spectroscopy reveals fast optical excitation-driven intramolecular dynamics of yellow fluorescent proteins,” Proc. Natl. Acad. USA 97, 151–156 (2000).
[CrossRef]

1999

P. Schwille, U. Haupts, S. Maiti, W. W. Webb, Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and two-photon excitation, Biophys. J. 77, 2251–2265 (1999).
[CrossRef] [PubMed]

J. Widengren, B. Terry, R. Rigler, “Protonation kinetics of GFP and FITC investigated by FCS—aspects of the use of fluorescent indicators for measuring pH,” Chem. Phys. 249, 259–271 (1999).
[CrossRef]

J. Widengren, U. Mets, R. Rigler, “Photodynamic properties of green fluorescent proteins investigated by fluorescence correlation spectroscopy,” Chem. Phys. 250, 171–186 (1999).
[CrossRef]

1998

U. Haupts, S. Maiti, P. Schwille, W. W. Webb, “Dynamics of fluorescence fluctuations in green fluorescent protein observed by fluorescence correlation spectroscopy,” Proc. Natl. Acad. Sci. USA 95, 13573–13578 (1998).
[CrossRef] [PubMed]

J. Mertz, “Molecular photodynamics involved in multi-photon excitation microscopy,” Eur. Phys. J. D. 3(24), 53–66 (1998).
[CrossRef]

C. Eggeling, J. Widengren, R. Rigler, C. A. M. SeidelG. G. Max-Planck-Institut fuer Biophysikalische Chemie, “Photobleaching of fluorescent dyes under conditions used for single-molecule detection: evidence of two-step photolysis,” Anal. Chem. 70, 2651–2659 (1998).
[CrossRef] [PubMed]

1997

C. Xu, W. W. Webb, “Multiphoton excitation of molecular fluorophores and nonlinear laser microscopy,” Top. Fluoresc. Spectrosc. 5, 471–540 (1997).
[CrossRef]

S. Maiti, U. Haupts, W. W. Webb, “Fluorescence correlation spectroscopy: diagnostics for sparse molecules,” Proc. Natl. Acad. Sci. USA 94, 11753–11757 (1997).
[PubMed]

1996

J. Widengren, R. Rigler, “Mechanisms of photobleaching investigated by fluorescence correlation spectroscopy,” Bioimaging 4(3), 149–157 (1996).
[CrossRef]

1995

K. M. Berland, P. T. C. So, E. Gratton, “Two-photon fluorescence correlation spectroscopy: method and application to the intracellular environment,” Biophys. J. 68, 694–701 (1995).
[CrossRef] [PubMed]

J. Widengren, U. Mets, R. Rigler, “Fluorescence correlation spectroscopy of triplet states in solution: a theoretical and experimental study,” J. Phys. Chem. 99, 13368–13379 (1995).
[CrossRef]

1994

J. Widengren, R. Rigler, U. Mets, “Triplet-state monitoring by fluorescence correlation spectroscopy,” J. Fluoresc. 4(3), 255–258 (1994).
[CrossRef]

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

1990

W. Denk, J. H. Strickler, W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science, 248, 73–76 (1990).
[CrossRef] [PubMed]

1974

E. L. Elson, D. Magde, “Fluorescence correlation spectroscopy. I. Conceptual basis and theory,” Biopolymers 13, 1–27 (1974).

D. Magde, E. L. Elson, W. W. Webb, “Fluorescence correlation spectroscopy. II. Experimental realization,” Biopolymers 13, 29–61 (1974).

1972

D. Magde, E. Elson, W. W. Webb, “Thermodynamic fluctuations in a reacting system. Measurement by fluorescence Correlation spectroscopy,” Phys. Rev. Lett. 29, 705–708 (1972).

Allo, N. W.

N. L. Thompson, A. M. Leito, N. W. Allo, “Recent advances in fluorescence correlation spectroscopy,” Curr. Opin. Struct. Biol. 12, 634–641 (2002).
[CrossRef] [PubMed]

Berland, K. M.

K. M. Berland, P. T. C. So, E. Gratton, “Two-photon fluorescence correlation spectroscopy: method and application to the intracellular environment,” Biophys. J. 68, 694–701 (1995).
[CrossRef] [PubMed]

Denk, W.

W. Denk, J. H. Strickler, W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science, 248, 73–76 (1990).
[CrossRef] [PubMed]

Dittrich, P.

F. Malvezzi-Campeggi, M. Jahnz, K. G. Heinze, P. Dittrich, P. Schwille, “Light-induced flickering of DsRed provides evidence for distinct and interconvertible fluorescent states,” Biophys. J. 81, 1776–1785 (2001).
[CrossRef] [PubMed]

Dittrich, P. S.

P. S. Dittrich, P. Schwille, “Photobleaching and stabilization of fluorophores used for single-molecule analysis with one- and two-photon excitation,” Appl. Phys. B 73, 829–873 (2001).
[CrossRef]

Eggeling, C.

C. Eggeling, J. Widengren, R. Rigler, C. A. M. SeidelG. G. Max-Planck-Institut fuer Biophysikalische Chemie, “Photobleaching of fluorescent dyes under conditions used for single-molecule detection: evidence of two-step photolysis,” Anal. Chem. 70, 2651–2659 (1998).
[CrossRef] [PubMed]

Eigen, M.

M. Eigen, 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, E. Elson, W. W. Webb, “Thermodynamic fluctuations in a reacting system. Measurement by fluorescence Correlation spectroscopy,” Phys. Rev. Lett. 29, 705–708 (1972).

Elson, E. L.

D. Magde, E. L. Elson, W. W. Webb, “Fluorescence correlation spectroscopy. II. Experimental realization,” Biopolymers 13, 29–61 (1974).

E. L. Elson, D. Magde, “Fluorescence correlation spectroscopy. I. Conceptual basis and theory,” Biopolymers 13, 1–27 (1974).

Gratton, E.

K. M. Berland, P. T. C. So, E. Gratton, “Two-photon fluorescence correlation spectroscopy: method and application to the intracellular environment,” Biophys. J. 68, 694–701 (1995).
[CrossRef] [PubMed]

Haupts, U.

P. Schwille, U. Haupts, S. Maiti, W. W. Webb, Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and two-photon excitation, Biophys. J. 77, 2251–2265 (1999).
[CrossRef] [PubMed]

U. Haupts, S. Maiti, P. Schwille, W. W. Webb, “Dynamics of fluorescence fluctuations in green fluorescent protein observed by fluorescence correlation spectroscopy,” Proc. Natl. Acad. Sci. USA 95, 13573–13578 (1998).
[CrossRef] [PubMed]

S. Maiti, U. Haupts, W. W. Webb, “Fluorescence correlation spectroscopy: diagnostics for sparse molecules,” Proc. Natl. Acad. Sci. USA 94, 11753–11757 (1997).
[PubMed]

Heikal, A. A.

S. T. Hess, S. Huang, A. A. Heikal, W. W. Webb, “Biological and chemical applications of fluorescence correlation spectroscopy: a review,” Biochemistry 41, 697–705 (2002).
[CrossRef] [PubMed]

Heikel, A. A.

P. Schulk, S. Kummer, A. A. Heikel, W. E. Moeiner, W. W. Webb, “Fluorescence Correlation Spectroscopy reveals fast optical excitation-driven intramolecular dynamics of yellow fluorescent proteins,” Proc. Natl. Acad. USA 97, 151–156 (2000).
[CrossRef]

Heinze, K. G.

F. Malvezzi-Campeggi, M. Jahnz, K. G. Heinze, P. Dittrich, P. Schwille, “Light-induced flickering of DsRed provides evidence for distinct and interconvertible fluorescent states,” Biophys. J. 81, 1776–1785 (2001).
[CrossRef] [PubMed]

Hess, S. T.

S. T. Hess, S. Huang, A. A. Heikal, W. W. Webb, “Biological and chemical applications of fluorescence correlation spectroscopy: a review,” Biochemistry 41, 697–705 (2002).
[CrossRef] [PubMed]

Huang, S.

S. T. Hess, S. Huang, A. A. Heikal, W. W. Webb, “Biological and chemical applications of fluorescence correlation spectroscopy: a review,” Biochemistry 41, 697–705 (2002).
[CrossRef] [PubMed]

Jahnz, M.

F. Malvezzi-Campeggi, M. Jahnz, K. G. Heinze, P. Dittrich, P. Schwille, “Light-induced flickering of DsRed provides evidence for distinct and interconvertible fluorescent states,” Biophys. J. 81, 1776–1785 (2001).
[CrossRef] [PubMed]

Kummer, S.

P. Schulk, S. Kummer, A. A. Heikel, W. E. Moeiner, W. W. Webb, “Fluorescence Correlation Spectroscopy reveals fast optical excitation-driven intramolecular dynamics of yellow fluorescent proteins,” Proc. Natl. Acad. USA 97, 151–156 (2000).
[CrossRef]

Leito, A. M.

N. L. Thompson, A. M. Leito, N. W. Allo, “Recent advances in fluorescence correlation spectroscopy,” Curr. Opin. Struct. Biol. 12, 634–641 (2002).
[CrossRef] [PubMed]

Magde, D.

E. L. Elson, D. Magde, “Fluorescence correlation spectroscopy. I. Conceptual basis and theory,” Biopolymers 13, 1–27 (1974).

D. Magde, E. L. Elson, W. W. Webb, “Fluorescence correlation spectroscopy. II. Experimental realization,” Biopolymers 13, 29–61 (1974).

D. Magde, E. Elson, W. W. Webb, “Thermodynamic fluctuations in a reacting system. Measurement by fluorescence Correlation spectroscopy,” Phys. Rev. Lett. 29, 705–708 (1972).

Maiti, S.

P. Schwille, U. Haupts, S. Maiti, W. W. Webb, Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and two-photon excitation, Biophys. J. 77, 2251–2265 (1999).
[CrossRef] [PubMed]

U. Haupts, S. Maiti, P. Schwille, W. W. Webb, “Dynamics of fluorescence fluctuations in green fluorescent protein observed by fluorescence correlation spectroscopy,” Proc. Natl. Acad. Sci. USA 95, 13573–13578 (1998).
[CrossRef] [PubMed]

S. Maiti, U. Haupts, W. W. Webb, “Fluorescence correlation spectroscopy: diagnostics for sparse molecules,” Proc. Natl. Acad. Sci. USA 94, 11753–11757 (1997).
[PubMed]

Malvezzi-Campeggi, F.

F. Malvezzi-Campeggi, M. Jahnz, K. G. Heinze, P. Dittrich, P. Schwille, “Light-induced flickering of DsRed provides evidence for distinct and interconvertible fluorescent states,” Biophys. J. 81, 1776–1785 (2001).
[CrossRef] [PubMed]

Mertz, J.

J. Mertz, “Molecular photodynamics involved in multi-photon excitation microscopy,” Eur. Phys. J. D. 3(24), 53–66 (1998).
[CrossRef]

Mets, U.

J. Widengren, U. Mets, R. Rigler, “Photodynamic properties of green fluorescent proteins investigated by fluorescence correlation spectroscopy,” Chem. Phys. 250, 171–186 (1999).
[CrossRef]

J. Widengren, U. Mets, R. Rigler, “Fluorescence correlation spectroscopy of triplet states in solution: a theoretical and experimental study,” J. Phys. Chem. 99, 13368–13379 (1995).
[CrossRef]

J. Widengren, R. Rigler, U. Mets, “Triplet-state monitoring by fluorescence correlation spectroscopy,” J. Fluoresc. 4(3), 255–258 (1994).
[CrossRef]

Moeiner, W. E.

P. Schulk, S. Kummer, A. A. Heikel, W. E. Moeiner, W. W. Webb, “Fluorescence Correlation Spectroscopy reveals fast optical excitation-driven intramolecular dynamics of yellow fluorescent proteins,” Proc. Natl. Acad. USA 97, 151–156 (2000).
[CrossRef]

Rigler, R.

T. Wohland, R. Rigler, H. Vogel, “The standard deviation in fluorescence correlation spectroscopy,” Biophys. J. 80, 2987–2999 (2001).
[CrossRef] [PubMed]

J. Widengren, B. Terry, R. Rigler, “Protonation kinetics of GFP and FITC investigated by FCS—aspects of the use of fluorescent indicators for measuring pH,” Chem. Phys. 249, 259–271 (1999).
[CrossRef]

J. Widengren, U. Mets, R. Rigler, “Photodynamic properties of green fluorescent proteins investigated by fluorescence correlation spectroscopy,” Chem. Phys. 250, 171–186 (1999).
[CrossRef]

C. Eggeling, J. Widengren, R. Rigler, C. A. M. SeidelG. G. Max-Planck-Institut fuer Biophysikalische Chemie, “Photobleaching of fluorescent dyes under conditions used for single-molecule detection: evidence of two-step photolysis,” Anal. Chem. 70, 2651–2659 (1998).
[CrossRef] [PubMed]

J. Widengren, R. Rigler, “Mechanisms of photobleaching investigated by fluorescence correlation spectroscopy,” Bioimaging 4(3), 149–157 (1996).
[CrossRef]

J. Widengren, U. Mets, R. Rigler, “Fluorescence correlation spectroscopy of triplet states in solution: a theoretical and experimental study,” J. Phys. Chem. 99, 13368–13379 (1995).
[CrossRef]

J. Widengren, R. Rigler, U. Mets, “Triplet-state monitoring by fluorescence correlation spectroscopy,” J. Fluoresc. 4(3), 255–258 (1994).
[CrossRef]

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

Schulk, P.

P. Schulk, S. Kummer, A. A. Heikel, W. E. Moeiner, W. W. Webb, “Fluorescence Correlation Spectroscopy reveals fast optical excitation-driven intramolecular dynamics of yellow fluorescent proteins,” Proc. Natl. Acad. USA 97, 151–156 (2000).
[CrossRef]

Schwille, P.

F. Malvezzi-Campeggi, M. Jahnz, K. G. Heinze, P. Dittrich, P. Schwille, “Light-induced flickering of DsRed provides evidence for distinct and interconvertible fluorescent states,” Biophys. J. 81, 1776–1785 (2001).
[CrossRef] [PubMed]

P. S. Dittrich, P. Schwille, “Photobleaching and stabilization of fluorophores used for single-molecule analysis with one- and two-photon excitation,” Appl. Phys. B 73, 829–873 (2001).
[CrossRef]

P. Schwille, U. Haupts, S. Maiti, W. W. Webb, Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and two-photon excitation, Biophys. J. 77, 2251–2265 (1999).
[CrossRef] [PubMed]

U. Haupts, S. Maiti, P. Schwille, W. W. Webb, “Dynamics of fluorescence fluctuations in green fluorescent protein observed by fluorescence correlation spectroscopy,” Proc. Natl. Acad. Sci. USA 95, 13573–13578 (1998).
[CrossRef] [PubMed]

Seidel, C. A. M.

C. Eggeling, J. Widengren, R. Rigler, C. A. M. SeidelG. G. Max-Planck-Institut fuer Biophysikalische Chemie, “Photobleaching of fluorescent dyes under conditions used for single-molecule detection: evidence of two-step photolysis,” Anal. Chem. 70, 2651–2659 (1998).
[CrossRef] [PubMed]

So, P. T. C.

K. M. Berland, P. T. C. So, E. Gratton, “Two-photon fluorescence correlation spectroscopy: method and application to the intracellular environment,” Biophys. J. 68, 694–701 (1995).
[CrossRef] [PubMed]

Strickler, J. H.

W. Denk, J. H. Strickler, W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science, 248, 73–76 (1990).
[CrossRef] [PubMed]

Terry, B.

J. Widengren, B. Terry, R. Rigler, “Protonation kinetics of GFP and FITC investigated by FCS—aspects of the use of fluorescent indicators for measuring pH,” Chem. Phys. 249, 259–271 (1999).
[CrossRef]

Thompson, N. L.

N. L. Thompson, A. M. Leito, N. W. Allo, “Recent advances in fluorescence correlation spectroscopy,” Curr. Opin. Struct. Biol. 12, 634–641 (2002).
[CrossRef] [PubMed]

N. L. Thompson, “Fluorescence correlation spectroscopy,” in Topics in Fluorescence Spectroscopy, J. R. Lakowicz, ed. (Plenum, New York, 1991), pp. 337–378.

Vogel, H.

T. Wohland, R. Rigler, H. Vogel, “The standard deviation in fluorescence correlation spectroscopy,” Biophys. J. 80, 2987–2999 (2001).
[CrossRef] [PubMed]

Webb, W. W.

S. T. Hess, S. Huang, A. A. Heikal, W. W. Webb, “Biological and chemical applications of fluorescence correlation spectroscopy: a review,” Biochemistry 41, 697–705 (2002).
[CrossRef] [PubMed]

P. Schulk, S. Kummer, A. A. Heikel, W. E. Moeiner, W. W. Webb, “Fluorescence Correlation Spectroscopy reveals fast optical excitation-driven intramolecular dynamics of yellow fluorescent proteins,” Proc. Natl. Acad. USA 97, 151–156 (2000).
[CrossRef]

P. Schwille, U. Haupts, S. Maiti, W. W. Webb, Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and two-photon excitation, Biophys. J. 77, 2251–2265 (1999).
[CrossRef] [PubMed]

U. Haupts, S. Maiti, P. Schwille, W. W. Webb, “Dynamics of fluorescence fluctuations in green fluorescent protein observed by fluorescence correlation spectroscopy,” Proc. Natl. Acad. Sci. USA 95, 13573–13578 (1998).
[CrossRef] [PubMed]

C. Xu, W. W. Webb, “Multiphoton excitation of molecular fluorophores and nonlinear laser microscopy,” Top. Fluoresc. Spectrosc. 5, 471–540 (1997).
[CrossRef]

S. Maiti, U. Haupts, W. W. Webb, “Fluorescence correlation spectroscopy: diagnostics for sparse molecules,” Proc. Natl. Acad. Sci. USA 94, 11753–11757 (1997).
[PubMed]

W. Denk, J. H. Strickler, W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science, 248, 73–76 (1990).
[CrossRef] [PubMed]

D. Magde, E. L. Elson, W. W. Webb, “Fluorescence correlation spectroscopy. II. Experimental realization,” Biopolymers 13, 29–61 (1974).

D. Magde, E. Elson, W. W. Webb, “Thermodynamic fluctuations in a reacting system. Measurement by fluorescence Correlation spectroscopy,” Phys. Rev. Lett. 29, 705–708 (1972).

Widengren, J.

J. Widengren, B. Terry, R. Rigler, “Protonation kinetics of GFP and FITC investigated by FCS—aspects of the use of fluorescent indicators for measuring pH,” Chem. Phys. 249, 259–271 (1999).
[CrossRef]

J. Widengren, U. Mets, R. Rigler, “Photodynamic properties of green fluorescent proteins investigated by fluorescence correlation spectroscopy,” Chem. Phys. 250, 171–186 (1999).
[CrossRef]

C. Eggeling, J. Widengren, R. Rigler, C. A. M. SeidelG. G. Max-Planck-Institut fuer Biophysikalische Chemie, “Photobleaching of fluorescent dyes under conditions used for single-molecule detection: evidence of two-step photolysis,” Anal. Chem. 70, 2651–2659 (1998).
[CrossRef] [PubMed]

J. Widengren, R. Rigler, “Mechanisms of photobleaching investigated by fluorescence correlation spectroscopy,” Bioimaging 4(3), 149–157 (1996).
[CrossRef]

J. Widengren, U. Mets, R. Rigler, “Fluorescence correlation spectroscopy of triplet states in solution: a theoretical and experimental study,” J. Phys. Chem. 99, 13368–13379 (1995).
[CrossRef]

J. Widengren, R. Rigler, U. Mets, “Triplet-state monitoring by fluorescence correlation spectroscopy,” J. Fluoresc. 4(3), 255–258 (1994).
[CrossRef]

Wohland, T.

T. Wohland, R. Rigler, H. Vogel, “The standard deviation in fluorescence correlation spectroscopy,” Biophys. J. 80, 2987–2999 (2001).
[CrossRef] [PubMed]

Xu, C.

C. Xu, W. W. Webb, “Multiphoton excitation of molecular fluorophores and nonlinear laser microscopy,” Top. Fluoresc. Spectrosc. 5, 471–540 (1997).
[CrossRef]

Anal. Chem.

C. Eggeling, J. Widengren, R. Rigler, C. A. M. SeidelG. G. Max-Planck-Institut fuer Biophysikalische Chemie, “Photobleaching of fluorescent dyes under conditions used for single-molecule detection: evidence of two-step photolysis,” Anal. Chem. 70, 2651–2659 (1998).
[CrossRef] [PubMed]

Appl. Phys. B

P. S. Dittrich, P. Schwille, “Photobleaching and stabilization of fluorophores used for single-molecule analysis with one- and two-photon excitation,” Appl. Phys. B 73, 829–873 (2001).
[CrossRef]

Biochemistry

S. T. Hess, S. Huang, A. A. Heikal, W. W. Webb, “Biological and chemical applications of fluorescence correlation spectroscopy: a review,” Biochemistry 41, 697–705 (2002).
[CrossRef] [PubMed]

Bioimaging

J. Widengren, R. Rigler, “Mechanisms of photobleaching investigated by fluorescence correlation spectroscopy,” Bioimaging 4(3), 149–157 (1996).
[CrossRef]

Biophys. J.

F. Malvezzi-Campeggi, M. Jahnz, K. G. Heinze, P. Dittrich, P. Schwille, “Light-induced flickering of DsRed provides evidence for distinct and interconvertible fluorescent states,” Biophys. J. 81, 1776–1785 (2001).
[CrossRef] [PubMed]

K. M. Berland, P. T. C. So, E. Gratton, “Two-photon fluorescence correlation spectroscopy: method and application to the intracellular environment,” Biophys. J. 68, 694–701 (1995).
[CrossRef] [PubMed]

P. Schwille, U. Haupts, S. Maiti, W. W. Webb, Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and two-photon excitation, Biophys. J. 77, 2251–2265 (1999).
[CrossRef] [PubMed]

T. Wohland, R. Rigler, H. Vogel, “The standard deviation in fluorescence correlation spectroscopy,” Biophys. J. 80, 2987–2999 (2001).
[CrossRef] [PubMed]

Biopolymers

E. L. Elson, D. Magde, “Fluorescence correlation spectroscopy. I. Conceptual basis and theory,” Biopolymers 13, 1–27 (1974).

D. Magde, E. L. Elson, W. W. Webb, “Fluorescence correlation spectroscopy. II. Experimental realization,” Biopolymers 13, 29–61 (1974).

Chem. Phys.

J. Widengren, B. Terry, R. Rigler, “Protonation kinetics of GFP and FITC investigated by FCS—aspects of the use of fluorescent indicators for measuring pH,” Chem. Phys. 249, 259–271 (1999).
[CrossRef]

J. Widengren, U. Mets, R. Rigler, “Photodynamic properties of green fluorescent proteins investigated by fluorescence correlation spectroscopy,” Chem. Phys. 250, 171–186 (1999).
[CrossRef]

Curr. Opin. Struct. Biol.

N. L. Thompson, A. M. Leito, N. W. Allo, “Recent advances in fluorescence correlation spectroscopy,” Curr. Opin. Struct. Biol. 12, 634–641 (2002).
[CrossRef] [PubMed]

Eur. Phys. J. D.

J. Mertz, “Molecular photodynamics involved in multi-photon excitation microscopy,” Eur. Phys. J. D. 3(24), 53–66 (1998).
[CrossRef]

J. Fluoresc.

J. Widengren, R. Rigler, U. Mets, “Triplet-state monitoring by fluorescence correlation spectroscopy,” J. Fluoresc. 4(3), 255–258 (1994).
[CrossRef]

J. Phys. Chem.

J. Widengren, U. Mets, R. Rigler, “Fluorescence correlation spectroscopy of triplet states in solution: a theoretical and experimental study,” J. Phys. Chem. 99, 13368–13379 (1995).
[CrossRef]

Phys. Rev. Lett.

D. Magde, E. Elson, W. W. Webb, “Thermodynamic fluctuations in a reacting system. Measurement by fluorescence Correlation spectroscopy,” Phys. Rev. Lett. 29, 705–708 (1972).

Proc. Natl. Acad. Sci. USA

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

S. Maiti, U. Haupts, W. W. Webb, “Fluorescence correlation spectroscopy: diagnostics for sparse molecules,” Proc. Natl. Acad. Sci. USA 94, 11753–11757 (1997).
[PubMed]

U. Haupts, S. Maiti, P. Schwille, W. W. Webb, “Dynamics of fluorescence fluctuations in green fluorescent protein observed by fluorescence correlation spectroscopy,” Proc. Natl. Acad. Sci. USA 95, 13573–13578 (1998).
[CrossRef] [PubMed]

Proc. Natl. Acad. USA

P. Schulk, S. Kummer, A. A. Heikel, W. E. Moeiner, W. W. Webb, “Fluorescence Correlation Spectroscopy reveals fast optical excitation-driven intramolecular dynamics of yellow fluorescent proteins,” Proc. Natl. Acad. USA 97, 151–156 (2000).
[CrossRef]

Science

W. Denk, J. H. Strickler, W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science, 248, 73–76 (1990).
[CrossRef] [PubMed]

Top. Fluoresc. Spectrosc.

C. Xu, W. W. Webb, “Multiphoton excitation of molecular fluorophores and nonlinear laser microscopy,” Top. Fluoresc. Spectrosc. 5, 471–540 (1997).
[CrossRef]

Other

N. L. Thompson, “Fluorescence correlation spectroscopy,” in Topics in Fluorescence Spectroscopy, J. R. Lakowicz, ed. (Plenum, New York, 1991), pp. 337–378.

R. Rigler, E. S. Elson, eds., Fluorescence Correlation Spectroscopy Theory and Applications (Springer, New York, 2001), Vol. 45, p. 486.

Our fitting routines are programmed with conditional statements in the fit function definitions such that the parameter alpha is assigned the value zero whenever the power is less than the saturation threshold parameter. This allows for simultaneous fitting of the entire curve, both above and below the saturation threshold, with asingle fitting function.

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