Abstract

In confocal fluorescence correlation microscopy (FCM) it is important to ensure that the correlation measurement is actually performed at the chosen location of the three-dimensional image of the specimen. We present a confocal FCM design that provides an automatic real-time readout of the location in the confocal microscopic image, which is aligned with the detector of the fluorescence correlation spectrometer. The design accomplishes this without using any special positioning device. The design is based on an apertured fluorescence detector placed close to the back aperture of the objective lens and can be easily incorporated into virtually any confocal microscope. We demonstrate the method by performing FCM measurements of a dye diffusing on a cell membrane.

© 2005 Optical Society of America

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  1. D. Magde, E. Elson, W. W. Webb, “Thermodynamic fluctuation in a reaction system — measurement by fluorescence correlation spectroscopy,” Phys. Rev. Lett. 29, 705–708 (1972)
    [CrossRef]
  2. E. Elson, D. Magde, “Fluorescence correlation spectroscopy: I. Conceptual basics and theory,” Biopolymers 13, 1–27 (1974).
    [CrossRef]
  3. D. Magde, E. Elson, W. W. Webb, “Fluorescence correlation spectroscopy: II. An experimental realization,” Biopolymers 13, 29–61 (1974).
    [CrossRef] [PubMed]
  4. S. Maiti, U. Haupts, W. W. Webb, “Fluorescence correlation spectroscopy: diagnostics for sparse molecules,” Proc. Natl. Acad. Sci. USA 94, 11753–11757 (1997).
  5. U. Haupts, S. Maiti, P. Schwille, W. W. Webb, “Dynamics of fluorescence fluctuations in green fluorescenct protein observed by fluorescence correlation spectroscopy,” Proc. Natl. Acad. Sci. USA 95, 13573–13578 (1998).
    [CrossRef]
  6. A. G. Palmer, N. L. Thompson, “Molecular aggregation characterized by high order autocorelation in fluorescence correlation spectroscopy,” Biophys. J. 52, 257–270 (1987).
  7. L. O. Tjernberg, A. Pramanik, S. Bjorling, P. Thyberg, J. Thyberg, C. Nordstedt, K. D. Berndt, L. Terenius, R. Rigler, “Amyloid beta-peptide polymerization studied using fluorescence correlation spectroscopy,” Chem. Biol. 6, 53–62 (1999).
    [CrossRef] [PubMed]
  8. P. Sengupta, K. Garai, B. Sahoo, Y. Shi, D. J. Callaway, S. Maiti, “The amyloid beta peptide [Abeta(1–40)] is thermodynamically soluble at physiological concentrations,” Biochemistry 42, 10506–10513 (2003).
    [CrossRef] [PubMed]
  9. E. L. Elson, J. Schlessinger, D. E. Koppel, D. Axelrod, W. W. Webb, “Measurement of lateral transport on cell surfaces,” Prog. Clin. Biol. Res. 9, 137–147 (1976).
    [PubMed]
  10. 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).
  11. P. Schwille, J. Korlach, W. W. Webb, “Fluorescence correlation spectroscopy with single-molecule sensitivity on cell and model membranes,” Cytometry 36, 176–182 (1999).
    [CrossRef] [PubMed]
  12. D. Magde, W. W. Webb, E. L. Elson, “Fluorescence correlation spectroscopy. III. Uniform translation and laminar flow,” Biopolymer 17, 361–376 (1978).
    [CrossRef]
  13. M. Brinkmeier, R. Rigler, “Flow analysis by means of fluorescence correlation spectroscopy,” Exp. Tech. Phys. 41, 205– 210 (1996).
  14. R. Brock, M. A. Hink, T. M. Jovin, “Fluorescence correlation microscopy of cells in the presence of autofluorescence,” Biophys. J. 75, 2547–2557 (1998).
  15. R. Brock, T. M. Jovin, “Fluorescence correlation microscopy (FCM): fluorescence correlation spectroscopy (FCS) in cell biology,” in Fluorescence Correlation Spectroscopy Theory and Applications. R. Rigler, E. S. Elson, eds. (Springer-Verlag, Berlin, 2001), p. 132–161.
    [CrossRef]
  16. E. L. Elson, “Fluorescence correlation spectroscopy measures molecular transport in cells,” Traffic 2, 789–796 (2001).
    [CrossRef] [PubMed]
  17. P. Sengupta, J. Balaji, S. Maiti, “Measuring diffusion in cell membranes by fluorescence correlation spectroscopy,” Methods 27, 374–387 (2002).
    [CrossRef] [PubMed]
  18. L. A. White, M. J. Eaton, M. C. Castro, K. J. Klose, M. Y. Globus, G. Shaw, S. R. Whittemore, “Distinct regulatory pathways control neurofilament expression and neurotransmitter synthesis in immortalized serotonergic neurons,” J Neurosci. 14, 6744–6753 (1994).
    [PubMed]
  19. P. Sengupta, K. Garai, J. Balaji, N. Periasamy, S. Maiti, “Measuring size distribution in highly heterogeneous systems with fluorescence correlation spectroscopy,” Biophys. J. 84, 1977–1984 (2003).
  20. N. O. Petersen, “Scanning fluorescence correlation spectroscopy. I. Theory and simulation of aggregation measurements,” Biophys. J. 49, 809–815 (1986).
  21. D. E. Koppel, F. Morgan, A. E. Cowan, J. H. Carson, “Scanning concentration correlation spectroscopy using the confocal laser microscope,” Biophys. J. 66, 502–507 (1994).

2003

P. Sengupta, K. Garai, B. Sahoo, Y. Shi, D. J. Callaway, S. Maiti, “The amyloid beta peptide [Abeta(1–40)] is thermodynamically soluble at physiological concentrations,” Biochemistry 42, 10506–10513 (2003).
[CrossRef] [PubMed]

P. Sengupta, K. Garai, J. Balaji, N. Periasamy, S. Maiti, “Measuring size distribution in highly heterogeneous systems with fluorescence correlation spectroscopy,” Biophys. J. 84, 1977–1984 (2003).

2002

P. Sengupta, J. Balaji, S. Maiti, “Measuring diffusion in cell membranes by fluorescence correlation spectroscopy,” Methods 27, 374–387 (2002).
[CrossRef] [PubMed]

2001

E. L. Elson, “Fluorescence correlation spectroscopy measures molecular transport in cells,” Traffic 2, 789–796 (2001).
[CrossRef] [PubMed]

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).

P. Schwille, J. Korlach, W. W. Webb, “Fluorescence correlation spectroscopy with single-molecule sensitivity on cell and model membranes,” Cytometry 36, 176–182 (1999).
[CrossRef] [PubMed]

L. O. Tjernberg, A. Pramanik, S. Bjorling, P. Thyberg, J. Thyberg, C. Nordstedt, K. D. Berndt, L. Terenius, R. Rigler, “Amyloid beta-peptide polymerization studied using fluorescence correlation spectroscopy,” Chem. Biol. 6, 53–62 (1999).
[CrossRef] [PubMed]

1998

R. Brock, M. A. Hink, T. M. Jovin, “Fluorescence correlation microscopy of cells in the presence of autofluorescence,” Biophys. J. 75, 2547–2557 (1998).

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

1997

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

1996

M. Brinkmeier, R. Rigler, “Flow analysis by means of fluorescence correlation spectroscopy,” Exp. Tech. Phys. 41, 205– 210 (1996).

1994

L. A. White, M. J. Eaton, M. C. Castro, K. J. Klose, M. Y. Globus, G. Shaw, S. R. Whittemore, “Distinct regulatory pathways control neurofilament expression and neurotransmitter synthesis in immortalized serotonergic neurons,” J Neurosci. 14, 6744–6753 (1994).
[PubMed]

D. E. Koppel, F. Morgan, A. E. Cowan, J. H. Carson, “Scanning concentration correlation spectroscopy using the confocal laser microscope,” Biophys. J. 66, 502–507 (1994).

1987

A. G. Palmer, N. L. Thompson, “Molecular aggregation characterized by high order autocorelation in fluorescence correlation spectroscopy,” Biophys. J. 52, 257–270 (1987).

1986

N. O. Petersen, “Scanning fluorescence correlation spectroscopy. I. Theory and simulation of aggregation measurements,” Biophys. J. 49, 809–815 (1986).

1978

D. Magde, W. W. Webb, E. L. Elson, “Fluorescence correlation spectroscopy. III. Uniform translation and laminar flow,” Biopolymer 17, 361–376 (1978).
[CrossRef]

1976

E. L. Elson, J. Schlessinger, D. E. Koppel, D. Axelrod, W. W. Webb, “Measurement of lateral transport on cell surfaces,” Prog. Clin. Biol. Res. 9, 137–147 (1976).
[PubMed]

1974

E. Elson, D. Magde, “Fluorescence correlation spectroscopy: I. Conceptual basics and theory,” Biopolymers 13, 1–27 (1974).
[CrossRef]

D. Magde, E. Elson, W. W. Webb, “Fluorescence correlation spectroscopy: II. An experimental realization,” Biopolymers 13, 29–61 (1974).
[CrossRef] [PubMed]

1972

D. Magde, E. Elson, W. W. Webb, “Thermodynamic fluctuation in a reaction system — measurement by fluorescence correlation spectroscopy,” Phys. Rev. Lett. 29, 705–708 (1972)
[CrossRef]

Axelrod, D.

E. L. Elson, J. Schlessinger, D. E. Koppel, D. Axelrod, W. W. Webb, “Measurement of lateral transport on cell surfaces,” Prog. Clin. Biol. Res. 9, 137–147 (1976).
[PubMed]

Balaji, J.

P. Sengupta, K. Garai, J. Balaji, N. Periasamy, S. Maiti, “Measuring size distribution in highly heterogeneous systems with fluorescence correlation spectroscopy,” Biophys. J. 84, 1977–1984 (2003).

P. Sengupta, J. Balaji, S. Maiti, “Measuring diffusion in cell membranes by fluorescence correlation spectroscopy,” Methods 27, 374–387 (2002).
[CrossRef] [PubMed]

Berndt, K. D.

L. O. Tjernberg, A. Pramanik, S. Bjorling, P. Thyberg, J. Thyberg, C. Nordstedt, K. D. Berndt, L. Terenius, R. Rigler, “Amyloid beta-peptide polymerization studied using fluorescence correlation spectroscopy,” Chem. Biol. 6, 53–62 (1999).
[CrossRef] [PubMed]

Bjorling, S.

L. O. Tjernberg, A. Pramanik, S. Bjorling, P. Thyberg, J. Thyberg, C. Nordstedt, K. D. Berndt, L. Terenius, R. Rigler, “Amyloid beta-peptide polymerization studied using fluorescence correlation spectroscopy,” Chem. Biol. 6, 53–62 (1999).
[CrossRef] [PubMed]

Brinkmeier, M.

M. Brinkmeier, R. Rigler, “Flow analysis by means of fluorescence correlation spectroscopy,” Exp. Tech. Phys. 41, 205– 210 (1996).

Brock, R.

R. Brock, M. A. Hink, T. M. Jovin, “Fluorescence correlation microscopy of cells in the presence of autofluorescence,” Biophys. J. 75, 2547–2557 (1998).

R. Brock, T. M. Jovin, “Fluorescence correlation microscopy (FCM): fluorescence correlation spectroscopy (FCS) in cell biology,” in Fluorescence Correlation Spectroscopy Theory and Applications. R. Rigler, E. S. Elson, eds. (Springer-Verlag, Berlin, 2001), p. 132–161.
[CrossRef]

Callaway, D. J.

P. Sengupta, K. Garai, B. Sahoo, Y. Shi, D. J. Callaway, S. Maiti, “The amyloid beta peptide [Abeta(1–40)] is thermodynamically soluble at physiological concentrations,” Biochemistry 42, 10506–10513 (2003).
[CrossRef] [PubMed]

Carson, J. H.

D. E. Koppel, F. Morgan, A. E. Cowan, J. H. Carson, “Scanning concentration correlation spectroscopy using the confocal laser microscope,” Biophys. J. 66, 502–507 (1994).

Castro, M. C.

L. A. White, M. J. Eaton, M. C. Castro, K. J. Klose, M. Y. Globus, G. Shaw, S. R. Whittemore, “Distinct regulatory pathways control neurofilament expression and neurotransmitter synthesis in immortalized serotonergic neurons,” J Neurosci. 14, 6744–6753 (1994).
[PubMed]

Cowan, A. E.

D. E. Koppel, F. Morgan, A. E. Cowan, J. H. Carson, “Scanning concentration correlation spectroscopy using the confocal laser microscope,” Biophys. J. 66, 502–507 (1994).

Eaton, M. J.

L. A. White, M. J. Eaton, M. C. Castro, K. J. Klose, M. Y. Globus, G. Shaw, S. R. Whittemore, “Distinct regulatory pathways control neurofilament expression and neurotransmitter synthesis in immortalized serotonergic neurons,” J Neurosci. 14, 6744–6753 (1994).
[PubMed]

Elson, E.

E. Elson, D. Magde, “Fluorescence correlation spectroscopy: I. Conceptual basics and theory,” Biopolymers 13, 1–27 (1974).
[CrossRef]

D. Magde, E. Elson, W. W. Webb, “Fluorescence correlation spectroscopy: II. An experimental realization,” Biopolymers 13, 29–61 (1974).
[CrossRef] [PubMed]

D. Magde, E. Elson, W. W. Webb, “Thermodynamic fluctuation in a reaction system — measurement by fluorescence correlation spectroscopy,” Phys. Rev. Lett. 29, 705–708 (1972)
[CrossRef]

Elson, E. L.

E. L. Elson, “Fluorescence correlation spectroscopy measures molecular transport in cells,” Traffic 2, 789–796 (2001).
[CrossRef] [PubMed]

D. Magde, W. W. Webb, E. L. Elson, “Fluorescence correlation spectroscopy. III. Uniform translation and laminar flow,” Biopolymer 17, 361–376 (1978).
[CrossRef]

E. L. Elson, J. Schlessinger, D. E. Koppel, D. Axelrod, W. W. Webb, “Measurement of lateral transport on cell surfaces,” Prog. Clin. Biol. Res. 9, 137–147 (1976).
[PubMed]

Garai, K.

P. Sengupta, K. Garai, B. Sahoo, Y. Shi, D. J. Callaway, S. Maiti, “The amyloid beta peptide [Abeta(1–40)] is thermodynamically soluble at physiological concentrations,” Biochemistry 42, 10506–10513 (2003).
[CrossRef] [PubMed]

P. Sengupta, K. Garai, J. Balaji, N. Periasamy, S. Maiti, “Measuring size distribution in highly heterogeneous systems with fluorescence correlation spectroscopy,” Biophys. J. 84, 1977–1984 (2003).

Globus, M. Y.

L. A. White, M. J. Eaton, M. C. Castro, K. J. Klose, M. Y. Globus, G. Shaw, S. R. Whittemore, “Distinct regulatory pathways control neurofilament expression and neurotransmitter synthesis in immortalized serotonergic neurons,” J Neurosci. 14, 6744–6753 (1994).
[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).

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

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

Hink, M. A.

R. Brock, M. A. Hink, T. M. Jovin, “Fluorescence correlation microscopy of cells in the presence of autofluorescence,” Biophys. J. 75, 2547–2557 (1998).

Jovin, T. M.

R. Brock, M. A. Hink, T. M. Jovin, “Fluorescence correlation microscopy of cells in the presence of autofluorescence,” Biophys. J. 75, 2547–2557 (1998).

R. Brock, T. M. Jovin, “Fluorescence correlation microscopy (FCM): fluorescence correlation spectroscopy (FCS) in cell biology,” in Fluorescence Correlation Spectroscopy Theory and Applications. R. Rigler, E. S. Elson, eds. (Springer-Verlag, Berlin, 2001), p. 132–161.
[CrossRef]

Klose, K. J.

L. A. White, M. J. Eaton, M. C. Castro, K. J. Klose, M. Y. Globus, G. Shaw, S. R. Whittemore, “Distinct regulatory pathways control neurofilament expression and neurotransmitter synthesis in immortalized serotonergic neurons,” J Neurosci. 14, 6744–6753 (1994).
[PubMed]

Koppel, D. E.

D. E. Koppel, F. Morgan, A. E. Cowan, J. H. Carson, “Scanning concentration correlation spectroscopy using the confocal laser microscope,” Biophys. J. 66, 502–507 (1994).

E. L. Elson, J. Schlessinger, D. E. Koppel, D. Axelrod, W. W. Webb, “Measurement of lateral transport on cell surfaces,” Prog. Clin. Biol. Res. 9, 137–147 (1976).
[PubMed]

Korlach, J.

P. Schwille, J. Korlach, W. W. Webb, “Fluorescence correlation spectroscopy with single-molecule sensitivity on cell and model membranes,” Cytometry 36, 176–182 (1999).
[CrossRef] [PubMed]

Magde, D.

D. Magde, W. W. Webb, E. L. Elson, “Fluorescence correlation spectroscopy. III. Uniform translation and laminar flow,” Biopolymer 17, 361–376 (1978).
[CrossRef]

E. Elson, D. Magde, “Fluorescence correlation spectroscopy: I. Conceptual basics and theory,” Biopolymers 13, 1–27 (1974).
[CrossRef]

D. Magde, E. Elson, W. W. Webb, “Fluorescence correlation spectroscopy: II. An experimental realization,” Biopolymers 13, 29–61 (1974).
[CrossRef] [PubMed]

D. Magde, E. Elson, W. W. Webb, “Thermodynamic fluctuation in a reaction system — measurement by fluorescence correlation spectroscopy,” Phys. Rev. Lett. 29, 705–708 (1972)
[CrossRef]

Maiti, S.

P. Sengupta, K. Garai, B. Sahoo, Y. Shi, D. J. Callaway, S. Maiti, “The amyloid beta peptide [Abeta(1–40)] is thermodynamically soluble at physiological concentrations,” Biochemistry 42, 10506–10513 (2003).
[CrossRef] [PubMed]

P. Sengupta, K. Garai, J. Balaji, N. Periasamy, S. Maiti, “Measuring size distribution in highly heterogeneous systems with fluorescence correlation spectroscopy,” Biophys. J. 84, 1977–1984 (2003).

P. Sengupta, J. Balaji, S. Maiti, “Measuring diffusion in cell membranes by fluorescence correlation spectroscopy,” Methods 27, 374–387 (2002).
[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).

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

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

Morgan, F.

D. E. Koppel, F. Morgan, A. E. Cowan, J. H. Carson, “Scanning concentration correlation spectroscopy using the confocal laser microscope,” Biophys. J. 66, 502–507 (1994).

Nordstedt, C.

L. O. Tjernberg, A. Pramanik, S. Bjorling, P. Thyberg, J. Thyberg, C. Nordstedt, K. D. Berndt, L. Terenius, R. Rigler, “Amyloid beta-peptide polymerization studied using fluorescence correlation spectroscopy,” Chem. Biol. 6, 53–62 (1999).
[CrossRef] [PubMed]

Palmer, A. G.

A. G. Palmer, N. L. Thompson, “Molecular aggregation characterized by high order autocorelation in fluorescence correlation spectroscopy,” Biophys. J. 52, 257–270 (1987).

Periasamy, N.

P. Sengupta, K. Garai, J. Balaji, N. Periasamy, S. Maiti, “Measuring size distribution in highly heterogeneous systems with fluorescence correlation spectroscopy,” Biophys. J. 84, 1977–1984 (2003).

Petersen, N. O.

N. O. Petersen, “Scanning fluorescence correlation spectroscopy. I. Theory and simulation of aggregation measurements,” Biophys. J. 49, 809–815 (1986).

Pramanik, A.

L. O. Tjernberg, A. Pramanik, S. Bjorling, P. Thyberg, J. Thyberg, C. Nordstedt, K. D. Berndt, L. Terenius, R. Rigler, “Amyloid beta-peptide polymerization studied using fluorescence correlation spectroscopy,” Chem. Biol. 6, 53–62 (1999).
[CrossRef] [PubMed]

Rigler, R.

L. O. Tjernberg, A. Pramanik, S. Bjorling, P. Thyberg, J. Thyberg, C. Nordstedt, K. D. Berndt, L. Terenius, R. Rigler, “Amyloid beta-peptide polymerization studied using fluorescence correlation spectroscopy,” Chem. Biol. 6, 53–62 (1999).
[CrossRef] [PubMed]

M. Brinkmeier, R. Rigler, “Flow analysis by means of fluorescence correlation spectroscopy,” Exp. Tech. Phys. 41, 205– 210 (1996).

Sahoo, B.

P. Sengupta, K. Garai, B. Sahoo, Y. Shi, D. J. Callaway, S. Maiti, “The amyloid beta peptide [Abeta(1–40)] is thermodynamically soluble at physiological concentrations,” Biochemistry 42, 10506–10513 (2003).
[CrossRef] [PubMed]

Schlessinger, J.

E. L. Elson, J. Schlessinger, D. E. Koppel, D. Axelrod, W. W. Webb, “Measurement of lateral transport on cell surfaces,” Prog. Clin. Biol. Res. 9, 137–147 (1976).
[PubMed]

Schwille, P.

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).

P. Schwille, J. Korlach, W. W. Webb, “Fluorescence correlation spectroscopy with single-molecule sensitivity on cell and model membranes,” Cytometry 36, 176–182 (1999).
[CrossRef] [PubMed]

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

Sengupta, P.

P. Sengupta, K. Garai, B. Sahoo, Y. Shi, D. J. Callaway, S. Maiti, “The amyloid beta peptide [Abeta(1–40)] is thermodynamically soluble at physiological concentrations,” Biochemistry 42, 10506–10513 (2003).
[CrossRef] [PubMed]

P. Sengupta, K. Garai, J. Balaji, N. Periasamy, S. Maiti, “Measuring size distribution in highly heterogeneous systems with fluorescence correlation spectroscopy,” Biophys. J. 84, 1977–1984 (2003).

P. Sengupta, J. Balaji, S. Maiti, “Measuring diffusion in cell membranes by fluorescence correlation spectroscopy,” Methods 27, 374–387 (2002).
[CrossRef] [PubMed]

Shaw, G.

L. A. White, M. J. Eaton, M. C. Castro, K. J. Klose, M. Y. Globus, G. Shaw, S. R. Whittemore, “Distinct regulatory pathways control neurofilament expression and neurotransmitter synthesis in immortalized serotonergic neurons,” J Neurosci. 14, 6744–6753 (1994).
[PubMed]

Shi, Y.

P. Sengupta, K. Garai, B. Sahoo, Y. Shi, D. J. Callaway, S. Maiti, “The amyloid beta peptide [Abeta(1–40)] is thermodynamically soluble at physiological concentrations,” Biochemistry 42, 10506–10513 (2003).
[CrossRef] [PubMed]

Terenius, L.

L. O. Tjernberg, A. Pramanik, S. Bjorling, P. Thyberg, J. Thyberg, C. Nordstedt, K. D. Berndt, L. Terenius, R. Rigler, “Amyloid beta-peptide polymerization studied using fluorescence correlation spectroscopy,” Chem. Biol. 6, 53–62 (1999).
[CrossRef] [PubMed]

Thompson, N. L.

A. G. Palmer, N. L. Thompson, “Molecular aggregation characterized by high order autocorelation in fluorescence correlation spectroscopy,” Biophys. J. 52, 257–270 (1987).

Thyberg, J.

L. O. Tjernberg, A. Pramanik, S. Bjorling, P. Thyberg, J. Thyberg, C. Nordstedt, K. D. Berndt, L. Terenius, R. Rigler, “Amyloid beta-peptide polymerization studied using fluorescence correlation spectroscopy,” Chem. Biol. 6, 53–62 (1999).
[CrossRef] [PubMed]

Thyberg, P.

L. O. Tjernberg, A. Pramanik, S. Bjorling, P. Thyberg, J. Thyberg, C. Nordstedt, K. D. Berndt, L. Terenius, R. Rigler, “Amyloid beta-peptide polymerization studied using fluorescence correlation spectroscopy,” Chem. Biol. 6, 53–62 (1999).
[CrossRef] [PubMed]

Tjernberg, L. O.

L. O. Tjernberg, A. Pramanik, S. Bjorling, P. Thyberg, J. Thyberg, C. Nordstedt, K. D. Berndt, L. Terenius, R. Rigler, “Amyloid beta-peptide polymerization studied using fluorescence correlation spectroscopy,” Chem. Biol. 6, 53–62 (1999).
[CrossRef] [PubMed]

Webb, W. W.

P. Schwille, J. Korlach, W. W. Webb, “Fluorescence correlation spectroscopy with single-molecule sensitivity on cell and model membranes,” Cytometry 36, 176–182 (1999).
[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).

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

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

D. Magde, W. W. Webb, E. L. Elson, “Fluorescence correlation spectroscopy. III. Uniform translation and laminar flow,” Biopolymer 17, 361–376 (1978).
[CrossRef]

E. L. Elson, J. Schlessinger, D. E. Koppel, D. Axelrod, W. W. Webb, “Measurement of lateral transport on cell surfaces,” Prog. Clin. Biol. Res. 9, 137–147 (1976).
[PubMed]

D. Magde, E. Elson, W. W. Webb, “Fluorescence correlation spectroscopy: II. An experimental realization,” Biopolymers 13, 29–61 (1974).
[CrossRef] [PubMed]

D. Magde, E. Elson, W. W. Webb, “Thermodynamic fluctuation in a reaction system — measurement by fluorescence correlation spectroscopy,” Phys. Rev. Lett. 29, 705–708 (1972)
[CrossRef]

White, L. A.

L. A. White, M. J. Eaton, M. C. Castro, K. J. Klose, M. Y. Globus, G. Shaw, S. R. Whittemore, “Distinct regulatory pathways control neurofilament expression and neurotransmitter synthesis in immortalized serotonergic neurons,” J Neurosci. 14, 6744–6753 (1994).
[PubMed]

Whittemore, S. R.

L. A. White, M. J. Eaton, M. C. Castro, K. J. Klose, M. Y. Globus, G. Shaw, S. R. Whittemore, “Distinct regulatory pathways control neurofilament expression and neurotransmitter synthesis in immortalized serotonergic neurons,” J Neurosci. 14, 6744–6753 (1994).
[PubMed]

Biochemistry

P. Sengupta, K. Garai, B. Sahoo, Y. Shi, D. J. Callaway, S. Maiti, “The amyloid beta peptide [Abeta(1–40)] is thermodynamically soluble at physiological concentrations,” Biochemistry 42, 10506–10513 (2003).
[CrossRef] [PubMed]

Biophys. J.

A. G. Palmer, N. L. Thompson, “Molecular aggregation characterized by high order autocorelation in fluorescence correlation spectroscopy,” Biophys. J. 52, 257–270 (1987).

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).

R. Brock, M. A. Hink, T. M. Jovin, “Fluorescence correlation microscopy of cells in the presence of autofluorescence,” Biophys. J. 75, 2547–2557 (1998).

P. Sengupta, K. Garai, J. Balaji, N. Periasamy, S. Maiti, “Measuring size distribution in highly heterogeneous systems with fluorescence correlation spectroscopy,” Biophys. J. 84, 1977–1984 (2003).

N. O. Petersen, “Scanning fluorescence correlation spectroscopy. I. Theory and simulation of aggregation measurements,” Biophys. J. 49, 809–815 (1986).

D. E. Koppel, F. Morgan, A. E. Cowan, J. H. Carson, “Scanning concentration correlation spectroscopy using the confocal laser microscope,” Biophys. J. 66, 502–507 (1994).

Biopolymer

D. Magde, W. W. Webb, E. L. Elson, “Fluorescence correlation spectroscopy. III. Uniform translation and laminar flow,” Biopolymer 17, 361–376 (1978).
[CrossRef]

Biopolymers

E. Elson, D. Magde, “Fluorescence correlation spectroscopy: I. Conceptual basics and theory,” Biopolymers 13, 1–27 (1974).
[CrossRef]

D. Magde, E. Elson, W. W. Webb, “Fluorescence correlation spectroscopy: II. An experimental realization,” Biopolymers 13, 29–61 (1974).
[CrossRef] [PubMed]

Chem. Biol.

L. O. Tjernberg, A. Pramanik, S. Bjorling, P. Thyberg, J. Thyberg, C. Nordstedt, K. D. Berndt, L. Terenius, R. Rigler, “Amyloid beta-peptide polymerization studied using fluorescence correlation spectroscopy,” Chem. Biol. 6, 53–62 (1999).
[CrossRef] [PubMed]

Cytometry

P. Schwille, J. Korlach, W. W. Webb, “Fluorescence correlation spectroscopy with single-molecule sensitivity on cell and model membranes,” Cytometry 36, 176–182 (1999).
[CrossRef] [PubMed]

Exp. Tech. Phys.

M. Brinkmeier, R. Rigler, “Flow analysis by means of fluorescence correlation spectroscopy,” Exp. Tech. Phys. 41, 205– 210 (1996).

J Neurosci.

L. A. White, M. J. Eaton, M. C. Castro, K. J. Klose, M. Y. Globus, G. Shaw, S. R. Whittemore, “Distinct regulatory pathways control neurofilament expression and neurotransmitter synthesis in immortalized serotonergic neurons,” J Neurosci. 14, 6744–6753 (1994).
[PubMed]

Methods

P. Sengupta, J. Balaji, S. Maiti, “Measuring diffusion in cell membranes by fluorescence correlation spectroscopy,” Methods 27, 374–387 (2002).
[CrossRef] [PubMed]

Phys. Rev. Lett.

D. Magde, E. Elson, W. W. Webb, “Thermodynamic fluctuation in a reaction system — measurement by fluorescence correlation spectroscopy,” Phys. Rev. Lett. 29, 705–708 (1972)
[CrossRef]

Proc. Natl. Acad. Sci. USA

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

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

Prog. Clin. Biol. Res.

E. L. Elson, J. Schlessinger, D. E. Koppel, D. Axelrod, W. W. Webb, “Measurement of lateral transport on cell surfaces,” Prog. Clin. Biol. Res. 9, 137–147 (1976).
[PubMed]

Traffic

E. L. Elson, “Fluorescence correlation spectroscopy measures molecular transport in cells,” Traffic 2, 789–796 (2001).
[CrossRef] [PubMed]

Other

R. Brock, T. M. Jovin, “Fluorescence correlation microscopy (FCM): fluorescence correlation spectroscopy (FCS) in cell biology,” in Fluorescence Correlation Spectroscopy Theory and Applications. R. Rigler, E. S. Elson, eds. (Springer-Verlag, Berlin, 2001), p. 132–161.
[CrossRef]

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Figures (3)

Fig. 1
Fig. 1

Schematic diagram of the confocal fluorescence correlation microscope. Fluorescence collected by the objective is split with a beam splitter. The reflected fraction is focused onto the fiber plane for FCS measurements and for generating the alignment spot in the confocal image. The transmitted fraction goes back to the confocal imaging detector.

Fig. 2
Fig. 2

Merged image from FCS and imaging detectors. The image obtained from the FCS detector is the spot shown in red and marked with an arrow. The confocal image of the cell membranes labeled with Nile Red is shown in green.

Fig. 3
Fig. 3

Correlation data and fits obtained from the specimen. FCS data obtained from Nile Red diffusing, fit with the dashed curve, in an aqueous solution and, fit with the dashed-dot curve, in the cell membrane. The fits are obtained by using the MEMFCS analysis routine. (Inset) Distribution of the diffusion times of Nile Red, dashed curve, in the solution and, dashed-dot curve, in the cell membrane given by the MEMFCS fit of the correlation data.

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