Abstract

We introduce two new alternative experimental realizations of dual focus fluorescence correlation spectroscopy (2fFCS), a method which allows for obtaining absolute diffusion coefficient of fast moving fluorescing molecules at nanomolar concentrations, based on fast polarization modulation of the excitation beam by a resonant electro-optical modulator. The first approach rotates every second linearly polarized laser pulse by 90 degrees to obtain independent intensity readout for both foci, similar to original design. The second approach combines polarization modulation of cw laser and fluorescence lifetime correlation spectroscopy (FLCS) like analysis to obtain clean correlation curves for both overlapping foci. We tested our new approaches with different lasers and samples, revealed a need for intensity cross-talk corrections by comparing the methods with each other and discussed experimental artifacts stemming from improper polarization alignment and detector afterpulsing. The advantages of our solutions are that the polarization rotation approach requires just one pulsed laser for each wavelength, that the polarization modulation approach even mitigates the need of pulsed lasers by using standard cw lasers and that it allows the DIC prism to be placed at an arbitrary angle. As a consequence the presented experimental solutions for 2fFCS can be more easily implemented into commercial laser scanning microscopes.

© 2014 Optical Society of America

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  1. D. Axelrod, D. E. Koppel, J. Schlessinger, E. Elson, and W. W. Webb, “Mobility measurement by analysis of fluorescence photobleaching recovery kinetics,” Biophys. J. 16(9), 1055–1069 (1976).
    [Crossref] [PubMed]
  2. D. Magde, E. L. Elson, and W. W. Webb, “Fluorescence correlation spectroscopy. II. An experimental realization,” Biopolymers 13(1), 29–61 (1974).
    [Crossref] [PubMed]
  3. N. L. Thompson, Topics in Fluorescence Spectroscopy (Plenum, 1991), pp. 337–378.
  4. M. J. Saxton, “Single-particle tracking: effects of corrals,” Biophys. J. 69(2), 389–398 (1995).
    [Crossref] [PubMed]
  5. P. Schwille, F. J. Meyer-Almes, and R. Rigler, “Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution,” Biophys. J. 72(4), 1878–1886 (1997).
    [Crossref] [PubMed]
  6. M. Böhmer, M. Wahl, H. J. Rahn, R. Erdmann, and J. Enderlein, “Time-resolved fluorescence correlation spectroscopy,” Chem. Phys. Lett. 353(5-6), 439–445 (2002).
    [Crossref]
  7. Z. Petrášek and P. Schwille, “Precise measurement of diffusion coefficients using scanning fluorescence correlation spectroscopy,” Biophys. J. 94(4), 1437–1448 (2008).
    [Crossref] [PubMed]
  8. R. Macháň and M. Hof, “Lipid diffusion in planar membranes investigated by fluorescence correlation spectroscopy,” Biochim. Biophys. Acta 1798(7), 1377–1391 (2010).
    [Crossref] [PubMed]
  9. T. Dertinger, V. Pacheco, I. von der Hocht, R. Hartmann, I. Gregor, and J. Enderlein, “Two-focus fluorescence correlation spectroscopy: A new tool for accurate and absolute diffusion measurements,” ChemPhysChem 8(3), 433–443 (2007).
    [Crossref] [PubMed]
  10. Y. Korlann, T. Dertinger, X. Michalet, S. Weiss, and J. Enderlein, “Measuring diffusion with polarization-modulation dual-focus fluorescence correlation spectroscopy,” Opt. Express 16(19), 14609–14616 (2008).
    [Crossref] [PubMed]
  11. S. Felekyan, S. Kalinin, H. Sanabria, A. Valeri, and C. A. M. Seidel, “Filtered FCS: species auto- and cross-correlation functions highlight binding and dynamics in biomolecules,” ChemPhysChem 13(4), 1036–1053 (2012).
    [Crossref] [PubMed]
  12. P. Kapusta, M. Wahl, A. Benda, M. Hof, and J. Enderlein, “Fluorescence lifetime correlation spectroscopy,” J. Fluoresc. 17(1), 43–48 (2007).
    [Crossref] [PubMed]
  13. P. Kapusta, R. Macháň, A. Benda, and M. Hof, “Fluorescence lifetime correlation spectroscopy (FLCS): concepts, applications and outlook,” Int. J. Mol. Sci. 13(10), 12890–12910 (2012).
    [Crossref] [PubMed]
  14. K. Akashi, H. Miyata, H. Itoh, and K. J. Kinosita., “Preparation of giant liposomes in physiological conditions and their characterization under an optical microscope,” Biophys. J. 71(6), 3242–3250 (1996).
    [Crossref] [PubMed]
  15. A. Benda, V. Fagul’ová, A. Deyneka, J. Enderlein, and M. Hof, “Fluorescence lifetime correlation spectroscopy combined with lifetime tuning: New perspectives in supported phospholipid bilayer research,” Langmuir 22(23), 9580–9585 (2006).
    [Crossref] [PubMed]
  16. J. Kriegsmann, I. Gregor, I. von der Hocht, J. Klare, M. Engelhard, J. Enderlein, and J. Fitter, “Translational diffusion and interaction of a photoreceptor and its cognate transducer observed in giant unilamellar vesicles by using dual-focus FCS,” ChemBioChem 10(11), 1823–1829 (2009).
    [Crossref] [PubMed]
  17. C. T. Culbertson, S. C. Jacobson, and J. Michael Ramsey, “Diffusion coefficient measurements in microfluidic devices,” Talanta 56(2), 365–373 (2002).
    [Crossref] [PubMed]
  18. A. Filippov, G. Orädd, and G. Lindblom, “The effect of cholesterol on the lateral diffusion of phospholipids in oriented bilayers,” Biophys. J. 84(5), 3079–3086 (2003).
    [Crossref] [PubMed]
  19. K. Weiss and J. Enderlein, “Lipid diffusion within black lipid membranes measured with dual-focus fluorescence correlation spectroscopy,” ChemPhysChem 13(4), 990–1000 (2012).
    [Crossref] [PubMed]
  20. A. Benda, M. Benes, V. Marecek, A. Lhotsky, W. T. Hermens, and M. Hof, “How to determine diffusion coefficients in planar phospholipid systems by confocal fluorescence correlation spectroscopy,” Langmuir 19(10), 4120–4126 (2003).
    [Crossref]
  21. T. Dertinger, A. Loman, B. Ewers, C. B. Müller, B. Krämer, and J. Enderlein, “The optics and performance of dual-focus fluorescence correlation spectroscopy,” Opt. Express 16(19), 14353–14368 (2008).
    [Crossref] [PubMed]
  22. J. Enderlein and I. Gregor, “Using fluorescence lifetime for discriminating detector afterpulsing in fluorescence-correlation spectroscopy,” Rev. Sci. Instrum. 76(3), 033102 (2005).
    [Crossref]

2012 (3)

S. Felekyan, S. Kalinin, H. Sanabria, A. Valeri, and C. A. M. Seidel, “Filtered FCS: species auto- and cross-correlation functions highlight binding and dynamics in biomolecules,” ChemPhysChem 13(4), 1036–1053 (2012).
[Crossref] [PubMed]

P. Kapusta, R. Macháň, A. Benda, and M. Hof, “Fluorescence lifetime correlation spectroscopy (FLCS): concepts, applications and outlook,” Int. J. Mol. Sci. 13(10), 12890–12910 (2012).
[Crossref] [PubMed]

K. Weiss and J. Enderlein, “Lipid diffusion within black lipid membranes measured with dual-focus fluorescence correlation spectroscopy,” ChemPhysChem 13(4), 990–1000 (2012).
[Crossref] [PubMed]

2010 (1)

R. Macháň and M. Hof, “Lipid diffusion in planar membranes investigated by fluorescence correlation spectroscopy,” Biochim. Biophys. Acta 1798(7), 1377–1391 (2010).
[Crossref] [PubMed]

2009 (1)

J. Kriegsmann, I. Gregor, I. von der Hocht, J. Klare, M. Engelhard, J. Enderlein, and J. Fitter, “Translational diffusion and interaction of a photoreceptor and its cognate transducer observed in giant unilamellar vesicles by using dual-focus FCS,” ChemBioChem 10(11), 1823–1829 (2009).
[Crossref] [PubMed]

2008 (3)

2007 (2)

T. Dertinger, V. Pacheco, I. von der Hocht, R. Hartmann, I. Gregor, and J. Enderlein, “Two-focus fluorescence correlation spectroscopy: A new tool for accurate and absolute diffusion measurements,” ChemPhysChem 8(3), 433–443 (2007).
[Crossref] [PubMed]

P. Kapusta, M. Wahl, A. Benda, M. Hof, and J. Enderlein, “Fluorescence lifetime correlation spectroscopy,” J. Fluoresc. 17(1), 43–48 (2007).
[Crossref] [PubMed]

2006 (1)

A. Benda, V. Fagul’ová, A. Deyneka, J. Enderlein, and M. Hof, “Fluorescence lifetime correlation spectroscopy combined with lifetime tuning: New perspectives in supported phospholipid bilayer research,” Langmuir 22(23), 9580–9585 (2006).
[Crossref] [PubMed]

2005 (1)

J. Enderlein and I. Gregor, “Using fluorescence lifetime for discriminating detector afterpulsing in fluorescence-correlation spectroscopy,” Rev. Sci. Instrum. 76(3), 033102 (2005).
[Crossref]

2003 (2)

A. Benda, M. Benes, V. Marecek, A. Lhotsky, W. T. Hermens, and M. Hof, “How to determine diffusion coefficients in planar phospholipid systems by confocal fluorescence correlation spectroscopy,” Langmuir 19(10), 4120–4126 (2003).
[Crossref]

A. Filippov, G. Orädd, and G. Lindblom, “The effect of cholesterol on the lateral diffusion of phospholipids in oriented bilayers,” Biophys. J. 84(5), 3079–3086 (2003).
[Crossref] [PubMed]

2002 (2)

C. T. Culbertson, S. C. Jacobson, and J. Michael Ramsey, “Diffusion coefficient measurements in microfluidic devices,” Talanta 56(2), 365–373 (2002).
[Crossref] [PubMed]

M. Böhmer, M. Wahl, H. J. Rahn, R. Erdmann, and J. Enderlein, “Time-resolved fluorescence correlation spectroscopy,” Chem. Phys. Lett. 353(5-6), 439–445 (2002).
[Crossref]

1997 (1)

P. Schwille, F. J. Meyer-Almes, and R. Rigler, “Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution,” Biophys. J. 72(4), 1878–1886 (1997).
[Crossref] [PubMed]

1996 (1)

K. Akashi, H. Miyata, H. Itoh, and K. J. Kinosita., “Preparation of giant liposomes in physiological conditions and their characterization under an optical microscope,” Biophys. J. 71(6), 3242–3250 (1996).
[Crossref] [PubMed]

1995 (1)

M. J. Saxton, “Single-particle tracking: effects of corrals,” Biophys. J. 69(2), 389–398 (1995).
[Crossref] [PubMed]

1976 (1)

D. Axelrod, D. E. Koppel, J. Schlessinger, E. Elson, and W. W. Webb, “Mobility measurement by analysis of fluorescence photobleaching recovery kinetics,” Biophys. J. 16(9), 1055–1069 (1976).
[Crossref] [PubMed]

1974 (1)

D. Magde, E. L. Elson, and W. W. Webb, “Fluorescence correlation spectroscopy. II. An experimental realization,” Biopolymers 13(1), 29–61 (1974).
[Crossref] [PubMed]

Akashi, K.

K. Akashi, H. Miyata, H. Itoh, and K. J. Kinosita., “Preparation of giant liposomes in physiological conditions and their characterization under an optical microscope,” Biophys. J. 71(6), 3242–3250 (1996).
[Crossref] [PubMed]

Axelrod, D.

D. Axelrod, D. E. Koppel, J. Schlessinger, E. Elson, and W. W. Webb, “Mobility measurement by analysis of fluorescence photobleaching recovery kinetics,” Biophys. J. 16(9), 1055–1069 (1976).
[Crossref] [PubMed]

Benda, A.

P. Kapusta, R. Macháň, A. Benda, and M. Hof, “Fluorescence lifetime correlation spectroscopy (FLCS): concepts, applications and outlook,” Int. J. Mol. Sci. 13(10), 12890–12910 (2012).
[Crossref] [PubMed]

P. Kapusta, M. Wahl, A. Benda, M. Hof, and J. Enderlein, “Fluorescence lifetime correlation spectroscopy,” J. Fluoresc. 17(1), 43–48 (2007).
[Crossref] [PubMed]

A. Benda, V. Fagul’ová, A. Deyneka, J. Enderlein, and M. Hof, “Fluorescence lifetime correlation spectroscopy combined with lifetime tuning: New perspectives in supported phospholipid bilayer research,” Langmuir 22(23), 9580–9585 (2006).
[Crossref] [PubMed]

A. Benda, M. Benes, V. Marecek, A. Lhotsky, W. T. Hermens, and M. Hof, “How to determine diffusion coefficients in planar phospholipid systems by confocal fluorescence correlation spectroscopy,” Langmuir 19(10), 4120–4126 (2003).
[Crossref]

Benes, M.

A. Benda, M. Benes, V. Marecek, A. Lhotsky, W. T. Hermens, and M. Hof, “How to determine diffusion coefficients in planar phospholipid systems by confocal fluorescence correlation spectroscopy,” Langmuir 19(10), 4120–4126 (2003).
[Crossref]

Böhmer, M.

M. Böhmer, M. Wahl, H. J. Rahn, R. Erdmann, and J. Enderlein, “Time-resolved fluorescence correlation spectroscopy,” Chem. Phys. Lett. 353(5-6), 439–445 (2002).
[Crossref]

Culbertson, C. T.

C. T. Culbertson, S. C. Jacobson, and J. Michael Ramsey, “Diffusion coefficient measurements in microfluidic devices,” Talanta 56(2), 365–373 (2002).
[Crossref] [PubMed]

Dertinger, T.

Deyneka, A.

A. Benda, V. Fagul’ová, A. Deyneka, J. Enderlein, and M. Hof, “Fluorescence lifetime correlation spectroscopy combined with lifetime tuning: New perspectives in supported phospholipid bilayer research,” Langmuir 22(23), 9580–9585 (2006).
[Crossref] [PubMed]

Elson, E.

D. Axelrod, D. E. Koppel, J. Schlessinger, E. Elson, and W. W. Webb, “Mobility measurement by analysis of fluorescence photobleaching recovery kinetics,” Biophys. J. 16(9), 1055–1069 (1976).
[Crossref] [PubMed]

Elson, E. L.

D. Magde, E. L. Elson, and W. W. Webb, “Fluorescence correlation spectroscopy. II. An experimental realization,” Biopolymers 13(1), 29–61 (1974).
[Crossref] [PubMed]

Enderlein, J.

K. Weiss and J. Enderlein, “Lipid diffusion within black lipid membranes measured with dual-focus fluorescence correlation spectroscopy,” ChemPhysChem 13(4), 990–1000 (2012).
[Crossref] [PubMed]

J. Kriegsmann, I. Gregor, I. von der Hocht, J. Klare, M. Engelhard, J. Enderlein, and J. Fitter, “Translational diffusion and interaction of a photoreceptor and its cognate transducer observed in giant unilamellar vesicles by using dual-focus FCS,” ChemBioChem 10(11), 1823–1829 (2009).
[Crossref] [PubMed]

Y. Korlann, T. Dertinger, X. Michalet, S. Weiss, and J. Enderlein, “Measuring diffusion with polarization-modulation dual-focus fluorescence correlation spectroscopy,” Opt. Express 16(19), 14609–14616 (2008).
[Crossref] [PubMed]

T. Dertinger, A. Loman, B. Ewers, C. B. Müller, B. Krämer, and J. Enderlein, “The optics and performance of dual-focus fluorescence correlation spectroscopy,” Opt. Express 16(19), 14353–14368 (2008).
[Crossref] [PubMed]

P. Kapusta, M. Wahl, A. Benda, M. Hof, and J. Enderlein, “Fluorescence lifetime correlation spectroscopy,” J. Fluoresc. 17(1), 43–48 (2007).
[Crossref] [PubMed]

T. Dertinger, V. Pacheco, I. von der Hocht, R. Hartmann, I. Gregor, and J. Enderlein, “Two-focus fluorescence correlation spectroscopy: A new tool for accurate and absolute diffusion measurements,” ChemPhysChem 8(3), 433–443 (2007).
[Crossref] [PubMed]

A. Benda, V. Fagul’ová, A. Deyneka, J. Enderlein, and M. Hof, “Fluorescence lifetime correlation spectroscopy combined with lifetime tuning: New perspectives in supported phospholipid bilayer research,” Langmuir 22(23), 9580–9585 (2006).
[Crossref] [PubMed]

J. Enderlein and I. Gregor, “Using fluorescence lifetime for discriminating detector afterpulsing in fluorescence-correlation spectroscopy,” Rev. Sci. Instrum. 76(3), 033102 (2005).
[Crossref]

M. Böhmer, M. Wahl, H. J. Rahn, R. Erdmann, and J. Enderlein, “Time-resolved fluorescence correlation spectroscopy,” Chem. Phys. Lett. 353(5-6), 439–445 (2002).
[Crossref]

Engelhard, M.

J. Kriegsmann, I. Gregor, I. von der Hocht, J. Klare, M. Engelhard, J. Enderlein, and J. Fitter, “Translational diffusion and interaction of a photoreceptor and its cognate transducer observed in giant unilamellar vesicles by using dual-focus FCS,” ChemBioChem 10(11), 1823–1829 (2009).
[Crossref] [PubMed]

Erdmann, R.

M. Böhmer, M. Wahl, H. J. Rahn, R. Erdmann, and J. Enderlein, “Time-resolved fluorescence correlation spectroscopy,” Chem. Phys. Lett. 353(5-6), 439–445 (2002).
[Crossref]

Ewers, B.

Fagul’ová, V.

A. Benda, V. Fagul’ová, A. Deyneka, J. Enderlein, and M. Hof, “Fluorescence lifetime correlation spectroscopy combined with lifetime tuning: New perspectives in supported phospholipid bilayer research,” Langmuir 22(23), 9580–9585 (2006).
[Crossref] [PubMed]

Felekyan, S.

S. Felekyan, S. Kalinin, H. Sanabria, A. Valeri, and C. A. M. Seidel, “Filtered FCS: species auto- and cross-correlation functions highlight binding and dynamics in biomolecules,” ChemPhysChem 13(4), 1036–1053 (2012).
[Crossref] [PubMed]

Filippov, A.

A. Filippov, G. Orädd, and G. Lindblom, “The effect of cholesterol on the lateral diffusion of phospholipids in oriented bilayers,” Biophys. J. 84(5), 3079–3086 (2003).
[Crossref] [PubMed]

Fitter, J.

J. Kriegsmann, I. Gregor, I. von der Hocht, J. Klare, M. Engelhard, J. Enderlein, and J. Fitter, “Translational diffusion and interaction of a photoreceptor and its cognate transducer observed in giant unilamellar vesicles by using dual-focus FCS,” ChemBioChem 10(11), 1823–1829 (2009).
[Crossref] [PubMed]

Gregor, I.

J. Kriegsmann, I. Gregor, I. von der Hocht, J. Klare, M. Engelhard, J. Enderlein, and J. Fitter, “Translational diffusion and interaction of a photoreceptor and its cognate transducer observed in giant unilamellar vesicles by using dual-focus FCS,” ChemBioChem 10(11), 1823–1829 (2009).
[Crossref] [PubMed]

T. Dertinger, V. Pacheco, I. von der Hocht, R. Hartmann, I. Gregor, and J. Enderlein, “Two-focus fluorescence correlation spectroscopy: A new tool for accurate and absolute diffusion measurements,” ChemPhysChem 8(3), 433–443 (2007).
[Crossref] [PubMed]

J. Enderlein and I. Gregor, “Using fluorescence lifetime for discriminating detector afterpulsing in fluorescence-correlation spectroscopy,” Rev. Sci. Instrum. 76(3), 033102 (2005).
[Crossref]

Hartmann, R.

T. Dertinger, V. Pacheco, I. von der Hocht, R. Hartmann, I. Gregor, and J. Enderlein, “Two-focus fluorescence correlation spectroscopy: A new tool for accurate and absolute diffusion measurements,” ChemPhysChem 8(3), 433–443 (2007).
[Crossref] [PubMed]

Hermens, W. T.

A. Benda, M. Benes, V. Marecek, A. Lhotsky, W. T. Hermens, and M. Hof, “How to determine diffusion coefficients in planar phospholipid systems by confocal fluorescence correlation spectroscopy,” Langmuir 19(10), 4120–4126 (2003).
[Crossref]

Hof, M.

P. Kapusta, R. Macháň, A. Benda, and M. Hof, “Fluorescence lifetime correlation spectroscopy (FLCS): concepts, applications and outlook,” Int. J. Mol. Sci. 13(10), 12890–12910 (2012).
[Crossref] [PubMed]

R. Macháň and M. Hof, “Lipid diffusion in planar membranes investigated by fluorescence correlation spectroscopy,” Biochim. Biophys. Acta 1798(7), 1377–1391 (2010).
[Crossref] [PubMed]

P. Kapusta, M. Wahl, A. Benda, M. Hof, and J. Enderlein, “Fluorescence lifetime correlation spectroscopy,” J. Fluoresc. 17(1), 43–48 (2007).
[Crossref] [PubMed]

A. Benda, V. Fagul’ová, A. Deyneka, J. Enderlein, and M. Hof, “Fluorescence lifetime correlation spectroscopy combined with lifetime tuning: New perspectives in supported phospholipid bilayer research,” Langmuir 22(23), 9580–9585 (2006).
[Crossref] [PubMed]

A. Benda, M. Benes, V. Marecek, A. Lhotsky, W. T. Hermens, and M. Hof, “How to determine diffusion coefficients in planar phospholipid systems by confocal fluorescence correlation spectroscopy,” Langmuir 19(10), 4120–4126 (2003).
[Crossref]

Itoh, H.

K. Akashi, H. Miyata, H. Itoh, and K. J. Kinosita., “Preparation of giant liposomes in physiological conditions and their characterization under an optical microscope,” Biophys. J. 71(6), 3242–3250 (1996).
[Crossref] [PubMed]

Jacobson, S. C.

C. T. Culbertson, S. C. Jacobson, and J. Michael Ramsey, “Diffusion coefficient measurements in microfluidic devices,” Talanta 56(2), 365–373 (2002).
[Crossref] [PubMed]

Kalinin, S.

S. Felekyan, S. Kalinin, H. Sanabria, A. Valeri, and C. A. M. Seidel, “Filtered FCS: species auto- and cross-correlation functions highlight binding and dynamics in biomolecules,” ChemPhysChem 13(4), 1036–1053 (2012).
[Crossref] [PubMed]

Kapusta, P.

P. Kapusta, R. Macháň, A. Benda, and M. Hof, “Fluorescence lifetime correlation spectroscopy (FLCS): concepts, applications and outlook,” Int. J. Mol. Sci. 13(10), 12890–12910 (2012).
[Crossref] [PubMed]

P. Kapusta, M. Wahl, A. Benda, M. Hof, and J. Enderlein, “Fluorescence lifetime correlation spectroscopy,” J. Fluoresc. 17(1), 43–48 (2007).
[Crossref] [PubMed]

Kinosita, K. J.

K. Akashi, H. Miyata, H. Itoh, and K. J. Kinosita., “Preparation of giant liposomes in physiological conditions and their characterization under an optical microscope,” Biophys. J. 71(6), 3242–3250 (1996).
[Crossref] [PubMed]

Klare, J.

J. Kriegsmann, I. Gregor, I. von der Hocht, J. Klare, M. Engelhard, J. Enderlein, and J. Fitter, “Translational diffusion and interaction of a photoreceptor and its cognate transducer observed in giant unilamellar vesicles by using dual-focus FCS,” ChemBioChem 10(11), 1823–1829 (2009).
[Crossref] [PubMed]

Koppel, D. E.

D. Axelrod, D. E. Koppel, J. Schlessinger, E. Elson, and W. W. Webb, “Mobility measurement by analysis of fluorescence photobleaching recovery kinetics,” Biophys. J. 16(9), 1055–1069 (1976).
[Crossref] [PubMed]

Korlann, Y.

Krämer, B.

Kriegsmann, J.

J. Kriegsmann, I. Gregor, I. von der Hocht, J. Klare, M. Engelhard, J. Enderlein, and J. Fitter, “Translational diffusion and interaction of a photoreceptor and its cognate transducer observed in giant unilamellar vesicles by using dual-focus FCS,” ChemBioChem 10(11), 1823–1829 (2009).
[Crossref] [PubMed]

Lhotsky, A.

A. Benda, M. Benes, V. Marecek, A. Lhotsky, W. T. Hermens, and M. Hof, “How to determine diffusion coefficients in planar phospholipid systems by confocal fluorescence correlation spectroscopy,” Langmuir 19(10), 4120–4126 (2003).
[Crossref]

Lindblom, G.

A. Filippov, G. Orädd, and G. Lindblom, “The effect of cholesterol on the lateral diffusion of phospholipids in oriented bilayers,” Biophys. J. 84(5), 3079–3086 (2003).
[Crossref] [PubMed]

Loman, A.

Machán, R.

P. Kapusta, R. Macháň, A. Benda, and M. Hof, “Fluorescence lifetime correlation spectroscopy (FLCS): concepts, applications and outlook,” Int. J. Mol. Sci. 13(10), 12890–12910 (2012).
[Crossref] [PubMed]

R. Macháň and M. Hof, “Lipid diffusion in planar membranes investigated by fluorescence correlation spectroscopy,” Biochim. Biophys. Acta 1798(7), 1377–1391 (2010).
[Crossref] [PubMed]

Magde, D.

D. Magde, E. L. Elson, and W. W. Webb, “Fluorescence correlation spectroscopy. II. An experimental realization,” Biopolymers 13(1), 29–61 (1974).
[Crossref] [PubMed]

Marecek, V.

A. Benda, M. Benes, V. Marecek, A. Lhotsky, W. T. Hermens, and M. Hof, “How to determine diffusion coefficients in planar phospholipid systems by confocal fluorescence correlation spectroscopy,” Langmuir 19(10), 4120–4126 (2003).
[Crossref]

Meyer-Almes, F. J.

P. Schwille, F. J. Meyer-Almes, and R. Rigler, “Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution,” Biophys. J. 72(4), 1878–1886 (1997).
[Crossref] [PubMed]

Michael Ramsey, J.

C. T. Culbertson, S. C. Jacobson, and J. Michael Ramsey, “Diffusion coefficient measurements in microfluidic devices,” Talanta 56(2), 365–373 (2002).
[Crossref] [PubMed]

Michalet, X.

Miyata, H.

K. Akashi, H. Miyata, H. Itoh, and K. J. Kinosita., “Preparation of giant liposomes in physiological conditions and their characterization under an optical microscope,” Biophys. J. 71(6), 3242–3250 (1996).
[Crossref] [PubMed]

Müller, C. B.

Orädd, G.

A. Filippov, G. Orädd, and G. Lindblom, “The effect of cholesterol on the lateral diffusion of phospholipids in oriented bilayers,” Biophys. J. 84(5), 3079–3086 (2003).
[Crossref] [PubMed]

Pacheco, V.

T. Dertinger, V. Pacheco, I. von der Hocht, R. Hartmann, I. Gregor, and J. Enderlein, “Two-focus fluorescence correlation spectroscopy: A new tool for accurate and absolute diffusion measurements,” ChemPhysChem 8(3), 433–443 (2007).
[Crossref] [PubMed]

Petrášek, Z.

Z. Petrášek and P. Schwille, “Precise measurement of diffusion coefficients using scanning fluorescence correlation spectroscopy,” Biophys. J. 94(4), 1437–1448 (2008).
[Crossref] [PubMed]

Rahn, H. J.

M. Böhmer, M. Wahl, H. J. Rahn, R. Erdmann, and J. Enderlein, “Time-resolved fluorescence correlation spectroscopy,” Chem. Phys. Lett. 353(5-6), 439–445 (2002).
[Crossref]

Rigler, R.

P. Schwille, F. J. Meyer-Almes, and R. Rigler, “Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution,” Biophys. J. 72(4), 1878–1886 (1997).
[Crossref] [PubMed]

Sanabria, H.

S. Felekyan, S. Kalinin, H. Sanabria, A. Valeri, and C. A. M. Seidel, “Filtered FCS: species auto- and cross-correlation functions highlight binding and dynamics in biomolecules,” ChemPhysChem 13(4), 1036–1053 (2012).
[Crossref] [PubMed]

Saxton, M. J.

M. J. Saxton, “Single-particle tracking: effects of corrals,” Biophys. J. 69(2), 389–398 (1995).
[Crossref] [PubMed]

Schlessinger, J.

D. Axelrod, D. E. Koppel, J. Schlessinger, E. Elson, and W. W. Webb, “Mobility measurement by analysis of fluorescence photobleaching recovery kinetics,” Biophys. J. 16(9), 1055–1069 (1976).
[Crossref] [PubMed]

Schwille, P.

Z. Petrášek and P. Schwille, “Precise measurement of diffusion coefficients using scanning fluorescence correlation spectroscopy,” Biophys. J. 94(4), 1437–1448 (2008).
[Crossref] [PubMed]

P. Schwille, F. J. Meyer-Almes, and R. Rigler, “Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution,” Biophys. J. 72(4), 1878–1886 (1997).
[Crossref] [PubMed]

Seidel, C. A. M.

S. Felekyan, S. Kalinin, H. Sanabria, A. Valeri, and C. A. M. Seidel, “Filtered FCS: species auto- and cross-correlation functions highlight binding and dynamics in biomolecules,” ChemPhysChem 13(4), 1036–1053 (2012).
[Crossref] [PubMed]

Valeri, A.

S. Felekyan, S. Kalinin, H. Sanabria, A. Valeri, and C. A. M. Seidel, “Filtered FCS: species auto- and cross-correlation functions highlight binding and dynamics in biomolecules,” ChemPhysChem 13(4), 1036–1053 (2012).
[Crossref] [PubMed]

von der Hocht, I.

J. Kriegsmann, I. Gregor, I. von der Hocht, J. Klare, M. Engelhard, J. Enderlein, and J. Fitter, “Translational diffusion and interaction of a photoreceptor and its cognate transducer observed in giant unilamellar vesicles by using dual-focus FCS,” ChemBioChem 10(11), 1823–1829 (2009).
[Crossref] [PubMed]

T. Dertinger, V. Pacheco, I. von der Hocht, R. Hartmann, I. Gregor, and J. Enderlein, “Two-focus fluorescence correlation spectroscopy: A new tool for accurate and absolute diffusion measurements,” ChemPhysChem 8(3), 433–443 (2007).
[Crossref] [PubMed]

Wahl, M.

P. Kapusta, M. Wahl, A. Benda, M. Hof, and J. Enderlein, “Fluorescence lifetime correlation spectroscopy,” J. Fluoresc. 17(1), 43–48 (2007).
[Crossref] [PubMed]

M. Böhmer, M. Wahl, H. J. Rahn, R. Erdmann, and J. Enderlein, “Time-resolved fluorescence correlation spectroscopy,” Chem. Phys. Lett. 353(5-6), 439–445 (2002).
[Crossref]

Webb, W. W.

D. Axelrod, D. E. Koppel, J. Schlessinger, E. Elson, and W. W. Webb, “Mobility measurement by analysis of fluorescence photobleaching recovery kinetics,” Biophys. J. 16(9), 1055–1069 (1976).
[Crossref] [PubMed]

D. Magde, E. L. Elson, and W. W. Webb, “Fluorescence correlation spectroscopy. II. An experimental realization,” Biopolymers 13(1), 29–61 (1974).
[Crossref] [PubMed]

Weiss, K.

K. Weiss and J. Enderlein, “Lipid diffusion within black lipid membranes measured with dual-focus fluorescence correlation spectroscopy,” ChemPhysChem 13(4), 990–1000 (2012).
[Crossref] [PubMed]

Weiss, S.

Biochim. Biophys. Acta (1)

R. Macháň and M. Hof, “Lipid diffusion in planar membranes investigated by fluorescence correlation spectroscopy,” Biochim. Biophys. Acta 1798(7), 1377–1391 (2010).
[Crossref] [PubMed]

Biophys. J. (6)

D. Axelrod, D. E. Koppel, J. Schlessinger, E. Elson, and W. W. Webb, “Mobility measurement by analysis of fluorescence photobleaching recovery kinetics,” Biophys. J. 16(9), 1055–1069 (1976).
[Crossref] [PubMed]

M. J. Saxton, “Single-particle tracking: effects of corrals,” Biophys. J. 69(2), 389–398 (1995).
[Crossref] [PubMed]

P. Schwille, F. J. Meyer-Almes, and R. Rigler, “Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution,” Biophys. J. 72(4), 1878–1886 (1997).
[Crossref] [PubMed]

Z. Petrášek and P. Schwille, “Precise measurement of diffusion coefficients using scanning fluorescence correlation spectroscopy,” Biophys. J. 94(4), 1437–1448 (2008).
[Crossref] [PubMed]

K. Akashi, H. Miyata, H. Itoh, and K. J. Kinosita., “Preparation of giant liposomes in physiological conditions and their characterization under an optical microscope,” Biophys. J. 71(6), 3242–3250 (1996).
[Crossref] [PubMed]

A. Filippov, G. Orädd, and G. Lindblom, “The effect of cholesterol on the lateral diffusion of phospholipids in oriented bilayers,” Biophys. J. 84(5), 3079–3086 (2003).
[Crossref] [PubMed]

Biopolymers (1)

D. Magde, E. L. Elson, and W. W. Webb, “Fluorescence correlation spectroscopy. II. An experimental realization,” Biopolymers 13(1), 29–61 (1974).
[Crossref] [PubMed]

Chem. Phys. Lett. (1)

M. Böhmer, M. Wahl, H. J. Rahn, R. Erdmann, and J. Enderlein, “Time-resolved fluorescence correlation spectroscopy,” Chem. Phys. Lett. 353(5-6), 439–445 (2002).
[Crossref]

ChemBioChem (1)

J. Kriegsmann, I. Gregor, I. von der Hocht, J. Klare, M. Engelhard, J. Enderlein, and J. Fitter, “Translational diffusion and interaction of a photoreceptor and its cognate transducer observed in giant unilamellar vesicles by using dual-focus FCS,” ChemBioChem 10(11), 1823–1829 (2009).
[Crossref] [PubMed]

ChemPhysChem (3)

T. Dertinger, V. Pacheco, I. von der Hocht, R. Hartmann, I. Gregor, and J. Enderlein, “Two-focus fluorescence correlation spectroscopy: A new tool for accurate and absolute diffusion measurements,” ChemPhysChem 8(3), 433–443 (2007).
[Crossref] [PubMed]

S. Felekyan, S. Kalinin, H. Sanabria, A. Valeri, and C. A. M. Seidel, “Filtered FCS: species auto- and cross-correlation functions highlight binding and dynamics in biomolecules,” ChemPhysChem 13(4), 1036–1053 (2012).
[Crossref] [PubMed]

K. Weiss and J. Enderlein, “Lipid diffusion within black lipid membranes measured with dual-focus fluorescence correlation spectroscopy,” ChemPhysChem 13(4), 990–1000 (2012).
[Crossref] [PubMed]

Int. J. Mol. Sci. (1)

P. Kapusta, R. Macháň, A. Benda, and M. Hof, “Fluorescence lifetime correlation spectroscopy (FLCS): concepts, applications and outlook,” Int. J. Mol. Sci. 13(10), 12890–12910 (2012).
[Crossref] [PubMed]

J. Fluoresc. (1)

P. Kapusta, M. Wahl, A. Benda, M. Hof, and J. Enderlein, “Fluorescence lifetime correlation spectroscopy,” J. Fluoresc. 17(1), 43–48 (2007).
[Crossref] [PubMed]

Langmuir (2)

A. Benda, V. Fagul’ová, A. Deyneka, J. Enderlein, and M. Hof, “Fluorescence lifetime correlation spectroscopy combined with lifetime tuning: New perspectives in supported phospholipid bilayer research,” Langmuir 22(23), 9580–9585 (2006).
[Crossref] [PubMed]

A. Benda, M. Benes, V. Marecek, A. Lhotsky, W. T. Hermens, and M. Hof, “How to determine diffusion coefficients in planar phospholipid systems by confocal fluorescence correlation spectroscopy,” Langmuir 19(10), 4120–4126 (2003).
[Crossref]

Opt. Express (2)

Rev. Sci. Instrum. (1)

J. Enderlein and I. Gregor, “Using fluorescence lifetime for discriminating detector afterpulsing in fluorescence-correlation spectroscopy,” Rev. Sci. Instrum. 76(3), 033102 (2005).
[Crossref]

Talanta (1)

C. T. Culbertson, S. C. Jacobson, and J. Michael Ramsey, “Diffusion coefficient measurements in microfluidic devices,” Talanta 56(2), 365–373 (2002).
[Crossref] [PubMed]

Other (1)

N. L. Thompson, Topics in Fluorescence Spectroscopy (Plenum, 1991), pp. 337–378.

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Figures (5)

Fig. 1
Fig. 1 Schematic illustration of the 2fFCS laser setup as modified in this contribution (DM corresponds to dichroic mirror). Inset: The EOM crystal periodically modulates the polarization state of the laser beam. The pulsed excitation (red lines) is synchronized with minima and maxima of polarization amplitudes for vertical and horizontal polarizations (green and blue lines), which results in discrete horizontal and vertical polarized excitation pulsed. In case of cw excitation EOM produces time varying elliptically polarized light (red spiral).

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Fig. 2
Fig. 2 (A) Theoretical time profile of excitation intensity in both foci (Eq. (9)) The red full lines show a time-profile for voltage amplitude equal to a quarter-wave voltage, the dotted blue lines for maximum plateau width voltage (1.128 times a quarter-wave voltage). The rectangles show time slots suitable for laser pulse positioning with less than 1% of cross-talk. The left rectangle is for a quarter-wave voltage and the right rectangle is for a maximum plateau width voltage. (B) Experimental time profiles of detected (B left) reflected light (diode laser 470 nm in cw mode) and (B right) fluorescence (Alexa 488) intensities for excitation polarized along both axes of DIC crystal. The experimental data are fitted by Eq. (10) (red lines). Fit parameters indicate that the zero voltage retardance of EO crystal is 0.62 ± 0.01, voltage amplitude is 0.79 ± 0.01 and Alexa 488 excited state lifetime 4.4 ns.

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Fig. 3
Fig. 3 Flowchart of data processing. (A) and (B) Area normalized histograms of photon arrival times for pulsed (A) and cw (B) acquisition (20 MHz sync frequency). The overall fluorescent signal (black line) is a linear combination of vertical polarization signal (red line), horizontal polarization signal (blue line) and of a constant afterpulsing and dark noise (green and brown). For cw excitation (B) the constant noise is a linear combination of vertical and horizontal polarization signal and cannot be separated. (C) and (D) Photon weighting filters for pulsed and cw acquisition, respectively, created from time resolved patterns (A) and (B). The sum of the patterns for each channel equals 1 (black line), keeping the overall intensity constant and showing that the overall signal is indeed a linear combination of the components. (E) and (F) Auto- and cross- correlation functions obtained by filtered correlation, globally fitted by Eq. (6), using the experimental standard error for weighting. Auto-correlation for focus 1 is obtained from vertical polarization filter, auto-correlation for focus 2 is obtained from horizontal polarization filter, and cross-correlation is obtained by cross-correlating filters for vertical and horizontal polarizations. Amplitudes of auto-correlations for different foci can differ due to different excitation light transmission efficiencies of vertically and horizontally polarized light through the optical system and the efficiency/sensitivity of detectors.

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Fig. 4
Fig. 4 (A) Lateral diffusion coefficients (D) determined for two different fluorescence dyes in SPBs, perylene (black squares) and BODIPY FL DHPE (red circles), in dependence on axial position in the detection volume. (B) Beam waist radii (ω0) as determined from the analysis of 2fFCS data (405 nm black squares and 470 nm red circles). Each point at particular axial position corresponds to D in Fig. 4(a).

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Fig. 5
Fig. 5 Comparison of normalized correlation curves obtained by pulsed and modulated 2fFCS for Alexa 488 in aqueous solution. The small intensity cross-talk between foci for pulsed 2fFCS causes the resulting correlation curves to be a mixture of auto- and cross-correlations instead of being pure auto- or cross-correlations (Eq. (8)). This results in a longer apparent diffusion time for auto-correlation curve and a higher amplitude for cross-correlation curve compared to cross-talk free curves obtained by modulation 2fFCS.

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Tables (1)

Tables Icon

Table 1 Diffusion coefficients for fluorescence standards in aqueous solution and lipid tracers in model bilayers (perylene (ex. 405 nm) and BODIPY FL DHPE (ex. 470 nm)). All values were achieved at 24°C. The error is expressed by standard deviation values (SD). * value corresponds to Atto488-carboxylic acid, ** value corresponds to Rhodamine B.

View Table

Equations (10)

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I j ( t )= ∑ k=1 n w k ( t ) p j k
w k ( t )= ∑ j=1 N f j k I j ( t )
f j k = ( [ M ⌢ T ⋅diag 〈 I j ( t ) 〉 t −1 ⋅ M ⌢ ] −1 ⋅ M ⌢ T ⋅diag 〈 I j ( t ) 〉 t −1 ) kj
M ^ jk = p j k
G kl ( τ )= 〈 w k ( t ) w l ( t+τ ) 〉 t 〈 w k ( t ) 〉 t 〈 w l ( t ) 〉 t = ∑ i=1 N ∑ j=1 N f i k f j l 〈 I i ( t ) I j ( t+τ ) 〉 t ∑ i=1 N ∑ j=1 N f i k f j l 〈 I i ( t ) 〉 t 〈 I j ( t ) 〉 t
g(t,δ)= g ∞ (δ)+2 ε 1 ε 2 c π Dt ∫ −∞ ∞ d z 1 ∫ −∞ ∞ d z 2 κ( z 1 )κ( z 2 ) 8Dt+ ω 2 ( z 1 )+ ω 2 ( z 2 ) exp[ − ( z 2 − z 1 ) 2 4Dt − 2 δ 2 8Dt+ ω 2 ( z 1 )+ ω 2 ( z 2 ) ]
g(t,δ)= g ∞ (δ)+ π ε 3 c 4 1 4Dt+ ω 2 (z) exp( − δ 2 4Dt+ ω 2 (z) ).
G A1 = (1− l 1 ) 2 + l 1 2 )g(t,0)+(2(1− l 1 ) l 1 )g(t,δ), G A2 = (1− l 2 ) 2 + l 2 2 )g(t,0)+(2(1− l 2 ) l 2 )g(t,δ), G C12 = G C21 =((1− l 1 ) l 2 +(1− l 2 ) l 1 )g(t,0)+((1− l 1 )(1− l 2 )+ l 1 l 2 )g(t,δ)
I f1 (t)= I 0 cos 2 ( 1 2 ( α+π V max V π sin( 2πf( t− t 0 ) ) ) ), I f2 (t)= I 0 sin 2 ( 1 2 ( α+π V max V π sin( 2πf( t− t 0 ) ) ) )
F f1 (t)= F 0 +( I 0 cos 2 ( 1 2 ( α+π V max V π sin( 2πf( t− t 0 ) ) ) ) )* e −t/ t 1 , F f2 (t)= F 0 +( I 0 sin 2 ( 1 2 ( α+π V max V π sin( 2πf( t− t 0 ) ) ) ) )* e −t/ t 1 .

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