Abstract

We discuss circular scanning Fluorescence Correlation Spectroscopy (sFCS) as a simple extension of standard FCS for accurate, robust and fast diffusion measurements on membranes. The implementation is based on a straightforward conversion of a conventional FCS instrument to a sFCS device by mounting a mirror onto a two-axis piezo scanner. The measurement volume is scanned in a circle with sub-micron radius, allowing the determination of diffusion coefficients and concentrations without any a priori knowledge of the size of the detection volume. This is highly important in measurements on two-dimensional surfaces, where the volume size, and therefore the quantitative outcome of the experiment, is determined by the relative position of the surface and the objective focus, a parameter difficult to control in practice. The technique is applied to diffusion measurements on model membrane systems: supported lipid bilayers and giant unilamellar vesicles. We show that the method is insensitive to membrane positioning and to disturbing processes on faster or slower time scales than diffusion, and yields accurate results even for fluctuating or drifting membranes. Its robustness, short measurement times, and small size of the probed area makes this technique particularly attractive for analyzing the properties of membranes and molecules diffusing and interacting within them.

© 2011 OSA

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  1. E. L. Elson and D. Magde, “Fluorescence correlation spectroscopy. I. Conceptual basis and theory,” Biopolymers 13, 1–27 (1974).
    [CrossRef]
  2. R. Rigler and E. S. Elson, eds., Fluorescence Correlation Spectroscopy. Theory and Application, Chemical Physics Series (Springer Verlag, Berlin, 2001), 1st ed.
    [CrossRef]
  3. K. Bacia and P. Schwille, “A dynamic view of cellular processes by in vivo fluorescence auto- and cross-correlation spectroscopy,” Methods 29, 74–85 (2003).
    [CrossRef] [PubMed]
  4. 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, 1055–1069 (1976).
    [CrossRef] [PubMed]
  5. M. J. Saxton and K. Jacobson, “Single-particle tracking: Applications to membrane dynamics,” Annu. Rev. Biophys. Biomol. Struct. 26, 373–399 (1997).
    [CrossRef] [PubMed]
  6. A. J. García-Sáez and P. Schwille, “Fluorescence correlation spectroscopy for the study of membrane dynamics and protein/lipid interactions,” Methods 46, 116–122 (2008).
    [CrossRef] [PubMed]
  7. R. Macháň and M. Hof, “Lipid diffusion in planar membranes investigated by fluorescence correlation spectroscopy,” Biochim. Biophys. Acta Biomembr. 1798, 1377–1391 (2010).
    [CrossRef]
  8. P. Schwille, F. J. Meyer-Almes, and R. Rigler, “Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution,” Biophys. J. 72, 1878–1886 (1997).
    [CrossRef] [PubMed]
  9. K. Bacia, S. A. Kim, and P. Schwille, “Fluorescence cross-correlation spectroscopy in living cells,” Nat. Methods 3, 83–89 (2006).
    [CrossRef] [PubMed]
  10. J. Enderlein, I. Gregor, D. Patra, and J. Fitter, “Art and artefacts of fluorescence correlation spectroscopy,” Curr. Pharm. Biotechnol. 5, 155–161 (2004).
    [CrossRef] [PubMed]
  11. S. Milon, R. Hovius, H. Vogel, and T. Wohland, “Factors influencing fluorescence correlation spectroscopy measurements on membranes: simulations and experiments,” Chem. Phys. 288, 171–186 (2003).
    [CrossRef]
  12. A. Benda, M. Beneš, V. Mareček, A. Lhotský, W. T. Hermens, and M. Hof, “How to determine diffusion coefficients in planar phospholipid systems by confocal fluorescence correlation spectroscopy,” Langmuir 19, 4120–4126 (2003).
    [CrossRef]
  13. E. Gielen, M. Vandeven, A. Margineanu, P. Dedecker, M. Van der Auweraer, Y. Engelborghs, J. Hofkens, and M. Ameloot, “On the use of z-scan fluorescence correlation experiments on giant unilamellar vesicles,” Chem. Phys. Lett. 469, 110–114 (2009).
    [CrossRef]
  14. 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 measements,” ChemPhysChem 8, 433–443 (2007).
    [CrossRef] [PubMed]
  15. Z. Petrášek, C. Hoege, A. Mashaghi, T. Ohrt, A. A. Hyman, and P. Schwille, “Characterization of protein dynamics in asymmetric cell division by scanning fluorescence correlation spectroscopy,” Biophys. J. 95, 5476–5486 (2008).
    [CrossRef]
  16. Z. Petrášek, C. Hoege, A. A. Hyman, and P. Schwille, “Two-photon fluorescence imaging and correlation analysis applied to protein dynamics in C. elegans embryo,” Proc. SPIE 6860, 68601L (2008).
    [CrossRef]
  17. J. Ries, S. Chiantia, and P. Schwille, “Accurate determination of membrane dynamics with line-scan FCS,” Biophys. J. 96, 1999–2008 (2009).
    [CrossRef] [PubMed]
  18. M. A. Digman, C. M. Brown, P. Sengupta, P. W. Wiseman, A. R. Horwitz, and E. Gratton, “Measuring fast dynamics in solutions and cells with a laser scanning microscope,” Biophys. J. 89, 1317–1327 (2005).
    [CrossRef] [PubMed]
  19. J. P. Skinner, Y. Chen, and J. D. Müller, “Position-sensitive scanning fluorescence correlation spectroscopy,” Biophys. J. 89, 1288–1301 (2005).
    [CrossRef] [PubMed]
  20. Z. Petrášek and P. Schwille, “Precise measurement of diffusion coefficients using scanning fluorescence correlation spectroscopy,” Biophys. J. 94, 1437–1448 (2008).
    [CrossRef]
  21. H. Qian and E. L. Elson, “Analysis of confocal laser-microscope optics for 3-D fluorescence correlation spectroscopy,” Appl. Opt. 30, 1185–1195 (1991).
    [CrossRef] [PubMed]
  22. S. T. Hess and W. W. Webb, “Focal volume optics and experimental artifacts in confocal fluorescence correlation spectroscopy,” Biophys. J. 83, 2300–2317 (2002).
    [CrossRef] [PubMed]
  23. M. Born and E. Wolf, Principles of Optics (Cambridge University Press, 1999), chap. 8, pp. 484–492, 7th ed.
  24. K. M. Berland, P. T. C. So, Y. Chen, W. W. Mantulin, and E. Gratton, “Scanning two-photon fluctuation correlation spectroscopy: Particle counting measurements for detection of molecular aggregation,” Biophys. J. 71, 410–420 (1996).
    [CrossRef] [PubMed]
  25. J. Widengren, U. Mets, and R. Rigler, “Fluorescence correlation spectroscopy of triplet states in solution: A theoretical and experimental study,” J. Phys. Chem. 99, 13368–13379 (1995).
    [CrossRef]
  26. S. Chiantia, N. Kahya, and P. Schwille, “Dehydration damage of domain-exhibiting supported bilayers: An AFM study on the protective effects of disaccharides and other stabilizing substances,” Langmuir 21, 6317–6323 (2005).
    [CrossRef] [PubMed]
  27. D. C. Carrer, E. Kummer, G. Chwastek, S. Chiantia, and P. Schwille, “Asymmetry determines the effects of natural ceramides on model membranes,” Soft Matter 5, 3279–3286 (2009).
    [CrossRef]
  28. N. Kahya, “Protein-protein and protein-lipid interactions in domain-assembly: Lessons from giant unilamellar vesicles,” Biochim. Biophys. Acta Biomembr. 1798, 1392–1398 (2010).
    [CrossRef]
  29. K. Bacia, D. Scherfeld, N. Kahya, and P. Schwille, “Fluorescence correlation spectroscopy relates rafts in model and native membranes,” Biophys. J. 87, 1034–1043 (2004).
    [CrossRef] [PubMed]
  30. M. Przybylo, J. Sýkora, J. Humpolíčková, A. Benda, A. Zan, and M. Hof, “Lipid diffusion in giant unilamellar vesicles is more than 2 times faster than in supported phospholipid bilayers under identical conditions,” Langmuir 22, 9096–9099 (2006).
    [CrossRef] [PubMed]
  31. S. Chiantia, J. Ries, N. Kahya, and P. Schwille, “Combined AFM and two-focus SFCS study of raft-exhibiting model membranes,” ChemPhysChem 7, 2409–2418 (2006).
    [CrossRef] [PubMed]
  32. U. Haupts, S. Maiti, P. Schwille, and W. W. Webb, “Dynamics of fluorescence fluctuations in green fluorescent protein observed by fluorescence correlation spectroscopy,” Proc. Natl. Acad. Sci. U. S. A. 95, 13573–13578 (1998).
    [CrossRef] [PubMed]
  33. J. Hendrix, C. Flors, P. Dedecker, J. Hofkens, and Y. Engelborghs, “Dark states in monomeric red fluorescent proteins studied by fluorescence correlation and single molecule spectroscopy,” Biophys. J. 94, 4103–4113 (2008).
    [CrossRef] [PubMed]
  34. J. Widengren and P. Schwille, “Characterization of photoinduced isomerization and back-isomerization of the cyanine dye Cy5 by fluorescence correlation spectroscopy,” J. Phys. Chem. A 104, 6416–6428 (2000).
    [CrossRef]
  35. K. Bacia and P. Schwille, “Practical guidelines for dual-color fluorescence cross-correlation spectroscopy,” Nature Protocols 2, 2842–2856 (2007).
    [CrossRef] [PubMed]
  36. E. P. Petrov and P. Schwille, “Fluorescence correlation spectroscopy on undulating membranes,” Biophys. J. 88, 524A–525A (2005).
  37. J. Evans, W. Gratzer, N. Mohandas, K. Parker, and J. Sleep, “Fluctuations of the red blood cell membrane: Relation to mechanical properties and lack of ATP dependence,” Biophys. J. 94, 4134–4144 (2008).
    [CrossRef] [PubMed]
  38. M. B. Schneider, J. T. Jenkins, and W. W. Webb, “Thermal fluctuations of large quasi-spherical bimolecular phospholipid-vesicles,” Journal De Physique 45, 1457–1472 (1984).
    [CrossRef]
  39. Y. Korlann, T. Dertinger, X. Michalet, S. Weiss, and J. Enderlein, “Measuring diffusion with polarization-modulation dual-focus fluorescence correlation spectroscopy,” Opt. Express 16, 14609–14616 (2008).
    [CrossRef] [PubMed]
  40. P. Ferrand, M. Pianta, A. Kress, A. Aillaud, H. Rigneault, and D. Marguet, “A versatile dual spot laser scanning confocal microscopy system for advanced fluorescence correlation spectroscopy analysis in living cell,” Rev. Sci. Instrum. 80, 083702 (2009).
    [CrossRef] [PubMed]
  41. C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, and S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457, 1159–1162 (2009).
    [CrossRef]

2010 (2)

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

N. Kahya, “Protein-protein and protein-lipid interactions in domain-assembly: Lessons from giant unilamellar vesicles,” Biochim. Biophys. Acta Biomembr. 1798, 1392–1398 (2010).
[CrossRef]

2009 (5)

E. Gielen, M. Vandeven, A. Margineanu, P. Dedecker, M. Van der Auweraer, Y. Engelborghs, J. Hofkens, and M. Ameloot, “On the use of z-scan fluorescence correlation experiments on giant unilamellar vesicles,” Chem. Phys. Lett. 469, 110–114 (2009).
[CrossRef]

J. Ries, S. Chiantia, and P. Schwille, “Accurate determination of membrane dynamics with line-scan FCS,” Biophys. J. 96, 1999–2008 (2009).
[CrossRef] [PubMed]

P. Ferrand, M. Pianta, A. Kress, A. Aillaud, H. Rigneault, and D. Marguet, “A versatile dual spot laser scanning confocal microscopy system for advanced fluorescence correlation spectroscopy analysis in living cell,” Rev. Sci. Instrum. 80, 083702 (2009).
[CrossRef] [PubMed]

C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, and S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457, 1159–1162 (2009).
[CrossRef]

D. C. Carrer, E. Kummer, G. Chwastek, S. Chiantia, and P. Schwille, “Asymmetry determines the effects of natural ceramides on model membranes,” Soft Matter 5, 3279–3286 (2009).
[CrossRef]

2008 (7)

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

Z. Petrášek, C. Hoege, A. Mashaghi, T. Ohrt, A. A. Hyman, and P. Schwille, “Characterization of protein dynamics in asymmetric cell division by scanning fluorescence correlation spectroscopy,” Biophys. J. 95, 5476–5486 (2008).
[CrossRef]

Z. Petrášek, C. Hoege, A. A. Hyman, and P. Schwille, “Two-photon fluorescence imaging and correlation analysis applied to protein dynamics in C. elegans embryo,” Proc. SPIE 6860, 68601L (2008).
[CrossRef]

A. J. García-Sáez and P. Schwille, “Fluorescence correlation spectroscopy for the study of membrane dynamics and protein/lipid interactions,” Methods 46, 116–122 (2008).
[CrossRef] [PubMed]

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

J. Hendrix, C. Flors, P. Dedecker, J. Hofkens, and Y. Engelborghs, “Dark states in monomeric red fluorescent proteins studied by fluorescence correlation and single molecule spectroscopy,” Biophys. J. 94, 4103–4113 (2008).
[CrossRef] [PubMed]

J. Evans, W. Gratzer, N. Mohandas, K. Parker, and J. Sleep, “Fluctuations of the red blood cell membrane: Relation to mechanical properties and lack of ATP dependence,” Biophys. J. 94, 4134–4144 (2008).
[CrossRef] [PubMed]

2007 (2)

K. Bacia and P. Schwille, “Practical guidelines for dual-color fluorescence cross-correlation spectroscopy,” Nature Protocols 2, 2842–2856 (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 measements,” ChemPhysChem 8, 433–443 (2007).
[CrossRef] [PubMed]

2006 (3)

K. Bacia, S. A. Kim, and P. Schwille, “Fluorescence cross-correlation spectroscopy in living cells,” Nat. Methods 3, 83–89 (2006).
[CrossRef] [PubMed]

M. Przybylo, J. Sýkora, J. Humpolíčková, A. Benda, A. Zan, and M. Hof, “Lipid diffusion in giant unilamellar vesicles is more than 2 times faster than in supported phospholipid bilayers under identical conditions,” Langmuir 22, 9096–9099 (2006).
[CrossRef] [PubMed]

S. Chiantia, J. Ries, N. Kahya, and P. Schwille, “Combined AFM and two-focus SFCS study of raft-exhibiting model membranes,” ChemPhysChem 7, 2409–2418 (2006).
[CrossRef] [PubMed]

2005 (4)

E. P. Petrov and P. Schwille, “Fluorescence correlation spectroscopy on undulating membranes,” Biophys. J. 88, 524A–525A (2005).

S. Chiantia, N. Kahya, and P. Schwille, “Dehydration damage of domain-exhibiting supported bilayers: An AFM study on the protective effects of disaccharides and other stabilizing substances,” Langmuir 21, 6317–6323 (2005).
[CrossRef] [PubMed]

M. A. Digman, C. M. Brown, P. Sengupta, P. W. Wiseman, A. R. Horwitz, and E. Gratton, “Measuring fast dynamics in solutions and cells with a laser scanning microscope,” Biophys. J. 89, 1317–1327 (2005).
[CrossRef] [PubMed]

J. P. Skinner, Y. Chen, and J. D. Müller, “Position-sensitive scanning fluorescence correlation spectroscopy,” Biophys. J. 89, 1288–1301 (2005).
[CrossRef] [PubMed]

2004 (2)

J. Enderlein, I. Gregor, D. Patra, and J. Fitter, “Art and artefacts of fluorescence correlation spectroscopy,” Curr. Pharm. Biotechnol. 5, 155–161 (2004).
[CrossRef] [PubMed]

K. Bacia, D. Scherfeld, N. Kahya, and P. Schwille, “Fluorescence correlation spectroscopy relates rafts in model and native membranes,” Biophys. J. 87, 1034–1043 (2004).
[CrossRef] [PubMed]

2003 (3)

S. Milon, R. Hovius, H. Vogel, and T. Wohland, “Factors influencing fluorescence correlation spectroscopy measurements on membranes: simulations and experiments,” Chem. Phys. 288, 171–186 (2003).
[CrossRef]

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

K. Bacia and P. Schwille, “A dynamic view of cellular processes by in vivo fluorescence auto- and cross-correlation spectroscopy,” Methods 29, 74–85 (2003).
[CrossRef] [PubMed]

2002 (1)

S. T. Hess and W. W. Webb, “Focal volume optics and experimental artifacts in confocal fluorescence correlation spectroscopy,” Biophys. J. 83, 2300–2317 (2002).
[CrossRef] [PubMed]

2000 (1)

J. Widengren and P. Schwille, “Characterization of photoinduced isomerization and back-isomerization of the cyanine dye Cy5 by fluorescence correlation spectroscopy,” J. Phys. Chem. A 104, 6416–6428 (2000).
[CrossRef]

1998 (1)

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

1997 (2)

M. J. Saxton and K. Jacobson, “Single-particle tracking: Applications to membrane dynamics,” Annu. Rev. Biophys. Biomol. Struct. 26, 373–399 (1997).
[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, 1878–1886 (1997).
[CrossRef] [PubMed]

1996 (1)

K. M. Berland, P. T. C. So, Y. Chen, W. W. Mantulin, and E. Gratton, “Scanning two-photon fluctuation correlation spectroscopy: Particle counting measurements for detection of molecular aggregation,” Biophys. J. 71, 410–420 (1996).
[CrossRef] [PubMed]

1995 (1)

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

1991 (1)

1984 (1)

M. B. Schneider, J. T. Jenkins, and W. W. Webb, “Thermal fluctuations of large quasi-spherical bimolecular phospholipid-vesicles,” Journal De Physique 45, 1457–1472 (1984).
[CrossRef]

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, 1055–1069 (1976).
[CrossRef] [PubMed]

1974 (1)

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

Aillaud, A.

P. Ferrand, M. Pianta, A. Kress, A. Aillaud, H. Rigneault, and D. Marguet, “A versatile dual spot laser scanning confocal microscopy system for advanced fluorescence correlation spectroscopy analysis in living cell,” Rev. Sci. Instrum. 80, 083702 (2009).
[CrossRef] [PubMed]

Ameloot, M.

E. Gielen, M. Vandeven, A. Margineanu, P. Dedecker, M. Van der Auweraer, Y. Engelborghs, J. Hofkens, and M. Ameloot, “On the use of z-scan fluorescence correlation experiments on giant unilamellar vesicles,” Chem. Phys. Lett. 469, 110–114 (2009).
[CrossRef]

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, 1055–1069 (1976).
[CrossRef] [PubMed]

Bacia, K.

K. Bacia and P. Schwille, “Practical guidelines for dual-color fluorescence cross-correlation spectroscopy,” Nature Protocols 2, 2842–2856 (2007).
[CrossRef] [PubMed]

K. Bacia, S. A. Kim, and P. Schwille, “Fluorescence cross-correlation spectroscopy in living cells,” Nat. Methods 3, 83–89 (2006).
[CrossRef] [PubMed]

K. Bacia, D. Scherfeld, N. Kahya, and P. Schwille, “Fluorescence correlation spectroscopy relates rafts in model and native membranes,” Biophys. J. 87, 1034–1043 (2004).
[CrossRef] [PubMed]

K. Bacia and P. Schwille, “A dynamic view of cellular processes by in vivo fluorescence auto- and cross-correlation spectroscopy,” Methods 29, 74–85 (2003).
[CrossRef] [PubMed]

Belov, V. N.

C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, and S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457, 1159–1162 (2009).
[CrossRef]

Benda, A.

M. Przybylo, J. Sýkora, J. Humpolíčková, A. Benda, A. Zan, and M. Hof, “Lipid diffusion in giant unilamellar vesicles is more than 2 times faster than in supported phospholipid bilayers under identical conditions,” Langmuir 22, 9096–9099 (2006).
[CrossRef] [PubMed]

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

Beneš, M.

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

Berland, K. M.

K. M. Berland, P. T. C. So, Y. Chen, W. W. Mantulin, and E. Gratton, “Scanning two-photon fluctuation correlation spectroscopy: Particle counting measurements for detection of molecular aggregation,” Biophys. J. 71, 410–420 (1996).
[CrossRef] [PubMed]

Born, M.

M. Born and E. Wolf, Principles of Optics (Cambridge University Press, 1999), chap. 8, pp. 484–492, 7th ed.

Brown, C. M.

M. A. Digman, C. M. Brown, P. Sengupta, P. W. Wiseman, A. R. Horwitz, and E. Gratton, “Measuring fast dynamics in solutions and cells with a laser scanning microscope,” Biophys. J. 89, 1317–1327 (2005).
[CrossRef] [PubMed]

Carrer, D. C.

D. C. Carrer, E. Kummer, G. Chwastek, S. Chiantia, and P. Schwille, “Asymmetry determines the effects of natural ceramides on model membranes,” Soft Matter 5, 3279–3286 (2009).
[CrossRef]

Chen, Y.

J. P. Skinner, Y. Chen, and J. D. Müller, “Position-sensitive scanning fluorescence correlation spectroscopy,” Biophys. J. 89, 1288–1301 (2005).
[CrossRef] [PubMed]

K. M. Berland, P. T. C. So, Y. Chen, W. W. Mantulin, and E. Gratton, “Scanning two-photon fluctuation correlation spectroscopy: Particle counting measurements for detection of molecular aggregation,” Biophys. J. 71, 410–420 (1996).
[CrossRef] [PubMed]

Chiantia, S.

J. Ries, S. Chiantia, and P. Schwille, “Accurate determination of membrane dynamics with line-scan FCS,” Biophys. J. 96, 1999–2008 (2009).
[CrossRef] [PubMed]

D. C. Carrer, E. Kummer, G. Chwastek, S. Chiantia, and P. Schwille, “Asymmetry determines the effects of natural ceramides on model membranes,” Soft Matter 5, 3279–3286 (2009).
[CrossRef]

S. Chiantia, J. Ries, N. Kahya, and P. Schwille, “Combined AFM and two-focus SFCS study of raft-exhibiting model membranes,” ChemPhysChem 7, 2409–2418 (2006).
[CrossRef] [PubMed]

S. Chiantia, N. Kahya, and P. Schwille, “Dehydration damage of domain-exhibiting supported bilayers: An AFM study on the protective effects of disaccharides and other stabilizing substances,” Langmuir 21, 6317–6323 (2005).
[CrossRef] [PubMed]

Chwastek, G.

D. C. Carrer, E. Kummer, G. Chwastek, S. Chiantia, and P. Schwille, “Asymmetry determines the effects of natural ceramides on model membranes,” Soft Matter 5, 3279–3286 (2009).
[CrossRef]

Dedecker, P.

E. Gielen, M. Vandeven, A. Margineanu, P. Dedecker, M. Van der Auweraer, Y. Engelborghs, J. Hofkens, and M. Ameloot, “On the use of z-scan fluorescence correlation experiments on giant unilamellar vesicles,” Chem. Phys. Lett. 469, 110–114 (2009).
[CrossRef]

J. Hendrix, C. Flors, P. Dedecker, J. Hofkens, and Y. Engelborghs, “Dark states in monomeric red fluorescent proteins studied by fluorescence correlation and single molecule spectroscopy,” Biophys. J. 94, 4103–4113 (2008).
[CrossRef] [PubMed]

Dertinger, T.

Y. Korlann, T. Dertinger, X. Michalet, S. Weiss, and J. Enderlein, “Measuring diffusion with polarization-modulation dual-focus fluorescence correlation spectroscopy,” Opt. Express 16, 14609–14616 (2008).
[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 measements,” ChemPhysChem 8, 433–443 (2007).
[CrossRef] [PubMed]

Digman, M. A.

M. A. Digman, C. M. Brown, P. Sengupta, P. W. Wiseman, A. R. Horwitz, and E. Gratton, “Measuring fast dynamics in solutions and cells with a laser scanning microscope,” Biophys. J. 89, 1317–1327 (2005).
[CrossRef] [PubMed]

Eggeling, C.

C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, and S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457, 1159–1162 (2009).
[CrossRef]

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, 1055–1069 (1976).
[CrossRef] [PubMed]

Elson, E. L.

H. Qian and E. L. Elson, “Analysis of confocal laser-microscope optics for 3-D fluorescence correlation spectroscopy,” Appl. Opt. 30, 1185–1195 (1991).
[CrossRef] [PubMed]

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

Enderlein, J.

Y. Korlann, T. Dertinger, X. Michalet, S. Weiss, and J. Enderlein, “Measuring diffusion with polarization-modulation dual-focus fluorescence correlation spectroscopy,” Opt. Express 16, 14609–14616 (2008).
[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 measements,” ChemPhysChem 8, 433–443 (2007).
[CrossRef] [PubMed]

J. Enderlein, I. Gregor, D. Patra, and J. Fitter, “Art and artefacts of fluorescence correlation spectroscopy,” Curr. Pharm. Biotechnol. 5, 155–161 (2004).
[CrossRef] [PubMed]

Engelborghs, Y.

E. Gielen, M. Vandeven, A. Margineanu, P. Dedecker, M. Van der Auweraer, Y. Engelborghs, J. Hofkens, and M. Ameloot, “On the use of z-scan fluorescence correlation experiments on giant unilamellar vesicles,” Chem. Phys. Lett. 469, 110–114 (2009).
[CrossRef]

J. Hendrix, C. Flors, P. Dedecker, J. Hofkens, and Y. Engelborghs, “Dark states in monomeric red fluorescent proteins studied by fluorescence correlation and single molecule spectroscopy,” Biophys. J. 94, 4103–4113 (2008).
[CrossRef] [PubMed]

Evans, J.

J. Evans, W. Gratzer, N. Mohandas, K. Parker, and J. Sleep, “Fluctuations of the red blood cell membrane: Relation to mechanical properties and lack of ATP dependence,” Biophys. J. 94, 4134–4144 (2008).
[CrossRef] [PubMed]

Ferrand, P.

P. Ferrand, M. Pianta, A. Kress, A. Aillaud, H. Rigneault, and D. Marguet, “A versatile dual spot laser scanning confocal microscopy system for advanced fluorescence correlation spectroscopy analysis in living cell,” Rev. Sci. Instrum. 80, 083702 (2009).
[CrossRef] [PubMed]

Fitter, J.

J. Enderlein, I. Gregor, D. Patra, and J. Fitter, “Art and artefacts of fluorescence correlation spectroscopy,” Curr. Pharm. Biotechnol. 5, 155–161 (2004).
[CrossRef] [PubMed]

Flors, C.

J. Hendrix, C. Flors, P. Dedecker, J. Hofkens, and Y. Engelborghs, “Dark states in monomeric red fluorescent proteins studied by fluorescence correlation and single molecule spectroscopy,” Biophys. J. 94, 4103–4113 (2008).
[CrossRef] [PubMed]

García-Sáez, A. J.

A. J. García-Sáez and P. Schwille, “Fluorescence correlation spectroscopy for the study of membrane dynamics and protein/lipid interactions,” Methods 46, 116–122 (2008).
[CrossRef] [PubMed]

Gielen, E.

E. Gielen, M. Vandeven, A. Margineanu, P. Dedecker, M. Van der Auweraer, Y. Engelborghs, J. Hofkens, and M. Ameloot, “On the use of z-scan fluorescence correlation experiments on giant unilamellar vesicles,” Chem. Phys. Lett. 469, 110–114 (2009).
[CrossRef]

Gratton, E.

M. A. Digman, C. M. Brown, P. Sengupta, P. W. Wiseman, A. R. Horwitz, and E. Gratton, “Measuring fast dynamics in solutions and cells with a laser scanning microscope,” Biophys. J. 89, 1317–1327 (2005).
[CrossRef] [PubMed]

K. M. Berland, P. T. C. So, Y. Chen, W. W. Mantulin, and E. Gratton, “Scanning two-photon fluctuation correlation spectroscopy: Particle counting measurements for detection of molecular aggregation,” Biophys. J. 71, 410–420 (1996).
[CrossRef] [PubMed]

Gratzer, W.

J. Evans, W. Gratzer, N. Mohandas, K. Parker, and J. Sleep, “Fluctuations of the red blood cell membrane: Relation to mechanical properties and lack of ATP dependence,” Biophys. J. 94, 4134–4144 (2008).
[CrossRef] [PubMed]

Gregor, I.

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 measements,” ChemPhysChem 8, 433–443 (2007).
[CrossRef] [PubMed]

J. Enderlein, I. Gregor, D. Patra, and J. Fitter, “Art and artefacts of fluorescence correlation spectroscopy,” Curr. Pharm. Biotechnol. 5, 155–161 (2004).
[CrossRef] [PubMed]

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 measements,” ChemPhysChem 8, 433–443 (2007).
[CrossRef] [PubMed]

Haupts, U.

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

Hein, B.

C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, and S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457, 1159–1162 (2009).
[CrossRef]

Hell, S. W.

C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, and S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457, 1159–1162 (2009).
[CrossRef]

Hendrix, J.

J. Hendrix, C. Flors, P. Dedecker, J. Hofkens, and Y. Engelborghs, “Dark states in monomeric red fluorescent proteins studied by fluorescence correlation and single molecule spectroscopy,” Biophys. J. 94, 4103–4113 (2008).
[CrossRef] [PubMed]

Hermens, W. T.

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

Hess, S. T.

S. T. Hess and W. W. Webb, “Focal volume optics and experimental artifacts in confocal fluorescence correlation spectroscopy,” Biophys. J. 83, 2300–2317 (2002).
[CrossRef] [PubMed]

Hoege, C.

Z. Petrášek, C. Hoege, A. A. Hyman, and P. Schwille, “Two-photon fluorescence imaging and correlation analysis applied to protein dynamics in C. elegans embryo,” Proc. SPIE 6860, 68601L (2008).
[CrossRef]

Z. Petrášek, C. Hoege, A. Mashaghi, T. Ohrt, A. A. Hyman, and P. Schwille, “Characterization of protein dynamics in asymmetric cell division by scanning fluorescence correlation spectroscopy,” Biophys. J. 95, 5476–5486 (2008).
[CrossRef]

Hof, M.

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

M. Przybylo, J. Sýkora, J. Humpolíčková, A. Benda, A. Zan, and M. Hof, “Lipid diffusion in giant unilamellar vesicles is more than 2 times faster than in supported phospholipid bilayers under identical conditions,” Langmuir 22, 9096–9099 (2006).
[CrossRef] [PubMed]

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

Hofkens, J.

E. Gielen, M. Vandeven, A. Margineanu, P. Dedecker, M. Van der Auweraer, Y. Engelborghs, J. Hofkens, and M. Ameloot, “On the use of z-scan fluorescence correlation experiments on giant unilamellar vesicles,” Chem. Phys. Lett. 469, 110–114 (2009).
[CrossRef]

J. Hendrix, C. Flors, P. Dedecker, J. Hofkens, and Y. Engelborghs, “Dark states in monomeric red fluorescent proteins studied by fluorescence correlation and single molecule spectroscopy,” Biophys. J. 94, 4103–4113 (2008).
[CrossRef] [PubMed]

Horwitz, A. R.

M. A. Digman, C. M. Brown, P. Sengupta, P. W. Wiseman, A. R. Horwitz, and E. Gratton, “Measuring fast dynamics in solutions and cells with a laser scanning microscope,” Biophys. J. 89, 1317–1327 (2005).
[CrossRef] [PubMed]

Hovius, R.

S. Milon, R. Hovius, H. Vogel, and T. Wohland, “Factors influencing fluorescence correlation spectroscopy measurements on membranes: simulations and experiments,” Chem. Phys. 288, 171–186 (2003).
[CrossRef]

Humpolícková, J.

M. Przybylo, J. Sýkora, J. Humpolíčková, A. Benda, A. Zan, and M. Hof, “Lipid diffusion in giant unilamellar vesicles is more than 2 times faster than in supported phospholipid bilayers under identical conditions,” Langmuir 22, 9096–9099 (2006).
[CrossRef] [PubMed]

Hyman, A. A.

Z. Petrášek, C. Hoege, A. Mashaghi, T. Ohrt, A. A. Hyman, and P. Schwille, “Characterization of protein dynamics in asymmetric cell division by scanning fluorescence correlation spectroscopy,” Biophys. J. 95, 5476–5486 (2008).
[CrossRef]

Z. Petrášek, C. Hoege, A. A. Hyman, and P. Schwille, “Two-photon fluorescence imaging and correlation analysis applied to protein dynamics in C. elegans embryo,” Proc. SPIE 6860, 68601L (2008).
[CrossRef]

Jacobson, K.

M. J. Saxton and K. Jacobson, “Single-particle tracking: Applications to membrane dynamics,” Annu. Rev. Biophys. Biomol. Struct. 26, 373–399 (1997).
[CrossRef] [PubMed]

Jenkins, J. T.

M. B. Schneider, J. T. Jenkins, and W. W. Webb, “Thermal fluctuations of large quasi-spherical bimolecular phospholipid-vesicles,” Journal De Physique 45, 1457–1472 (1984).
[CrossRef]

Kahya, N.

N. Kahya, “Protein-protein and protein-lipid interactions in domain-assembly: Lessons from giant unilamellar vesicles,” Biochim. Biophys. Acta Biomembr. 1798, 1392–1398 (2010).
[CrossRef]

S. Chiantia, J. Ries, N. Kahya, and P. Schwille, “Combined AFM and two-focus SFCS study of raft-exhibiting model membranes,” ChemPhysChem 7, 2409–2418 (2006).
[CrossRef] [PubMed]

S. Chiantia, N. Kahya, and P. Schwille, “Dehydration damage of domain-exhibiting supported bilayers: An AFM study on the protective effects of disaccharides and other stabilizing substances,” Langmuir 21, 6317–6323 (2005).
[CrossRef] [PubMed]

K. Bacia, D. Scherfeld, N. Kahya, and P. Schwille, “Fluorescence correlation spectroscopy relates rafts in model and native membranes,” Biophys. J. 87, 1034–1043 (2004).
[CrossRef] [PubMed]

Kim, S. A.

K. Bacia, S. A. Kim, and P. Schwille, “Fluorescence cross-correlation spectroscopy in living cells,” Nat. Methods 3, 83–89 (2006).
[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, 1055–1069 (1976).
[CrossRef] [PubMed]

Korlann, Y.

Kress, A.

P. Ferrand, M. Pianta, A. Kress, A. Aillaud, H. Rigneault, and D. Marguet, “A versatile dual spot laser scanning confocal microscopy system for advanced fluorescence correlation spectroscopy analysis in living cell,” Rev. Sci. Instrum. 80, 083702 (2009).
[CrossRef] [PubMed]

Kummer, E.

D. C. Carrer, E. Kummer, G. Chwastek, S. Chiantia, and P. Schwille, “Asymmetry determines the effects of natural ceramides on model membranes,” Soft Matter 5, 3279–3286 (2009).
[CrossRef]

Lhotský, A.

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

Machán, R.

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

Magde, D.

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

Maiti, S.

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

Mantulin, W. W.

K. M. Berland, P. T. C. So, Y. Chen, W. W. Mantulin, and E. Gratton, “Scanning two-photon fluctuation correlation spectroscopy: Particle counting measurements for detection of molecular aggregation,” Biophys. J. 71, 410–420 (1996).
[CrossRef] [PubMed]

Marecek, V.

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

Margineanu, A.

E. Gielen, M. Vandeven, A. Margineanu, P. Dedecker, M. Van der Auweraer, Y. Engelborghs, J. Hofkens, and M. Ameloot, “On the use of z-scan fluorescence correlation experiments on giant unilamellar vesicles,” Chem. Phys. Lett. 469, 110–114 (2009).
[CrossRef]

Marguet, D.

P. Ferrand, M. Pianta, A. Kress, A. Aillaud, H. Rigneault, and D. Marguet, “A versatile dual spot laser scanning confocal microscopy system for advanced fluorescence correlation spectroscopy analysis in living cell,” Rev. Sci. Instrum. 80, 083702 (2009).
[CrossRef] [PubMed]

Mashaghi, A.

Z. Petrášek, C. Hoege, A. Mashaghi, T. Ohrt, A. A. Hyman, and P. Schwille, “Characterization of protein dynamics in asymmetric cell division by scanning fluorescence correlation spectroscopy,” Biophys. J. 95, 5476–5486 (2008).
[CrossRef]

Medda, R.

C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, and S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457, 1159–1162 (2009).
[CrossRef]

Mets, U.

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

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, 1878–1886 (1997).
[CrossRef] [PubMed]

Michalet, X.

Milon, S.

S. Milon, R. Hovius, H. Vogel, and T. Wohland, “Factors influencing fluorescence correlation spectroscopy measurements on membranes: simulations and experiments,” Chem. Phys. 288, 171–186 (2003).
[CrossRef]

Mohandas, N.

J. Evans, W. Gratzer, N. Mohandas, K. Parker, and J. Sleep, “Fluctuations of the red blood cell membrane: Relation to mechanical properties and lack of ATP dependence,” Biophys. J. 94, 4134–4144 (2008).
[CrossRef] [PubMed]

Müller, J. D.

J. P. Skinner, Y. Chen, and J. D. Müller, “Position-sensitive scanning fluorescence correlation spectroscopy,” Biophys. J. 89, 1288–1301 (2005).
[CrossRef] [PubMed]

Ohrt, T.

Z. Petrášek, C. Hoege, A. Mashaghi, T. Ohrt, A. A. Hyman, and P. Schwille, “Characterization of protein dynamics in asymmetric cell division by scanning fluorescence correlation spectroscopy,” Biophys. J. 95, 5476–5486 (2008).
[CrossRef]

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 measements,” ChemPhysChem 8, 433–443 (2007).
[CrossRef] [PubMed]

Parker, K.

J. Evans, W. Gratzer, N. Mohandas, K. Parker, and J. Sleep, “Fluctuations of the red blood cell membrane: Relation to mechanical properties and lack of ATP dependence,” Biophys. J. 94, 4134–4144 (2008).
[CrossRef] [PubMed]

Patra, D.

J. Enderlein, I. Gregor, D. Patra, and J. Fitter, “Art and artefacts of fluorescence correlation spectroscopy,” Curr. Pharm. Biotechnol. 5, 155–161 (2004).
[CrossRef] [PubMed]

Petrášek, Z.

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

Z. Petrášek, C. Hoege, A. A. Hyman, and P. Schwille, “Two-photon fluorescence imaging and correlation analysis applied to protein dynamics in C. elegans embryo,” Proc. SPIE 6860, 68601L (2008).
[CrossRef]

Z. Petrášek, C. Hoege, A. Mashaghi, T. Ohrt, A. A. Hyman, and P. Schwille, “Characterization of protein dynamics in asymmetric cell division by scanning fluorescence correlation spectroscopy,” Biophys. J. 95, 5476–5486 (2008).
[CrossRef]

Petrov, E. P.

E. P. Petrov and P. Schwille, “Fluorescence correlation spectroscopy on undulating membranes,” Biophys. J. 88, 524A–525A (2005).

Pianta, M.

P. Ferrand, M. Pianta, A. Kress, A. Aillaud, H. Rigneault, and D. Marguet, “A versatile dual spot laser scanning confocal microscopy system for advanced fluorescence correlation spectroscopy analysis in living cell,” Rev. Sci. Instrum. 80, 083702 (2009).
[CrossRef] [PubMed]

Polyakova, S.

C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, and S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457, 1159–1162 (2009).
[CrossRef]

Przybylo, M.

M. Przybylo, J. Sýkora, J. Humpolíčková, A. Benda, A. Zan, and M. Hof, “Lipid diffusion in giant unilamellar vesicles is more than 2 times faster than in supported phospholipid bilayers under identical conditions,” Langmuir 22, 9096–9099 (2006).
[CrossRef] [PubMed]

Qian, H.

Ries, J.

J. Ries, S. Chiantia, and P. Schwille, “Accurate determination of membrane dynamics with line-scan FCS,” Biophys. J. 96, 1999–2008 (2009).
[CrossRef] [PubMed]

S. Chiantia, J. Ries, N. Kahya, and P. Schwille, “Combined AFM and two-focus SFCS study of raft-exhibiting model membranes,” ChemPhysChem 7, 2409–2418 (2006).
[CrossRef] [PubMed]

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, 1878–1886 (1997).
[CrossRef] [PubMed]

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

Rigneault, H.

P. Ferrand, M. Pianta, A. Kress, A. Aillaud, H. Rigneault, and D. Marguet, “A versatile dual spot laser scanning confocal microscopy system for advanced fluorescence correlation spectroscopy analysis in living cell,” Rev. Sci. Instrum. 80, 083702 (2009).
[CrossRef] [PubMed]

Ringemann, C.

C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, and S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457, 1159–1162 (2009).
[CrossRef]

Sandhoff, K.

C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, and S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457, 1159–1162 (2009).
[CrossRef]

Saxton, M. J.

M. J. Saxton and K. Jacobson, “Single-particle tracking: Applications to membrane dynamics,” Annu. Rev. Biophys. Biomol. Struct. 26, 373–399 (1997).
[CrossRef] [PubMed]

Scherfeld, D.

K. Bacia, D. Scherfeld, N. Kahya, and P. Schwille, “Fluorescence correlation spectroscopy relates rafts in model and native membranes,” Biophys. J. 87, 1034–1043 (2004).
[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, 1055–1069 (1976).
[CrossRef] [PubMed]

Schneider, M. B.

M. B. Schneider, J. T. Jenkins, and W. W. Webb, “Thermal fluctuations of large quasi-spherical bimolecular phospholipid-vesicles,” Journal De Physique 45, 1457–1472 (1984).
[CrossRef]

Schönle, A.

C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, and S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457, 1159–1162 (2009).
[CrossRef]

Schwarzmann, G.

C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, and S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457, 1159–1162 (2009).
[CrossRef]

Schwille, P.

J. Ries, S. Chiantia, and P. Schwille, “Accurate determination of membrane dynamics with line-scan FCS,” Biophys. J. 96, 1999–2008 (2009).
[CrossRef] [PubMed]

D. C. Carrer, E. Kummer, G. Chwastek, S. Chiantia, and P. Schwille, “Asymmetry determines the effects of natural ceramides on model membranes,” Soft Matter 5, 3279–3286 (2009).
[CrossRef]

A. J. García-Sáez and P. Schwille, “Fluorescence correlation spectroscopy for the study of membrane dynamics and protein/lipid interactions,” Methods 46, 116–122 (2008).
[CrossRef] [PubMed]

Z. Petrášek, C. Hoege, A. A. Hyman, and P. Schwille, “Two-photon fluorescence imaging and correlation analysis applied to protein dynamics in C. elegans embryo,” Proc. SPIE 6860, 68601L (2008).
[CrossRef]

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

Z. Petrášek, C. Hoege, A. Mashaghi, T. Ohrt, A. A. Hyman, and P. Schwille, “Characterization of protein dynamics in asymmetric cell division by scanning fluorescence correlation spectroscopy,” Biophys. J. 95, 5476–5486 (2008).
[CrossRef]

K. Bacia and P. Schwille, “Practical guidelines for dual-color fluorescence cross-correlation spectroscopy,” Nature Protocols 2, 2842–2856 (2007).
[CrossRef] [PubMed]

K. Bacia, S. A. Kim, and P. Schwille, “Fluorescence cross-correlation spectroscopy in living cells,” Nat. Methods 3, 83–89 (2006).
[CrossRef] [PubMed]

S. Chiantia, J. Ries, N. Kahya, and P. Schwille, “Combined AFM and two-focus SFCS study of raft-exhibiting model membranes,” ChemPhysChem 7, 2409–2418 (2006).
[CrossRef] [PubMed]

S. Chiantia, N. Kahya, and P. Schwille, “Dehydration damage of domain-exhibiting supported bilayers: An AFM study on the protective effects of disaccharides and other stabilizing substances,” Langmuir 21, 6317–6323 (2005).
[CrossRef] [PubMed]

E. P. Petrov and P. Schwille, “Fluorescence correlation spectroscopy on undulating membranes,” Biophys. J. 88, 524A–525A (2005).

K. Bacia, D. Scherfeld, N. Kahya, and P. Schwille, “Fluorescence correlation spectroscopy relates rafts in model and native membranes,” Biophys. J. 87, 1034–1043 (2004).
[CrossRef] [PubMed]

K. Bacia and P. Schwille, “A dynamic view of cellular processes by in vivo fluorescence auto- and cross-correlation spectroscopy,” Methods 29, 74–85 (2003).
[CrossRef] [PubMed]

J. Widengren and P. Schwille, “Characterization of photoinduced isomerization and back-isomerization of the cyanine dye Cy5 by fluorescence correlation spectroscopy,” J. Phys. Chem. A 104, 6416–6428 (2000).
[CrossRef]

U. Haupts, S. Maiti, P. Schwille, and W. W. Webb, “Dynamics of fluorescence fluctuations in green fluorescent protein observed by fluorescence correlation spectroscopy,” Proc. Natl. Acad. Sci. U. S. A. 95, 13573–13578 (1998).
[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, 1878–1886 (1997).
[CrossRef] [PubMed]

Sengupta, P.

M. A. Digman, C. M. Brown, P. Sengupta, P. W. Wiseman, A. R. Horwitz, and E. Gratton, “Measuring fast dynamics in solutions and cells with a laser scanning microscope,” Biophys. J. 89, 1317–1327 (2005).
[CrossRef] [PubMed]

Skinner, J. P.

J. P. Skinner, Y. Chen, and J. D. Müller, “Position-sensitive scanning fluorescence correlation spectroscopy,” Biophys. J. 89, 1288–1301 (2005).
[CrossRef] [PubMed]

Sleep, J.

J. Evans, W. Gratzer, N. Mohandas, K. Parker, and J. Sleep, “Fluctuations of the red blood cell membrane: Relation to mechanical properties and lack of ATP dependence,” Biophys. J. 94, 4134–4144 (2008).
[CrossRef] [PubMed]

So, P. T. C.

K. M. Berland, P. T. C. So, Y. Chen, W. W. Mantulin, and E. Gratton, “Scanning two-photon fluctuation correlation spectroscopy: Particle counting measurements for detection of molecular aggregation,” Biophys. J. 71, 410–420 (1996).
[CrossRef] [PubMed]

Sýkora, J.

M. Przybylo, J. Sýkora, J. Humpolíčková, A. Benda, A. Zan, and M. Hof, “Lipid diffusion in giant unilamellar vesicles is more than 2 times faster than in supported phospholipid bilayers under identical conditions,” Langmuir 22, 9096–9099 (2006).
[CrossRef] [PubMed]

Van der Auweraer, M.

E. Gielen, M. Vandeven, A. Margineanu, P. Dedecker, M. Van der Auweraer, Y. Engelborghs, J. Hofkens, and M. Ameloot, “On the use of z-scan fluorescence correlation experiments on giant unilamellar vesicles,” Chem. Phys. Lett. 469, 110–114 (2009).
[CrossRef]

Vandeven, M.

E. Gielen, M. Vandeven, A. Margineanu, P. Dedecker, M. Van der Auweraer, Y. Engelborghs, J. Hofkens, and M. Ameloot, “On the use of z-scan fluorescence correlation experiments on giant unilamellar vesicles,” Chem. Phys. Lett. 469, 110–114 (2009).
[CrossRef]

Vogel, H.

S. Milon, R. Hovius, H. Vogel, and T. Wohland, “Factors influencing fluorescence correlation spectroscopy measurements on membranes: simulations and experiments,” Chem. Phys. 288, 171–186 (2003).
[CrossRef]

von der Hocht, I.

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 measements,” ChemPhysChem 8, 433–443 (2007).
[CrossRef] [PubMed]

von Middendorff, C.

C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, and S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457, 1159–1162 (2009).
[CrossRef]

Webb, W. W.

S. T. Hess and W. W. Webb, “Focal volume optics and experimental artifacts in confocal fluorescence correlation spectroscopy,” Biophys. J. 83, 2300–2317 (2002).
[CrossRef] [PubMed]

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

M. B. Schneider, J. T. Jenkins, and W. W. Webb, “Thermal fluctuations of large quasi-spherical bimolecular phospholipid-vesicles,” Journal De Physique 45, 1457–1472 (1984).
[CrossRef]

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, 1055–1069 (1976).
[CrossRef] [PubMed]

Weiss, S.

Widengren, J.

J. Widengren and P. Schwille, “Characterization of photoinduced isomerization and back-isomerization of the cyanine dye Cy5 by fluorescence correlation spectroscopy,” J. Phys. Chem. A 104, 6416–6428 (2000).
[CrossRef]

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

Wiseman, P. W.

M. A. Digman, C. M. Brown, P. Sengupta, P. W. Wiseman, A. R. Horwitz, and E. Gratton, “Measuring fast dynamics in solutions and cells with a laser scanning microscope,” Biophys. J. 89, 1317–1327 (2005).
[CrossRef] [PubMed]

Wohland, T.

S. Milon, R. Hovius, H. Vogel, and T. Wohland, “Factors influencing fluorescence correlation spectroscopy measurements on membranes: simulations and experiments,” Chem. Phys. 288, 171–186 (2003).
[CrossRef]

Wolf, E.

M. Born and E. Wolf, Principles of Optics (Cambridge University Press, 1999), chap. 8, pp. 484–492, 7th ed.

Zan, A.

M. Przybylo, J. Sýkora, J. Humpolíčková, A. Benda, A. Zan, and M. Hof, “Lipid diffusion in giant unilamellar vesicles is more than 2 times faster than in supported phospholipid bilayers under identical conditions,” Langmuir 22, 9096–9099 (2006).
[CrossRef] [PubMed]

Annu. Rev. Biophys. Biomol. Struct. (1)

M. J. Saxton and K. Jacobson, “Single-particle tracking: Applications to membrane dynamics,” Annu. Rev. Biophys. Biomol. Struct. 26, 373–399 (1997).
[CrossRef] [PubMed]

Appl. Opt. (1)

Biochim. Biophys. Acta Biomembr. (2)

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

N. Kahya, “Protein-protein and protein-lipid interactions in domain-assembly: Lessons from giant unilamellar vesicles,” Biochim. Biophys. Acta Biomembr. 1798, 1392–1398 (2010).
[CrossRef]

Biophys. J. (13)

K. Bacia, D. Scherfeld, N. Kahya, and P. Schwille, “Fluorescence correlation spectroscopy relates rafts in model and native membranes,” Biophys. J. 87, 1034–1043 (2004).
[CrossRef] [PubMed]

Z. Petrášek, C. Hoege, A. Mashaghi, T. Ohrt, A. A. Hyman, and P. Schwille, “Characterization of protein dynamics in asymmetric cell division by scanning fluorescence correlation spectroscopy,” Biophys. J. 95, 5476–5486 (2008).
[CrossRef]

K. M. Berland, P. T. C. So, Y. Chen, W. W. Mantulin, and E. Gratton, “Scanning two-photon fluctuation correlation spectroscopy: Particle counting measurements for detection of molecular aggregation,” Biophys. J. 71, 410–420 (1996).
[CrossRef] [PubMed]

J. Hendrix, C. Flors, P. Dedecker, J. Hofkens, and Y. Engelborghs, “Dark states in monomeric red fluorescent proteins studied by fluorescence correlation and single molecule spectroscopy,” Biophys. J. 94, 4103–4113 (2008).
[CrossRef] [PubMed]

E. P. Petrov and P. Schwille, “Fluorescence correlation spectroscopy on undulating membranes,” Biophys. J. 88, 524A–525A (2005).

J. Evans, W. Gratzer, N. Mohandas, K. Parker, and J. Sleep, “Fluctuations of the red blood cell membrane: Relation to mechanical properties and lack of ATP dependence,” Biophys. J. 94, 4134–4144 (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, 1878–1886 (1997).
[CrossRef] [PubMed]

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, 1055–1069 (1976).
[CrossRef] [PubMed]

J. Ries, S. Chiantia, and P. Schwille, “Accurate determination of membrane dynamics with line-scan FCS,” Biophys. J. 96, 1999–2008 (2009).
[CrossRef] [PubMed]

M. A. Digman, C. M. Brown, P. Sengupta, P. W. Wiseman, A. R. Horwitz, and E. Gratton, “Measuring fast dynamics in solutions and cells with a laser scanning microscope,” Biophys. J. 89, 1317–1327 (2005).
[CrossRef] [PubMed]

J. P. Skinner, Y. Chen, and J. D. Müller, “Position-sensitive scanning fluorescence correlation spectroscopy,” Biophys. J. 89, 1288–1301 (2005).
[CrossRef] [PubMed]

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

S. T. Hess and W. W. Webb, “Focal volume optics and experimental artifacts in confocal fluorescence correlation spectroscopy,” Biophys. J. 83, 2300–2317 (2002).
[CrossRef] [PubMed]

Biopolymers (1)

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

Chem. Phys. (1)

S. Milon, R. Hovius, H. Vogel, and T. Wohland, “Factors influencing fluorescence correlation spectroscopy measurements on membranes: simulations and experiments,” Chem. Phys. 288, 171–186 (2003).
[CrossRef]

Chem. Phys. Lett. (1)

E. Gielen, M. Vandeven, A. Margineanu, P. Dedecker, M. Van der Auweraer, Y. Engelborghs, J. Hofkens, and M. Ameloot, “On the use of z-scan fluorescence correlation experiments on giant unilamellar vesicles,” Chem. Phys. Lett. 469, 110–114 (2009).
[CrossRef]

ChemPhysChem (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 measements,” ChemPhysChem 8, 433–443 (2007).
[CrossRef] [PubMed]

S. Chiantia, J. Ries, N. Kahya, and P. Schwille, “Combined AFM and two-focus SFCS study of raft-exhibiting model membranes,” ChemPhysChem 7, 2409–2418 (2006).
[CrossRef] [PubMed]

Curr. Pharm. Biotechnol. (1)

J. Enderlein, I. Gregor, D. Patra, and J. Fitter, “Art and artefacts of fluorescence correlation spectroscopy,” Curr. Pharm. Biotechnol. 5, 155–161 (2004).
[CrossRef] [PubMed]

J. Phys. Chem. (1)

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

J. Phys. Chem. A (1)

J. Widengren and P. Schwille, “Characterization of photoinduced isomerization and back-isomerization of the cyanine dye Cy5 by fluorescence correlation spectroscopy,” J. Phys. Chem. A 104, 6416–6428 (2000).
[CrossRef]

Journal De Physique (1)

M. B. Schneider, J. T. Jenkins, and W. W. Webb, “Thermal fluctuations of large quasi-spherical bimolecular phospholipid-vesicles,” Journal De Physique 45, 1457–1472 (1984).
[CrossRef]

Langmuir (3)

S. Chiantia, N. Kahya, and P. Schwille, “Dehydration damage of domain-exhibiting supported bilayers: An AFM study on the protective effects of disaccharides and other stabilizing substances,” Langmuir 21, 6317–6323 (2005).
[CrossRef] [PubMed]

M. Przybylo, J. Sýkora, J. Humpolíčková, A. Benda, A. Zan, and M. Hof, “Lipid diffusion in giant unilamellar vesicles is more than 2 times faster than in supported phospholipid bilayers under identical conditions,” Langmuir 22, 9096–9099 (2006).
[CrossRef] [PubMed]

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

Methods (2)

K. Bacia and P. Schwille, “A dynamic view of cellular processes by in vivo fluorescence auto- and cross-correlation spectroscopy,” Methods 29, 74–85 (2003).
[CrossRef] [PubMed]

A. J. García-Sáez and P. Schwille, “Fluorescence correlation spectroscopy for the study of membrane dynamics and protein/lipid interactions,” Methods 46, 116–122 (2008).
[CrossRef] [PubMed]

Nat. Methods (1)

K. Bacia, S. A. Kim, and P. Schwille, “Fluorescence cross-correlation spectroscopy in living cells,” Nat. Methods 3, 83–89 (2006).
[CrossRef] [PubMed]

Nature (1)

C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, and S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457, 1159–1162 (2009).
[CrossRef]

Nature Protocols (1)

K. Bacia and P. Schwille, “Practical guidelines for dual-color fluorescence cross-correlation spectroscopy,” Nature Protocols 2, 2842–2856 (2007).
[CrossRef] [PubMed]

Opt. Express (1)

Proc. Natl. Acad. Sci. U. S. A. (1)

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

Proc. SPIE (1)

Z. Petrášek, C. Hoege, A. A. Hyman, and P. Schwille, “Two-photon fluorescence imaging and correlation analysis applied to protein dynamics in C. elegans embryo,” Proc. SPIE 6860, 68601L (2008).
[CrossRef]

Rev. Sci. Instrum. (1)

P. Ferrand, M. Pianta, A. Kress, A. Aillaud, H. Rigneault, and D. Marguet, “A versatile dual spot laser scanning confocal microscopy system for advanced fluorescence correlation spectroscopy analysis in living cell,” Rev. Sci. Instrum. 80, 083702 (2009).
[CrossRef] [PubMed]

Soft Matter (1)

D. C. Carrer, E. Kummer, G. Chwastek, S. Chiantia, and P. Schwille, “Asymmetry determines the effects of natural ceramides on model membranes,” Soft Matter 5, 3279–3286 (2009).
[CrossRef]

Other (2)

R. Rigler and E. S. Elson, eds., Fluorescence Correlation Spectroscopy. Theory and Application, Chemical Physics Series (Springer Verlag, Berlin, 2001), 1st ed.
[CrossRef]

M. Born and E. Wolf, Principles of Optics (Cambridge University Press, 1999), chap. 8, pp. 484–492, 7th ed.

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Figures (6)

Fig. 1
Fig. 1

The principle of circular scanning FCS on a 2D surface (membrane). A: The position of the objective focal plane with respect to the membrane plane affects not only the size a of the illuminated area on the membrane (the measurement volume Veff), but also the volume (area) overlap between any two different beam positions (here shown for the maximum possible distance, the scan circle diameter 2R). Left: the membrane coincides with the objective focus; Right: the membrane is below the objective focus. B: The axial displacement of the membrane away from the focus influences the autocorrelation in a complex way, changing not only the amplitude g(0) and the diffusion time τD, but also affecting the shape of the correlation peaks. The distinct ways of how variations in D and a affect the correlation allows the determination of the diffusion coefficient D and the volume size a from a single autocorrelation curve. C: The χ r 2 map as a function of D and a with a single minimum calculated from an experimental sFCS curve, demonstrating that D and a are uncorrelated. D: The χ r 2 map for a standard FCS curve with a minimum for all values of D where D = a2D, showing that D and a are fully correlated.

Fig. 2
Fig. 2

The experimental setup for scanning FCS. A: The standard FCS setup is modified by mounting the last mirror before the back objective entry onto a two-axis piezo scanner. B: The scanner is used to move the beam uniformly, with a known frequency f, in a circle of radius R comparable to the size a of the focused beam. The distance d between two focus positions separated by a lag time τ varies between 0 and 2R, and is uniquely determined by the relation d = 2Rsin(πfτ).

Fig. 3
Fig. 3

The dependence of the autocorrelation curves and the fit parameters on the membrane position relative to the focal plane. The measurements with the membrane near the focal plane (A) and away from the focal plane (B) result in clearly different autocorrelations, with the fitting yielding different amplitudes g(0), diffusion coefficients τD, and volume sizes a, but equal diffusion coefficients D. A series of measurements at different axial positions results in diffusion coefficient (C) and concentration (F) practically independent of the axial position, and the volume size (D) and diffusion time (E) exhibit a clear minimum when the membrane coincides with the objective focal plane. The inset in B shows the top pole of the GUV with the circular scan path (scale bar 1μm). The sample was a GUV prepared with DOPC and DiO.

Fig. 4
Fig. 4

The recovered diffusion coefficient is independent of the interval (τ1,τ2) in which the autocorrelation curve is analyzed. A: The range of intervals in which the data were analyzed had a variable lower bound τ1 varying from 10 μs to 20 ms and the upper bound τ2 = 30 ms (C), or a fixed lower bound τ1 = 10μs and a variable upper bound varying from 5 ms to 300 ms (D). The mean diffusion coefficient and its standard deviation σD calculated from 20 measurements are practically independent of the fitting range, both when the analysis is limited to either the short (C) or the longer (D) time scales. Sample: DOPC SLB with Bodipy-Chol. B: In circular scanning FCS, even a narrow fitting interval (2.5, 17.5) ms, that is, three scan periods, produces a deep, well defined minimum in χ r 2, sufficiently restricting the diffusion coefficient (solid line). In standard FCS, the same narrow fitting interval leads to a very shallow minimum and is not sufficient to constrain D (dashed line). Sample: DOPC GUV with Bodipy-Chol.

Fig. 5
Fig. 5

A slow axial drift has a minimal effect on the sFCS results. A: relative diffusion coefficients D/D0, relative concentrations c/c0, and volume sizes a obtained from fits of simulated data; where the actual volume size parameter a continuously varied in the range (0.12,0.12 + Δa)μm. The model parameters: D = 5μm2s−1, a0 = 0.12μm, f = 200 Hz, R = 0.4μm. The dashed lines indicate the range of volume sizes a covered by a given value Δa. B: Experimental autocorrelation curves with (blue) and without (black) circular scanning, where the objective focal plane was moved during the measurement uniformly along the optical axis by 1.8 μm, passing through the membrane. Both curves were fit in the range (0.01, 10) ms. The curve obtained without scanning was shifted down by Δg(0) = 0.004 for clarity. The inset shows the fluorescence intensity F(t) during the measurement.

Fig. 6
Fig. 6

The effect of membrane fluctuations on the sFCS results. Without circular scanning (A), and in the presence of axial fluctuations the autocorrelation curve (green) deviates significantly from the curve measured in the absence of axial fluctuations (blue), and cannot be described by a simple diffusion model. With circular scanning (C and D), the autocorrelation with axial fluctuations (D) still deviates from that without fluctuations (C), however, a fit of the initial part of the curve in the interval (0.01, 10) ms yields the same diffusion coefficient as obtained when the fluctuations are absent. The inset in C: SLB with the circular scan path. B: A measurement performed on the top pole of a fluctuating GUV with a fit of the initial part of the curve yields the expected diffusion coefficient (DOPC with DiO). The inset shows the GUV fluctuations (scale bars: 1μm).

Equations (7)

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

W ex ( r ) = a 0 2 a 2 ( z ) e x 2 + y 2 2 a 2 ( z ) ,
a 2 ( z ) = a 0 2 + b 2 z 2 , b = λ 4 π n a 0 ,
W ( x , y ) = e x 2 + y 2 2 a 2 .
g ( τ ) = 1 c V eff 1 1 + τ τ D e R 2 sin 2 ( π f τ ) a 2 ( 1 + τ τ D ) ,
τ D = a 2 / D .
c = 1 g ( 0 ) V eff = 1 4 π a 2 g ( 0 ) .
g 12 ( 0 ) g 1 ( 0 ) = 2 a 1 2 a 1 2 + a 2 2 c 12 c 2 + c 12

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