Abstract

We present a fluorescence correlation spectroscopy (FCS) approach to obtain spectral cross-talk free auto- and cross-correlation functions for probes with highly overlapping emission spectra. Confocal microscopes with either a hyperspectral EM-CCD or six-channel PMT array spectral detection were used, followed by a photon filtering correlation approach that results in spectral unmixing. The method is highly sensitive and can distinguish between Atto488 and Oregon Green 488 signals so that auto-correlation curves can be fitted without the need for cross-talk correction. We also applied the approach to the membrane dye Laurdan whose emission is dependent on the lipid order within the bilayer. With fluorescence spectral correlation spectroscopy (FSCS), we could obtain spectral cross-talk free auto- and cross-correlation functions corresponding to Laurdan located in liquid ordered and liquid disordered phases.

© 2014 Optical Society of America

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  1. E. L. Elson, “Fluorescence correlation spectroscopy: past, present, future,” Biophys. J. 101(12), 2855–2870 (2011).
    [CrossRef] [PubMed]
  2. Y. Tian, M. M. Martinez, D. Pappas, “Fluorescence correlation spectroscopy: a review of biochemical and microfluidic applications,” Appl. Spectrosc. 65(4), 115A–124A (2011).
    [CrossRef] [PubMed]
  3. P. Schwille, F. J. Meyer-Almes, R. Rigler, “Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution,” Biophys. J. 72(4), 1878–1886 (1997).
    [CrossRef] [PubMed]
  4. H. Park, C. Pack, M. Kinjo, B. K. Kaang, “In vivo quantitative analysis of PKA subunit interaction and cAMP level by dual color fluorescence cross correlation spectroscopy,” Mol. Cells 26(1), 87–92 (2008).
    [PubMed]
  5. K. G. Heinze, A. Koltermann, P. Schwille, “Simultaneous two-photon excitation of distinct labels for dual-color fluorescence crosscorrelation analysis,” Proc. Natl. Acad. Sci. U.S.A. 97(19), 10377–10382 (2000).
    [CrossRef] [PubMed]
  6. K. Bacia, Z. Petrášek, P. Schwille, “Correcting for spectral cross-talk in dual-color fluorescence cross-correlation spectroscopy,” ChemPhysChem 13(5), 1221–1231 (2012).
    [CrossRef] [PubMed]
  7. B. K. Müller, E. Zaychikov, C. Bräuchle, D. C. Lamb, “Pulsed interleaved excitation,” Biophys. J. 89(5), 3508–3522 (2005).
    [CrossRef] [PubMed]
  8. M. Burkhardt, K. G. Heinze, P. Schwille, “Four-color fluorescence correlation spectroscopy realized in a grating-based detection platform,” Opt. Lett. 30(17), 2266–2268 (2005).
    [CrossRef] [PubMed]
  9. M. J. Previte, S. Pelet, K. H. Kim, C. Buehler, P. T. So, “Spectrally resolved fluorescence correlation spectroscopy based on global analysis,” Anal. Chem. 80(9), 3277–3284 (2008).
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  10. F. Bestvater, Z. Seghiri, M. S. Kang, N. Gröner, J. Y. Lee, K. B. Im, M. Wachsmuth, “EMCCD-based spectrally resolved fluorescence correlation spectroscopy,” Opt. Express 18(23), 23818–23828 (2010).
    [CrossRef] [PubMed]
  11. M. Böhmer, M. Wahl, H.-J. Rahn, R. Erdmann, J. Enderlein, “Time-resolved fluorescence correlation spectroscopy,” Chem. Phys. Lett. 353(5-6), 439–445 (2002).
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  12. P. Kapusta, M. Wahl, A. Benda, M. Hof, J. Enderlein, “Fluorescence lifetime correlation spectroscopy,” J. Fluoresc. 17(1), 43–48 (2006).
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  13. P. Kapusta, R. Macháň, A. Benda, M. Hof, “Fluorescence lifetime correlation spectroscopy (FLCS): concepts, applications and outlook,” Int. J. Mol. Sci. 13(12), 12890–12910 (2012).
    [CrossRef] [PubMed]
  14. J. Humpolíčková, A. Benda, R. Macháň, J. Enderlein, M. Hof, “Dynamic saturation optical microscopy: employing dark-state formation kinetics for resolution enhancement,” Phys. Chem. Chem. Phys. 12(39), 12457–12465 (2010).
    [CrossRef] [PubMed]
  15. J. Humpolícková, A. Benda, J. Enderlein, “Optical saturation as a versatile tool to enhance resolution in confocal microscopy,” Biophys. J. 97(9), 2623–2629 (2009).
    [CrossRef] [PubMed]
  16. L. Beranová, J. Humpolíčková, J. Sýkora, A. Benda, L. Cwiklik, P. Jurkiewicz, G. Gröbner, M. Hof, “Effect of heavy water on phospholipid membranes: experimental confirmation of molecular dynamics simulations,” Phys. Chem. Chem. Phys. 14(42), 14516–14522 (2012).
    [CrossRef] [PubMed]
  17. A. G. Basden, C. A. Haniff, C. D. Mackay, “Photon counting strategies with low-light-level CCDs,” Mon. Not. R. Astron. Soc. 345(3), 985–991 (2003).
    [CrossRef]
  18. O. Daigle, C. Carignan, S. Blais-Ouellette, “Faint flux performance of an EMCCD,” Proc. SPIE 6276, 62761F (2006).
    [CrossRef]
  19. J. R. Unruh, E. Gratton, “Analysis of molecular concentration and brightness from fluorescence fluctuation data with an electron multiplied CCD camera,” Biophys. J. 95(11), 5385–5398 (2008).
    [CrossRef] [PubMed]
  20. J. Humpolícková, L. Beranová, M. Stĕpánek, A. Benda, K. Procházka, M. Hof, “Fluorescence lifetime correlation spectroscopy reveals compaction mechanism of 10 and 49 kbp DNA and differences between polycation and cationic surfactant,” J. Phys. Chem. B 112(51), 16823–16829 (2008).
    [CrossRef] [PubMed]
  21. A. Benda, “Spectral fluorescence correlation spectroscopy with EM-CCD camera,” presented at 15th International Workshop on “Single Molecule Spectroscopy and Ultrasensitive Analysis in the Life Sciences”, Berlin, Germany, 15–18 Sept. 2009.
  22. A. Benda, “Spectral fluorescence correlation spectroscopy with EM-CCD camera,” presented at Methods and Applications in Fluorescence 11, Budapest, Hungary, 6–9 Sept. 2009.
  23. B. Kannan, J. Y. Har, P. Liu, I. Maruyama, J. L. Ding, T. Wohland, “Electron multiplying charge-coupled device camera based fluorescence correlation spectroscopy,” Anal. Chem. 78(10), 3444–3451 (2006).
    [CrossRef] [PubMed]
  24. M. Burkhardt, P. Schwille, “Electron multiplying CCD based detection for spatially resolved fluorescence correlation spectroscopy,” Opt. Express 14(12), 5013–5020 (2006).
    [CrossRef] [PubMed]
  25. A. S. Klymchenko, Y. Mely, “Fluorescent environment-sensitive dyes as reporters of biomolecular interactions,” Prog. Mol. Biol. Transl. Sci. 113, 35–58 (2013).
    [CrossRef] [PubMed]
  26. K. Gaus, T. Zech, T. Harder, “Visualizing membrane microdomains by Laurdan 2-photon microscopy,” Mol. Membr. Biol. 23(1), 41–48 (2006).
    [CrossRef] [PubMed]
  27. T. Parasassi, F. Conti, E. Gratton, “Fluorophores in a polar medium: time dependence of emission spectra detected by multifrequency phase and modulation fluorometry,” Cell. Mol. Biol. 32(1), 99–102 (1986).
    [PubMed]
  28. L. A. Bagatolli, T. Parasassi, G. D. Fidelio, E. Gratton, “A model for the interaction of 6-lauroyl-2-(N,N-dimethylamino)naphthalene with lipid environments: implications for spectral properties,” Photochem. Photobiol. 70(4), 557–564 (1999).
    [CrossRef] [PubMed]
  29. P. Jurkiewicz, L. Cwiklik, P. Jungwirth, M. Hof, “Lipid hydration and mobility: an interplay between fluorescence solvent relaxation experiments and molecular dynamics simulations,” Biochimie 94(1), 26–32 (2012).
    [CrossRef] [PubMed]
  30. D. M. Owen, C. Rentero, A. Magenau, A. Abu-Siniyeh, K. Gaus, “Quantitative imaging of membrane lipid order in cells and organisms,” Nat. Protoc. 7(1), 24–35 (2011).
    [CrossRef] [PubMed]
  31. P. Jurkiewicz, J. Sýkora, A. Olzyńska, J. Humpolícková, M. Hof, “Solvent relaxation in phospholipid bilayers: principles and recent applications,” J. Fluoresc. 15(6), 883–894 (2005).
    [CrossRef] [PubMed]
  32. T. Parasassi, E. Gratton, H. Zajicek, M. Levi, W. Yu, “Detecting membrane lipid microdomains by two-photon fluorescence microscopy,” IEEE Eng. Med. Biol. Mag. 18(5), 92–99 (1999).
    [CrossRef] [PubMed]
  33. N. Kahya, D. Scherfeld, K. Bacia, B. Poolman, P. Schwille, “Probing lipid mobility of raft-exhibiting model membranes by fluorescence correlation spectroscopy,” J. Biol. Chem. 278(30), 28109–28115 (2003).
    [CrossRef] [PubMed]

2013 (1)

A. S. Klymchenko, Y. Mely, “Fluorescent environment-sensitive dyes as reporters of biomolecular interactions,” Prog. Mol. Biol. Transl. Sci. 113, 35–58 (2013).
[CrossRef] [PubMed]

2012 (4)

P. Kapusta, R. Macháň, A. Benda, M. Hof, “Fluorescence lifetime correlation spectroscopy (FLCS): concepts, applications and outlook,” Int. J. Mol. Sci. 13(12), 12890–12910 (2012).
[CrossRef] [PubMed]

L. Beranová, J. Humpolíčková, J. Sýkora, A. Benda, L. Cwiklik, P. Jurkiewicz, G. Gröbner, M. Hof, “Effect of heavy water on phospholipid membranes: experimental confirmation of molecular dynamics simulations,” Phys. Chem. Chem. Phys. 14(42), 14516–14522 (2012).
[CrossRef] [PubMed]

K. Bacia, Z. Petrášek, P. Schwille, “Correcting for spectral cross-talk in dual-color fluorescence cross-correlation spectroscopy,” ChemPhysChem 13(5), 1221–1231 (2012).
[CrossRef] [PubMed]

P. Jurkiewicz, L. Cwiklik, P. Jungwirth, M. Hof, “Lipid hydration and mobility: an interplay between fluorescence solvent relaxation experiments and molecular dynamics simulations,” Biochimie 94(1), 26–32 (2012).
[CrossRef] [PubMed]

2011 (3)

D. M. Owen, C. Rentero, A. Magenau, A. Abu-Siniyeh, K. Gaus, “Quantitative imaging of membrane lipid order in cells and organisms,” Nat. Protoc. 7(1), 24–35 (2011).
[CrossRef] [PubMed]

Y. Tian, M. M. Martinez, D. Pappas, “Fluorescence correlation spectroscopy: a review of biochemical and microfluidic applications,” Appl. Spectrosc. 65(4), 115A–124A (2011).
[CrossRef] [PubMed]

E. L. Elson, “Fluorescence correlation spectroscopy: past, present, future,” Biophys. J. 101(12), 2855–2870 (2011).
[CrossRef] [PubMed]

2010 (2)

J. Humpolíčková, A. Benda, R. Macháň, J. Enderlein, M. Hof, “Dynamic saturation optical microscopy: employing dark-state formation kinetics for resolution enhancement,” Phys. Chem. Chem. Phys. 12(39), 12457–12465 (2010).
[CrossRef] [PubMed]

F. Bestvater, Z. Seghiri, M. S. Kang, N. Gröner, J. Y. Lee, K. B. Im, M. Wachsmuth, “EMCCD-based spectrally resolved fluorescence correlation spectroscopy,” Opt. Express 18(23), 23818–23828 (2010).
[CrossRef] [PubMed]

2009 (1)

J. Humpolícková, A. Benda, J. Enderlein, “Optical saturation as a versatile tool to enhance resolution in confocal microscopy,” Biophys. J. 97(9), 2623–2629 (2009).
[CrossRef] [PubMed]

2008 (4)

J. R. Unruh, E. Gratton, “Analysis of molecular concentration and brightness from fluorescence fluctuation data with an electron multiplied CCD camera,” Biophys. J. 95(11), 5385–5398 (2008).
[CrossRef] [PubMed]

J. Humpolícková, L. Beranová, M. Stĕpánek, A. Benda, K. Procházka, M. Hof, “Fluorescence lifetime correlation spectroscopy reveals compaction mechanism of 10 and 49 kbp DNA and differences between polycation and cationic surfactant,” J. Phys. Chem. B 112(51), 16823–16829 (2008).
[CrossRef] [PubMed]

M. J. Previte, S. Pelet, K. H. Kim, C. Buehler, P. T. So, “Spectrally resolved fluorescence correlation spectroscopy based on global analysis,” Anal. Chem. 80(9), 3277–3284 (2008).
[CrossRef] [PubMed]

H. Park, C. Pack, M. Kinjo, B. K. Kaang, “In vivo quantitative analysis of PKA subunit interaction and cAMP level by dual color fluorescence cross correlation spectroscopy,” Mol. Cells 26(1), 87–92 (2008).
[PubMed]

2006 (5)

P. Kapusta, M. Wahl, A. Benda, M. Hof, J. Enderlein, “Fluorescence lifetime correlation spectroscopy,” J. Fluoresc. 17(1), 43–48 (2006).
[CrossRef] [PubMed]

B. Kannan, J. Y. Har, P. Liu, I. Maruyama, J. L. Ding, T. Wohland, “Electron multiplying charge-coupled device camera based fluorescence correlation spectroscopy,” Anal. Chem. 78(10), 3444–3451 (2006).
[CrossRef] [PubMed]

K. Gaus, T. Zech, T. Harder, “Visualizing membrane microdomains by Laurdan 2-photon microscopy,” Mol. Membr. Biol. 23(1), 41–48 (2006).
[CrossRef] [PubMed]

M. Burkhardt, P. Schwille, “Electron multiplying CCD based detection for spatially resolved fluorescence correlation spectroscopy,” Opt. Express 14(12), 5013–5020 (2006).
[CrossRef] [PubMed]

O. Daigle, C. Carignan, S. Blais-Ouellette, “Faint flux performance of an EMCCD,” Proc. SPIE 6276, 62761F (2006).
[CrossRef]

2005 (3)

M. Burkhardt, K. G. Heinze, P. Schwille, “Four-color fluorescence correlation spectroscopy realized in a grating-based detection platform,” Opt. Lett. 30(17), 2266–2268 (2005).
[CrossRef] [PubMed]

P. Jurkiewicz, J. Sýkora, A. Olzyńska, J. Humpolícková, M. Hof, “Solvent relaxation in phospholipid bilayers: principles and recent applications,” J. Fluoresc. 15(6), 883–894 (2005).
[CrossRef] [PubMed]

B. K. Müller, E. Zaychikov, C. Bräuchle, D. C. Lamb, “Pulsed interleaved excitation,” Biophys. J. 89(5), 3508–3522 (2005).
[CrossRef] [PubMed]

2003 (2)

A. G. Basden, C. A. Haniff, C. D. Mackay, “Photon counting strategies with low-light-level CCDs,” Mon. Not. R. Astron. Soc. 345(3), 985–991 (2003).
[CrossRef]

N. Kahya, D. Scherfeld, K. Bacia, B. Poolman, P. Schwille, “Probing lipid mobility of raft-exhibiting model membranes by fluorescence correlation spectroscopy,” J. Biol. Chem. 278(30), 28109–28115 (2003).
[CrossRef] [PubMed]

2002 (1)

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

2000 (1)

K. G. Heinze, A. Koltermann, P. Schwille, “Simultaneous two-photon excitation of distinct labels for dual-color fluorescence crosscorrelation analysis,” Proc. Natl. Acad. Sci. U.S.A. 97(19), 10377–10382 (2000).
[CrossRef] [PubMed]

1999 (2)

L. A. Bagatolli, T. Parasassi, G. D. Fidelio, E. Gratton, “A model for the interaction of 6-lauroyl-2-(N,N-dimethylamino)naphthalene with lipid environments: implications for spectral properties,” Photochem. Photobiol. 70(4), 557–564 (1999).
[CrossRef] [PubMed]

T. Parasassi, E. Gratton, H. Zajicek, M. Levi, W. Yu, “Detecting membrane lipid microdomains by two-photon fluorescence microscopy,” IEEE Eng. Med. Biol. Mag. 18(5), 92–99 (1999).
[CrossRef] [PubMed]

1997 (1)

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

1986 (1)

T. Parasassi, F. Conti, E. Gratton, “Fluorophores in a polar medium: time dependence of emission spectra detected by multifrequency phase and modulation fluorometry,” Cell. Mol. Biol. 32(1), 99–102 (1986).
[PubMed]

Abu-Siniyeh, A.

D. M. Owen, C. Rentero, A. Magenau, A. Abu-Siniyeh, K. Gaus, “Quantitative imaging of membrane lipid order in cells and organisms,” Nat. Protoc. 7(1), 24–35 (2011).
[CrossRef] [PubMed]

Bacia, K.

K. Bacia, Z. Petrášek, P. Schwille, “Correcting for spectral cross-talk in dual-color fluorescence cross-correlation spectroscopy,” ChemPhysChem 13(5), 1221–1231 (2012).
[CrossRef] [PubMed]

N. Kahya, D. Scherfeld, K. Bacia, B. Poolman, P. Schwille, “Probing lipid mobility of raft-exhibiting model membranes by fluorescence correlation spectroscopy,” J. Biol. Chem. 278(30), 28109–28115 (2003).
[CrossRef] [PubMed]

Bagatolli, L. A.

L. A. Bagatolli, T. Parasassi, G. D. Fidelio, E. Gratton, “A model for the interaction of 6-lauroyl-2-(N,N-dimethylamino)naphthalene with lipid environments: implications for spectral properties,” Photochem. Photobiol. 70(4), 557–564 (1999).
[CrossRef] [PubMed]

Basden, A. G.

A. G. Basden, C. A. Haniff, C. D. Mackay, “Photon counting strategies with low-light-level CCDs,” Mon. Not. R. Astron. Soc. 345(3), 985–991 (2003).
[CrossRef]

Benda, A.

P. Kapusta, R. Macháň, A. Benda, M. Hof, “Fluorescence lifetime correlation spectroscopy (FLCS): concepts, applications and outlook,” Int. J. Mol. Sci. 13(12), 12890–12910 (2012).
[CrossRef] [PubMed]

L. Beranová, J. Humpolíčková, J. Sýkora, A. Benda, L. Cwiklik, P. Jurkiewicz, G. Gröbner, M. Hof, “Effect of heavy water on phospholipid membranes: experimental confirmation of molecular dynamics simulations,” Phys. Chem. Chem. Phys. 14(42), 14516–14522 (2012).
[CrossRef] [PubMed]

J. Humpolíčková, A. Benda, R. Macháň, J. Enderlein, M. Hof, “Dynamic saturation optical microscopy: employing dark-state formation kinetics for resolution enhancement,” Phys. Chem. Chem. Phys. 12(39), 12457–12465 (2010).
[CrossRef] [PubMed]

J. Humpolícková, A. Benda, J. Enderlein, “Optical saturation as a versatile tool to enhance resolution in confocal microscopy,” Biophys. J. 97(9), 2623–2629 (2009).
[CrossRef] [PubMed]

J. Humpolícková, L. Beranová, M. Stĕpánek, A. Benda, K. Procházka, M. Hof, “Fluorescence lifetime correlation spectroscopy reveals compaction mechanism of 10 and 49 kbp DNA and differences between polycation and cationic surfactant,” J. Phys. Chem. B 112(51), 16823–16829 (2008).
[CrossRef] [PubMed]

P. Kapusta, M. Wahl, A. Benda, M. Hof, J. Enderlein, “Fluorescence lifetime correlation spectroscopy,” J. Fluoresc. 17(1), 43–48 (2006).
[CrossRef] [PubMed]

Beranová, L.

L. Beranová, J. Humpolíčková, J. Sýkora, A. Benda, L. Cwiklik, P. Jurkiewicz, G. Gröbner, M. Hof, “Effect of heavy water on phospholipid membranes: experimental confirmation of molecular dynamics simulations,” Phys. Chem. Chem. Phys. 14(42), 14516–14522 (2012).
[CrossRef] [PubMed]

J. Humpolícková, L. Beranová, M. Stĕpánek, A. Benda, K. Procházka, M. Hof, “Fluorescence lifetime correlation spectroscopy reveals compaction mechanism of 10 and 49 kbp DNA and differences between polycation and cationic surfactant,” J. Phys. Chem. B 112(51), 16823–16829 (2008).
[CrossRef] [PubMed]

Bestvater, F.

Blais-Ouellette, S.

O. Daigle, C. Carignan, S. Blais-Ouellette, “Faint flux performance of an EMCCD,” Proc. SPIE 6276, 62761F (2006).
[CrossRef]

Böhmer, M.

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

Bräuchle, C.

B. K. Müller, E. Zaychikov, C. Bräuchle, D. C. Lamb, “Pulsed interleaved excitation,” Biophys. J. 89(5), 3508–3522 (2005).
[CrossRef] [PubMed]

Buehler, C.

M. J. Previte, S. Pelet, K. H. Kim, C. Buehler, P. T. So, “Spectrally resolved fluorescence correlation spectroscopy based on global analysis,” Anal. Chem. 80(9), 3277–3284 (2008).
[CrossRef] [PubMed]

Burkhardt, M.

Carignan, C.

O. Daigle, C. Carignan, S. Blais-Ouellette, “Faint flux performance of an EMCCD,” Proc. SPIE 6276, 62761F (2006).
[CrossRef]

Conti, F.

T. Parasassi, F. Conti, E. Gratton, “Fluorophores in a polar medium: time dependence of emission spectra detected by multifrequency phase and modulation fluorometry,” Cell. Mol. Biol. 32(1), 99–102 (1986).
[PubMed]

Cwiklik, L.

P. Jurkiewicz, L. Cwiklik, P. Jungwirth, M. Hof, “Lipid hydration and mobility: an interplay between fluorescence solvent relaxation experiments and molecular dynamics simulations,” Biochimie 94(1), 26–32 (2012).
[CrossRef] [PubMed]

L. Beranová, J. Humpolíčková, J. Sýkora, A. Benda, L. Cwiklik, P. Jurkiewicz, G. Gröbner, M. Hof, “Effect of heavy water on phospholipid membranes: experimental confirmation of molecular dynamics simulations,” Phys. Chem. Chem. Phys. 14(42), 14516–14522 (2012).
[CrossRef] [PubMed]

Daigle, O.

O. Daigle, C. Carignan, S. Blais-Ouellette, “Faint flux performance of an EMCCD,” Proc. SPIE 6276, 62761F (2006).
[CrossRef]

Ding, J. L.

B. Kannan, J. Y. Har, P. Liu, I. Maruyama, J. L. Ding, T. Wohland, “Electron multiplying charge-coupled device camera based fluorescence correlation spectroscopy,” Anal. Chem. 78(10), 3444–3451 (2006).
[CrossRef] [PubMed]

Elson, E. L.

E. L. Elson, “Fluorescence correlation spectroscopy: past, present, future,” Biophys. J. 101(12), 2855–2870 (2011).
[CrossRef] [PubMed]

Enderlein, J.

J. Humpolíčková, A. Benda, R. Macháň, J. Enderlein, M. Hof, “Dynamic saturation optical microscopy: employing dark-state formation kinetics for resolution enhancement,” Phys. Chem. Chem. Phys. 12(39), 12457–12465 (2010).
[CrossRef] [PubMed]

J. Humpolícková, A. Benda, J. Enderlein, “Optical saturation as a versatile tool to enhance resolution in confocal microscopy,” Biophys. J. 97(9), 2623–2629 (2009).
[CrossRef] [PubMed]

P. Kapusta, M. Wahl, A. Benda, M. Hof, J. Enderlein, “Fluorescence lifetime correlation spectroscopy,” J. Fluoresc. 17(1), 43–48 (2006).
[CrossRef] [PubMed]

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

Erdmann, R.

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

Fidelio, G. D.

L. A. Bagatolli, T. Parasassi, G. D. Fidelio, E. Gratton, “A model for the interaction of 6-lauroyl-2-(N,N-dimethylamino)naphthalene with lipid environments: implications for spectral properties,” Photochem. Photobiol. 70(4), 557–564 (1999).
[CrossRef] [PubMed]

Gaus, K.

D. M. Owen, C. Rentero, A. Magenau, A. Abu-Siniyeh, K. Gaus, “Quantitative imaging of membrane lipid order in cells and organisms,” Nat. Protoc. 7(1), 24–35 (2011).
[CrossRef] [PubMed]

K. Gaus, T. Zech, T. Harder, “Visualizing membrane microdomains by Laurdan 2-photon microscopy,” Mol. Membr. Biol. 23(1), 41–48 (2006).
[CrossRef] [PubMed]

Gratton, E.

J. R. Unruh, E. Gratton, “Analysis of molecular concentration and brightness from fluorescence fluctuation data with an electron multiplied CCD camera,” Biophys. J. 95(11), 5385–5398 (2008).
[CrossRef] [PubMed]

T. Parasassi, E. Gratton, H. Zajicek, M. Levi, W. Yu, “Detecting membrane lipid microdomains by two-photon fluorescence microscopy,” IEEE Eng. Med. Biol. Mag. 18(5), 92–99 (1999).
[CrossRef] [PubMed]

L. A. Bagatolli, T. Parasassi, G. D. Fidelio, E. Gratton, “A model for the interaction of 6-lauroyl-2-(N,N-dimethylamino)naphthalene with lipid environments: implications for spectral properties,” Photochem. Photobiol. 70(4), 557–564 (1999).
[CrossRef] [PubMed]

T. Parasassi, F. Conti, E. Gratton, “Fluorophores in a polar medium: time dependence of emission spectra detected by multifrequency phase and modulation fluorometry,” Cell. Mol. Biol. 32(1), 99–102 (1986).
[PubMed]

Gröbner, G.

L. Beranová, J. Humpolíčková, J. Sýkora, A. Benda, L. Cwiklik, P. Jurkiewicz, G. Gröbner, M. Hof, “Effect of heavy water on phospholipid membranes: experimental confirmation of molecular dynamics simulations,” Phys. Chem. Chem. Phys. 14(42), 14516–14522 (2012).
[CrossRef] [PubMed]

Gröner, N.

Haniff, C. A.

A. G. Basden, C. A. Haniff, C. D. Mackay, “Photon counting strategies with low-light-level CCDs,” Mon. Not. R. Astron. Soc. 345(3), 985–991 (2003).
[CrossRef]

Har, J. Y.

B. Kannan, J. Y. Har, P. Liu, I. Maruyama, J. L. Ding, T. Wohland, “Electron multiplying charge-coupled device camera based fluorescence correlation spectroscopy,” Anal. Chem. 78(10), 3444–3451 (2006).
[CrossRef] [PubMed]

Harder, T.

K. Gaus, T. Zech, T. Harder, “Visualizing membrane microdomains by Laurdan 2-photon microscopy,” Mol. Membr. Biol. 23(1), 41–48 (2006).
[CrossRef] [PubMed]

Heinze, K. G.

M. Burkhardt, K. G. Heinze, P. Schwille, “Four-color fluorescence correlation spectroscopy realized in a grating-based detection platform,” Opt. Lett. 30(17), 2266–2268 (2005).
[CrossRef] [PubMed]

K. G. Heinze, A. Koltermann, P. Schwille, “Simultaneous two-photon excitation of distinct labels for dual-color fluorescence crosscorrelation analysis,” Proc. Natl. Acad. Sci. U.S.A. 97(19), 10377–10382 (2000).
[CrossRef] [PubMed]

Hof, M.

L. Beranová, J. Humpolíčková, J. Sýkora, A. Benda, L. Cwiklik, P. Jurkiewicz, G. Gröbner, M. Hof, “Effect of heavy water on phospholipid membranes: experimental confirmation of molecular dynamics simulations,” Phys. Chem. Chem. Phys. 14(42), 14516–14522 (2012).
[CrossRef] [PubMed]

P. Jurkiewicz, L. Cwiklik, P. Jungwirth, M. Hof, “Lipid hydration and mobility: an interplay between fluorescence solvent relaxation experiments and molecular dynamics simulations,” Biochimie 94(1), 26–32 (2012).
[CrossRef] [PubMed]

P. Kapusta, R. Macháň, A. Benda, M. Hof, “Fluorescence lifetime correlation spectroscopy (FLCS): concepts, applications and outlook,” Int. J. Mol. Sci. 13(12), 12890–12910 (2012).
[CrossRef] [PubMed]

J. Humpolíčková, A. Benda, R. Macháň, J. Enderlein, M. Hof, “Dynamic saturation optical microscopy: employing dark-state formation kinetics for resolution enhancement,” Phys. Chem. Chem. Phys. 12(39), 12457–12465 (2010).
[CrossRef] [PubMed]

J. Humpolícková, L. Beranová, M. Stĕpánek, A. Benda, K. Procházka, M. Hof, “Fluorescence lifetime correlation spectroscopy reveals compaction mechanism of 10 and 49 kbp DNA and differences between polycation and cationic surfactant,” J. Phys. Chem. B 112(51), 16823–16829 (2008).
[CrossRef] [PubMed]

P. Kapusta, M. Wahl, A. Benda, M. Hof, J. Enderlein, “Fluorescence lifetime correlation spectroscopy,” J. Fluoresc. 17(1), 43–48 (2006).
[CrossRef] [PubMed]

P. Jurkiewicz, J. Sýkora, A. Olzyńska, J. Humpolícková, M. Hof, “Solvent relaxation in phospholipid bilayers: principles and recent applications,” J. Fluoresc. 15(6), 883–894 (2005).
[CrossRef] [PubMed]

Humpolícková, J.

L. Beranová, J. Humpolíčková, J. Sýkora, A. Benda, L. Cwiklik, P. Jurkiewicz, G. Gröbner, M. Hof, “Effect of heavy water on phospholipid membranes: experimental confirmation of molecular dynamics simulations,” Phys. Chem. Chem. Phys. 14(42), 14516–14522 (2012).
[CrossRef] [PubMed]

J. Humpolíčková, A. Benda, R. Macháň, J. Enderlein, M. Hof, “Dynamic saturation optical microscopy: employing dark-state formation kinetics for resolution enhancement,” Phys. Chem. Chem. Phys. 12(39), 12457–12465 (2010).
[CrossRef] [PubMed]

J. Humpolícková, A. Benda, J. Enderlein, “Optical saturation as a versatile tool to enhance resolution in confocal microscopy,” Biophys. J. 97(9), 2623–2629 (2009).
[CrossRef] [PubMed]

J. Humpolícková, L. Beranová, M. Stĕpánek, A. Benda, K. Procházka, M. Hof, “Fluorescence lifetime correlation spectroscopy reveals compaction mechanism of 10 and 49 kbp DNA and differences between polycation and cationic surfactant,” J. Phys. Chem. B 112(51), 16823–16829 (2008).
[CrossRef] [PubMed]

P. Jurkiewicz, J. Sýkora, A. Olzyńska, J. Humpolícková, M. Hof, “Solvent relaxation in phospholipid bilayers: principles and recent applications,” J. Fluoresc. 15(6), 883–894 (2005).
[CrossRef] [PubMed]

Im, K. B.

Jungwirth, P.

P. Jurkiewicz, L. Cwiklik, P. Jungwirth, M. Hof, “Lipid hydration and mobility: an interplay between fluorescence solvent relaxation experiments and molecular dynamics simulations,” Biochimie 94(1), 26–32 (2012).
[CrossRef] [PubMed]

Jurkiewicz, P.

P. Jurkiewicz, L. Cwiklik, P. Jungwirth, M. Hof, “Lipid hydration and mobility: an interplay between fluorescence solvent relaxation experiments and molecular dynamics simulations,” Biochimie 94(1), 26–32 (2012).
[CrossRef] [PubMed]

L. Beranová, J. Humpolíčková, J. Sýkora, A. Benda, L. Cwiklik, P. Jurkiewicz, G. Gröbner, M. Hof, “Effect of heavy water on phospholipid membranes: experimental confirmation of molecular dynamics simulations,” Phys. Chem. Chem. Phys. 14(42), 14516–14522 (2012).
[CrossRef] [PubMed]

P. Jurkiewicz, J. Sýkora, A. Olzyńska, J. Humpolícková, M. Hof, “Solvent relaxation in phospholipid bilayers: principles and recent applications,” J. Fluoresc. 15(6), 883–894 (2005).
[CrossRef] [PubMed]

Kaang, B. K.

H. Park, C. Pack, M. Kinjo, B. K. Kaang, “In vivo quantitative analysis of PKA subunit interaction and cAMP level by dual color fluorescence cross correlation spectroscopy,” Mol. Cells 26(1), 87–92 (2008).
[PubMed]

Kahya, N.

N. Kahya, D. Scherfeld, K. Bacia, B. Poolman, P. Schwille, “Probing lipid mobility of raft-exhibiting model membranes by fluorescence correlation spectroscopy,” J. Biol. Chem. 278(30), 28109–28115 (2003).
[CrossRef] [PubMed]

Kang, M. S.

Kannan, B.

B. Kannan, J. Y. Har, P. Liu, I. Maruyama, J. L. Ding, T. Wohland, “Electron multiplying charge-coupled device camera based fluorescence correlation spectroscopy,” Anal. Chem. 78(10), 3444–3451 (2006).
[CrossRef] [PubMed]

Kapusta, P.

P. Kapusta, R. Macháň, A. Benda, M. Hof, “Fluorescence lifetime correlation spectroscopy (FLCS): concepts, applications and outlook,” Int. J. Mol. Sci. 13(12), 12890–12910 (2012).
[CrossRef] [PubMed]

P. Kapusta, M. Wahl, A. Benda, M. Hof, J. Enderlein, “Fluorescence lifetime correlation spectroscopy,” J. Fluoresc. 17(1), 43–48 (2006).
[CrossRef] [PubMed]

Kim, K. H.

M. J. Previte, S. Pelet, K. H. Kim, C. Buehler, P. T. So, “Spectrally resolved fluorescence correlation spectroscopy based on global analysis,” Anal. Chem. 80(9), 3277–3284 (2008).
[CrossRef] [PubMed]

Kinjo, M.

H. Park, C. Pack, M. Kinjo, B. K. Kaang, “In vivo quantitative analysis of PKA subunit interaction and cAMP level by dual color fluorescence cross correlation spectroscopy,” Mol. Cells 26(1), 87–92 (2008).
[PubMed]

Klymchenko, A. S.

A. S. Klymchenko, Y. Mely, “Fluorescent environment-sensitive dyes as reporters of biomolecular interactions,” Prog. Mol. Biol. Transl. Sci. 113, 35–58 (2013).
[CrossRef] [PubMed]

Koltermann, A.

K. G. Heinze, A. Koltermann, P. Schwille, “Simultaneous two-photon excitation of distinct labels for dual-color fluorescence crosscorrelation analysis,” Proc. Natl. Acad. Sci. U.S.A. 97(19), 10377–10382 (2000).
[CrossRef] [PubMed]

Lamb, D. C.

B. K. Müller, E. Zaychikov, C. Bräuchle, D. C. Lamb, “Pulsed interleaved excitation,” Biophys. J. 89(5), 3508–3522 (2005).
[CrossRef] [PubMed]

Lee, J. Y.

Levi, M.

T. Parasassi, E. Gratton, H. Zajicek, M. Levi, W. Yu, “Detecting membrane lipid microdomains by two-photon fluorescence microscopy,” IEEE Eng. Med. Biol. Mag. 18(5), 92–99 (1999).
[CrossRef] [PubMed]

Liu, P.

B. Kannan, J. Y. Har, P. Liu, I. Maruyama, J. L. Ding, T. Wohland, “Electron multiplying charge-coupled device camera based fluorescence correlation spectroscopy,” Anal. Chem. 78(10), 3444–3451 (2006).
[CrossRef] [PubMed]

Machán, R.

P. Kapusta, R. Macháň, A. Benda, M. Hof, “Fluorescence lifetime correlation spectroscopy (FLCS): concepts, applications and outlook,” Int. J. Mol. Sci. 13(12), 12890–12910 (2012).
[CrossRef] [PubMed]

J. Humpolíčková, A. Benda, R. Macháň, J. Enderlein, M. Hof, “Dynamic saturation optical microscopy: employing dark-state formation kinetics for resolution enhancement,” Phys. Chem. Chem. Phys. 12(39), 12457–12465 (2010).
[CrossRef] [PubMed]

Mackay, C. D.

A. G. Basden, C. A. Haniff, C. D. Mackay, “Photon counting strategies with low-light-level CCDs,” Mon. Not. R. Astron. Soc. 345(3), 985–991 (2003).
[CrossRef]

Magenau, A.

D. M. Owen, C. Rentero, A. Magenau, A. Abu-Siniyeh, K. Gaus, “Quantitative imaging of membrane lipid order in cells and organisms,” Nat. Protoc. 7(1), 24–35 (2011).
[CrossRef] [PubMed]

Martinez, M. M.

Maruyama, I.

B. Kannan, J. Y. Har, P. Liu, I. Maruyama, J. L. Ding, T. Wohland, “Electron multiplying charge-coupled device camera based fluorescence correlation spectroscopy,” Anal. Chem. 78(10), 3444–3451 (2006).
[CrossRef] [PubMed]

Mely, Y.

A. S. Klymchenko, Y. Mely, “Fluorescent environment-sensitive dyes as reporters of biomolecular interactions,” Prog. Mol. Biol. Transl. Sci. 113, 35–58 (2013).
[CrossRef] [PubMed]

Meyer-Almes, F. J.

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

Müller, B. K.

B. K. Müller, E. Zaychikov, C. Bräuchle, D. C. Lamb, “Pulsed interleaved excitation,” Biophys. J. 89(5), 3508–3522 (2005).
[CrossRef] [PubMed]

Olzynska, A.

P. Jurkiewicz, J. Sýkora, A. Olzyńska, J. Humpolícková, M. Hof, “Solvent relaxation in phospholipid bilayers: principles and recent applications,” J. Fluoresc. 15(6), 883–894 (2005).
[CrossRef] [PubMed]

Owen, D. M.

D. M. Owen, C. Rentero, A. Magenau, A. Abu-Siniyeh, K. Gaus, “Quantitative imaging of membrane lipid order in cells and organisms,” Nat. Protoc. 7(1), 24–35 (2011).
[CrossRef] [PubMed]

Pack, C.

H. Park, C. Pack, M. Kinjo, B. K. Kaang, “In vivo quantitative analysis of PKA subunit interaction and cAMP level by dual color fluorescence cross correlation spectroscopy,” Mol. Cells 26(1), 87–92 (2008).
[PubMed]

Pappas, D.

Parasassi, T.

L. A. Bagatolli, T. Parasassi, G. D. Fidelio, E. Gratton, “A model for the interaction of 6-lauroyl-2-(N,N-dimethylamino)naphthalene with lipid environments: implications for spectral properties,” Photochem. Photobiol. 70(4), 557–564 (1999).
[CrossRef] [PubMed]

T. Parasassi, E. Gratton, H. Zajicek, M. Levi, W. Yu, “Detecting membrane lipid microdomains by two-photon fluorescence microscopy,” IEEE Eng. Med. Biol. Mag. 18(5), 92–99 (1999).
[CrossRef] [PubMed]

T. Parasassi, F. Conti, E. Gratton, “Fluorophores in a polar medium: time dependence of emission spectra detected by multifrequency phase and modulation fluorometry,” Cell. Mol. Biol. 32(1), 99–102 (1986).
[PubMed]

Park, H.

H. Park, C. Pack, M. Kinjo, B. K. Kaang, “In vivo quantitative analysis of PKA subunit interaction and cAMP level by dual color fluorescence cross correlation spectroscopy,” Mol. Cells 26(1), 87–92 (2008).
[PubMed]

Pelet, S.

M. J. Previte, S. Pelet, K. H. Kim, C. Buehler, P. T. So, “Spectrally resolved fluorescence correlation spectroscopy based on global analysis,” Anal. Chem. 80(9), 3277–3284 (2008).
[CrossRef] [PubMed]

Petrášek, Z.

K. Bacia, Z. Petrášek, P. Schwille, “Correcting for spectral cross-talk in dual-color fluorescence cross-correlation spectroscopy,” ChemPhysChem 13(5), 1221–1231 (2012).
[CrossRef] [PubMed]

Poolman, B.

N. Kahya, D. Scherfeld, K. Bacia, B. Poolman, P. Schwille, “Probing lipid mobility of raft-exhibiting model membranes by fluorescence correlation spectroscopy,” J. Biol. Chem. 278(30), 28109–28115 (2003).
[CrossRef] [PubMed]

Previte, M. J.

M. J. Previte, S. Pelet, K. H. Kim, C. Buehler, P. T. So, “Spectrally resolved fluorescence correlation spectroscopy based on global analysis,” Anal. Chem. 80(9), 3277–3284 (2008).
[CrossRef] [PubMed]

Procházka, K.

J. Humpolícková, L. Beranová, M. Stĕpánek, A. Benda, K. Procházka, M. Hof, “Fluorescence lifetime correlation spectroscopy reveals compaction mechanism of 10 and 49 kbp DNA and differences between polycation and cationic surfactant,” J. Phys. Chem. B 112(51), 16823–16829 (2008).
[CrossRef] [PubMed]

Rahn, H.-J.

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

Rentero, C.

D. M. Owen, C. Rentero, A. Magenau, A. Abu-Siniyeh, K. Gaus, “Quantitative imaging of membrane lipid order in cells and organisms,” Nat. Protoc. 7(1), 24–35 (2011).
[CrossRef] [PubMed]

Rigler, R.

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

Scherfeld, D.

N. Kahya, D. Scherfeld, K. Bacia, B. Poolman, P. Schwille, “Probing lipid mobility of raft-exhibiting model membranes by fluorescence correlation spectroscopy,” J. Biol. Chem. 278(30), 28109–28115 (2003).
[CrossRef] [PubMed]

Schwille, P.

K. Bacia, Z. Petrášek, P. Schwille, “Correcting for spectral cross-talk in dual-color fluorescence cross-correlation spectroscopy,” ChemPhysChem 13(5), 1221–1231 (2012).
[CrossRef] [PubMed]

M. Burkhardt, P. Schwille, “Electron multiplying CCD based detection for spatially resolved fluorescence correlation spectroscopy,” Opt. Express 14(12), 5013–5020 (2006).
[CrossRef] [PubMed]

M. Burkhardt, K. G. Heinze, P. Schwille, “Four-color fluorescence correlation spectroscopy realized in a grating-based detection platform,” Opt. Lett. 30(17), 2266–2268 (2005).
[CrossRef] [PubMed]

N. Kahya, D. Scherfeld, K. Bacia, B. Poolman, P. Schwille, “Probing lipid mobility of raft-exhibiting model membranes by fluorescence correlation spectroscopy,” J. Biol. Chem. 278(30), 28109–28115 (2003).
[CrossRef] [PubMed]

K. G. Heinze, A. Koltermann, P. Schwille, “Simultaneous two-photon excitation of distinct labels for dual-color fluorescence crosscorrelation analysis,” Proc. Natl. Acad. Sci. U.S.A. 97(19), 10377–10382 (2000).
[CrossRef] [PubMed]

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

Seghiri, Z.

So, P. T.

M. J. Previte, S. Pelet, K. H. Kim, C. Buehler, P. T. So, “Spectrally resolved fluorescence correlation spectroscopy based on global analysis,” Anal. Chem. 80(9), 3277–3284 (2008).
[CrossRef] [PubMed]

Stepánek, M.

J. Humpolícková, L. Beranová, M. Stĕpánek, A. Benda, K. Procházka, M. Hof, “Fluorescence lifetime correlation spectroscopy reveals compaction mechanism of 10 and 49 kbp DNA and differences between polycation and cationic surfactant,” J. Phys. Chem. B 112(51), 16823–16829 (2008).
[CrossRef] [PubMed]

Sýkora, J.

L. Beranová, J. Humpolíčková, J. Sýkora, A. Benda, L. Cwiklik, P. Jurkiewicz, G. Gröbner, M. Hof, “Effect of heavy water on phospholipid membranes: experimental confirmation of molecular dynamics simulations,” Phys. Chem. Chem. Phys. 14(42), 14516–14522 (2012).
[CrossRef] [PubMed]

P. Jurkiewicz, J. Sýkora, A. Olzyńska, J. Humpolícková, M. Hof, “Solvent relaxation in phospholipid bilayers: principles and recent applications,” J. Fluoresc. 15(6), 883–894 (2005).
[CrossRef] [PubMed]

Tian, Y.

Unruh, J. R.

J. R. Unruh, E. Gratton, “Analysis of molecular concentration and brightness from fluorescence fluctuation data with an electron multiplied CCD camera,” Biophys. J. 95(11), 5385–5398 (2008).
[CrossRef] [PubMed]

Wachsmuth, M.

Wahl, M.

P. Kapusta, M. Wahl, A. Benda, M. Hof, J. Enderlein, “Fluorescence lifetime correlation spectroscopy,” J. Fluoresc. 17(1), 43–48 (2006).
[CrossRef] [PubMed]

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

Wohland, T.

B. Kannan, J. Y. Har, P. Liu, I. Maruyama, J. L. Ding, T. Wohland, “Electron multiplying charge-coupled device camera based fluorescence correlation spectroscopy,” Anal. Chem. 78(10), 3444–3451 (2006).
[CrossRef] [PubMed]

Yu, W.

T. Parasassi, E. Gratton, H. Zajicek, M. Levi, W. Yu, “Detecting membrane lipid microdomains by two-photon fluorescence microscopy,” IEEE Eng. Med. Biol. Mag. 18(5), 92–99 (1999).
[CrossRef] [PubMed]

Zajicek, H.

T. Parasassi, E. Gratton, H. Zajicek, M. Levi, W. Yu, “Detecting membrane lipid microdomains by two-photon fluorescence microscopy,” IEEE Eng. Med. Biol. Mag. 18(5), 92–99 (1999).
[CrossRef] [PubMed]

Zaychikov, E.

B. K. Müller, E. Zaychikov, C. Bräuchle, D. C. Lamb, “Pulsed interleaved excitation,” Biophys. J. 89(5), 3508–3522 (2005).
[CrossRef] [PubMed]

Zech, T.

K. Gaus, T. Zech, T. Harder, “Visualizing membrane microdomains by Laurdan 2-photon microscopy,” Mol. Membr. Biol. 23(1), 41–48 (2006).
[CrossRef] [PubMed]

Anal. Chem. (2)

M. J. Previte, S. Pelet, K. H. Kim, C. Buehler, P. T. So, “Spectrally resolved fluorescence correlation spectroscopy based on global analysis,” Anal. Chem. 80(9), 3277–3284 (2008).
[CrossRef] [PubMed]

B. Kannan, J. Y. Har, P. Liu, I. Maruyama, J. L. Ding, T. Wohland, “Electron multiplying charge-coupled device camera based fluorescence correlation spectroscopy,” Anal. Chem. 78(10), 3444–3451 (2006).
[CrossRef] [PubMed]

Appl. Spectrosc. (1)

Biochimie (1)

P. Jurkiewicz, L. Cwiklik, P. Jungwirth, M. Hof, “Lipid hydration and mobility: an interplay between fluorescence solvent relaxation experiments and molecular dynamics simulations,” Biochimie 94(1), 26–32 (2012).
[CrossRef] [PubMed]

Biophys. J. (5)

J. R. Unruh, E. Gratton, “Analysis of molecular concentration and brightness from fluorescence fluctuation data with an electron multiplied CCD camera,” Biophys. J. 95(11), 5385–5398 (2008).
[CrossRef] [PubMed]

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

E. L. Elson, “Fluorescence correlation spectroscopy: past, present, future,” Biophys. J. 101(12), 2855–2870 (2011).
[CrossRef] [PubMed]

B. K. Müller, E. Zaychikov, C. Bräuchle, D. C. Lamb, “Pulsed interleaved excitation,” Biophys. J. 89(5), 3508–3522 (2005).
[CrossRef] [PubMed]

J. Humpolícková, A. Benda, J. Enderlein, “Optical saturation as a versatile tool to enhance resolution in confocal microscopy,” Biophys. J. 97(9), 2623–2629 (2009).
[CrossRef] [PubMed]

Cell. Mol. Biol. (1)

T. Parasassi, F. Conti, E. Gratton, “Fluorophores in a polar medium: time dependence of emission spectra detected by multifrequency phase and modulation fluorometry,” Cell. Mol. Biol. 32(1), 99–102 (1986).
[PubMed]

Chem. Phys. Lett. (1)

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

ChemPhysChem (1)

K. Bacia, Z. Petrášek, P. Schwille, “Correcting for spectral cross-talk in dual-color fluorescence cross-correlation spectroscopy,” ChemPhysChem 13(5), 1221–1231 (2012).
[CrossRef] [PubMed]

IEEE Eng. Med. Biol. Mag. (1)

T. Parasassi, E. Gratton, H. Zajicek, M. Levi, W. Yu, “Detecting membrane lipid microdomains by two-photon fluorescence microscopy,” IEEE Eng. Med. Biol. Mag. 18(5), 92–99 (1999).
[CrossRef] [PubMed]

Int. J. Mol. Sci. (1)

P. Kapusta, R. Macháň, A. Benda, M. Hof, “Fluorescence lifetime correlation spectroscopy (FLCS): concepts, applications and outlook,” Int. J. Mol. Sci. 13(12), 12890–12910 (2012).
[CrossRef] [PubMed]

J. Biol. Chem. (1)

N. Kahya, D. Scherfeld, K. Bacia, B. Poolman, P. Schwille, “Probing lipid mobility of raft-exhibiting model membranes by fluorescence correlation spectroscopy,” J. Biol. Chem. 278(30), 28109–28115 (2003).
[CrossRef] [PubMed]

J. Fluoresc. (2)

P. Jurkiewicz, J. Sýkora, A. Olzyńska, J. Humpolícková, M. Hof, “Solvent relaxation in phospholipid bilayers: principles and recent applications,” J. Fluoresc. 15(6), 883–894 (2005).
[CrossRef] [PubMed]

P. Kapusta, M. Wahl, A. Benda, M. Hof, J. Enderlein, “Fluorescence lifetime correlation spectroscopy,” J. Fluoresc. 17(1), 43–48 (2006).
[CrossRef] [PubMed]

J. Phys. Chem. B (1)

J. Humpolícková, L. Beranová, M. Stĕpánek, A. Benda, K. Procházka, M. Hof, “Fluorescence lifetime correlation spectroscopy reveals compaction mechanism of 10 and 49 kbp DNA and differences between polycation and cationic surfactant,” J. Phys. Chem. B 112(51), 16823–16829 (2008).
[CrossRef] [PubMed]

Mol. Cells (1)

H. Park, C. Pack, M. Kinjo, B. K. Kaang, “In vivo quantitative analysis of PKA subunit interaction and cAMP level by dual color fluorescence cross correlation spectroscopy,” Mol. Cells 26(1), 87–92 (2008).
[PubMed]

Mol. Membr. Biol. (1)

K. Gaus, T. Zech, T. Harder, “Visualizing membrane microdomains by Laurdan 2-photon microscopy,” Mol. Membr. Biol. 23(1), 41–48 (2006).
[CrossRef] [PubMed]

Mon. Not. R. Astron. Soc. (1)

A. G. Basden, C. A. Haniff, C. D. Mackay, “Photon counting strategies with low-light-level CCDs,” Mon. Not. R. Astron. Soc. 345(3), 985–991 (2003).
[CrossRef]

Nat. Protoc. (1)

D. M. Owen, C. Rentero, A. Magenau, A. Abu-Siniyeh, K. Gaus, “Quantitative imaging of membrane lipid order in cells and organisms,” Nat. Protoc. 7(1), 24–35 (2011).
[CrossRef] [PubMed]

Opt. Express (2)

Opt. Lett. (1)

Photochem. Photobiol. (1)

L. A. Bagatolli, T. Parasassi, G. D. Fidelio, E. Gratton, “A model for the interaction of 6-lauroyl-2-(N,N-dimethylamino)naphthalene with lipid environments: implications for spectral properties,” Photochem. Photobiol. 70(4), 557–564 (1999).
[CrossRef] [PubMed]

Phys. Chem. Chem. Phys. (2)

J. Humpolíčková, A. Benda, R. Macháň, J. Enderlein, M. Hof, “Dynamic saturation optical microscopy: employing dark-state formation kinetics for resolution enhancement,” Phys. Chem. Chem. Phys. 12(39), 12457–12465 (2010).
[CrossRef] [PubMed]

L. Beranová, J. Humpolíčková, J. Sýkora, A. Benda, L. Cwiklik, P. Jurkiewicz, G. Gröbner, M. Hof, “Effect of heavy water on phospholipid membranes: experimental confirmation of molecular dynamics simulations,” Phys. Chem. Chem. Phys. 14(42), 14516–14522 (2012).
[CrossRef] [PubMed]

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

K. G. Heinze, A. Koltermann, P. Schwille, “Simultaneous two-photon excitation of distinct labels for dual-color fluorescence crosscorrelation analysis,” Proc. Natl. Acad. Sci. U.S.A. 97(19), 10377–10382 (2000).
[CrossRef] [PubMed]

Proc. SPIE (1)

O. Daigle, C. Carignan, S. Blais-Ouellette, “Faint flux performance of an EMCCD,” Proc. SPIE 6276, 62761F (2006).
[CrossRef]

Prog. Mol. Biol. Transl. Sci. (1)

A. S. Klymchenko, Y. Mely, “Fluorescent environment-sensitive dyes as reporters of biomolecular interactions,” Prog. Mol. Biol. Transl. Sci. 113, 35–58 (2013).
[CrossRef] [PubMed]

Other (2)

A. Benda, “Spectral fluorescence correlation spectroscopy with EM-CCD camera,” presented at 15th International Workshop on “Single Molecule Spectroscopy and Ultrasensitive Analysis in the Life Sciences”, Berlin, Germany, 15–18 Sept. 2009.

A. Benda, “Spectral fluorescence correlation spectroscopy with EM-CCD camera,” presented at Methods and Applications in Fluorescence 11, Budapest, Hungary, 6–9 Sept. 2009.

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Figures (4)

Fig. 1
Fig. 1

Principle of fluorescence spectral correlation spectroscopy (FSCS) data acquisition and analysis. (A) FSCS setup is based on a standard confocal microscope with a single excitation wavelength. The emitted fluorescence is re-collimated after passing through a pinhole, dispersed via a prism or grid and focused onto a line array of single photon counting detectors (APDs, PMTs or EM-CCD). Each detected photon n is assigned a spectral detection channel λn and detection time tn relative to the start of the acquisition. Data is saved in time-tagged spectrally resolved (TTSR) format. (B) Emission spectra were reconstructed from TTSR data obtained for 100 nm diameter DOPC vesicles labelled with BODIPY FL-DOPE at ratio 1:1000 (red line), 200 nm diameter DOPC vesicles labelled with DiO at ratio 1:1000 (blue line), a 3:1 mixture of 100 nm BODIPY FL-DOPE labelled vesicles with 200 nm DiO labelled vesicles (green line) and the detector’s background signal (black line). The data was obtained with an EM-CCD camera in single photon counting mode. (C) Photon weighting filters were calculated from the spectral patterns shown in B according to Eq. (3). Note the sum of all filters (brown line) is equal to 1 for each spectral channel. (D) Spectral cross-talk free autocorrelation functions of each component (DiO – blue line, BODIPY FL-DOPE – red line and EM-CCD background – black line) and their cross-correlation functions (only DiO versus BODIPY FL-DOPE cross-correlation is shown – magenta line) were obtained from the TTSR data of the 3:1 mixture of 100 nm BODIPY FL-DOPE labelled vesicles with 200 nm DiO labelled vesicles (green line in B) after applying the photon weighting filters according to Eq. (4).

Fig. 2
Fig. 2

Fluorescence spectral correlation spectroscopy (FSCS) with an EM-CCD camera. (A) An EM-CCD camera (128x128 pixels) was used in continuous crop mode to obtain a 62.5 kHz line spectral readout of 128 pixels with single photon sensitivity (EM gain 1000). A typical single line readout is shown (black line). Intensities above the defined threshold (red line) were considered as single photon events. (B) Comparison of experimental spectra of Alexa488 in water (1 nM at 10 µW – red line, 1, 2, 4 and 8 nM at 40 µW – blue, cyan, magenta and dark yellow lines, respectively, 488 nm excitation) with the spectrum of EM-CCD background (black line. Measured with a closed shutter). The high read-out background (integral intensity 375 kHz from all 128 channels) makes this detection unsuitable for single molecule experiments (1 nM Alexa488 at 10 µW 488 nm excitation has an integral fluorescence intensity of 5.4 kHz after background subtraction – red line minus black line). The rise of background with increasing pixel number is indicative of readout-induced noise (C) Amplitude-normalized autocorrelation curves measured for 100 nm diameter DOPC vesicles labelled with BODIPY FL-DOPE at ratio 1:1000 (red line), 200 nm diameter DOPC vesicles labelled with DiO at ratio 1:1000 (blue line) and BODIPY FL-DOPE (red dots) and DiO (blue dots) autocorrelation curves obtained from a 3:1 molar mixture of 100 nm BODIPY FL-DOPE-labelled vesicles and 200 nm DiO-labelled vesicles, indicating the successful separation of the diffusion times for 100 nm and 200 nm vesicles despite the spectral overlap of BODIPY FL-DOPE and DiO.(D) Autocorrelation curves of DiO, obtained from a 3:1 molar mixture of 100 nm BODIPY FL-DOPE-labelled vesicles with 200 nm DiO labelled vesicles at various binning of spectral channels. Number of channels are 128 (black line,) 32 (red line), 16 (blue line), 8 (cyan line) and 5 (magenta line).

Fig. 3
Fig. 3

Fluorescence spectral correlation spectroscopy (FSCS) with spectral GaAsP detectors. (A) Emission spectra for free Atto488 in solution (red line), 100 nm DOPC vesicles labelled with Oregon Green 488-DOPE at ratio 1:10 000 (OG488, blue line) and a 1:1 volume mixture of 100 nm OG488-labelled vesicles with free Atto488 in solution (green line) were reconstructed from six spectral channel TTSR data acquired with an LSM780 confocal microscope. (B) Photon weighting filters were calculated for Atto488 (red line) and OG488 (blue line) according to Eq. (1). The sum of filters (brown line) adds up to 1. (C) Autocorrelation curve for a 1:1 volume mixture of 100 nm OG488-labelled vesicles with free Atto488 in solution without any filtering applied (green line) and autocorrelation curves of Atto488 (red line) and OG488 (blue line) and cross-correlation curve between Atto488 and OG488 (magenta line) from the same TTSR data after applying the photon weighting filters shown in (B). Note the lack of cross-correlation, indicating successful unmixing of the Atto488 and OG488 signals. (D) Auto-correlation curves for Atto488 in solution (red line), 100 nm OG488-labelled vesicles (blue line) and FSCS auto-correlation curves for Atto488 (red dots) and OG488 (blue dots) from a 1:1 volume mixture of 100 nm OG488-labelled vesicles with free Atto488 in solution. Both direct FCS measurement and FSCS measurement in a mixture returned diffusion times of 40 µs for free Atto488 in solution and 580 µs for OG488 in vesicles. The filtered auto-correlation curves are noisier due to the inherent uncertainty caused by the statistical photon weighting.

Fig. 4
Fig. 4

Fluorescence spectral correlation spectroscopy (FSCS) analysis of large unilamellar vesicles (LUVs) suspensions stained with environmentally sensitive membrane dye Laurdan. Laurdan is a membrane dye whose emission spectrum depends on the lipid phase. (A) 100 nm vesicles in liquid disordered phase (DOPC, red line), liquid ordered phase (70:30 molar mixture of SM:Chol, blue line) and with coexisting liquid ordered/disordered phase (50:35:15 molar mixture of DOPC:SM:Chol, cyan line) were stained with Laurdan before extrusion at molar ratio of 1:100 dye:lipid. Laurdan emission spectra from the three different types of lipid vesicles, plus a 1:1 volume mixture (green line) of liquid disordered phase vesicles and liquid ordered phase vesicles are shown. Data were reconstructed from six spectral channel TTSR data acquired with an LSM780 confocal microscope (405 nm excitation). (B) Photon weighting filters were calculated for spectral patterns devised from Laurdan in disordered (red line) and Laurdan in ordered (blue line), both for suspension of LUVs with coexisting liquid ordered/disordered phase (full square) and for mixture of monophasic vesicles (hollow square) according to Eq. (1). The sum of filters (black lines) is 1. (C) FSCS analysis of a 1:1 volume mixture of liquid disordered phase vesicles and liquid ordered phase vesicles (green in A) returned auto-correlation curves for Laurdan in liquid disordered phase pattern (red line) and Laurdan in liquid disordered phase pattern (blue line). As anticipated, no cross-correlation between the phases (magenta line) was detected. (D) FSCS analysis of the Laurdan-labelled vesicles with coexisting liquid ordered and liquid disordered phases (cyan line in A) returned auto-correlation curves for Laurdan in liquid disordered phase pattern (red line), liquid ordered phase pattern (blue) and the cross-correlation curve between the two patterns (magenta line). The high amplitude of the cross-correlation curve indicates that the patterns fluctuate together, which is expected since both lipid phases are located together in each vesicle.

Equations (6)

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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( τ )=1+ 1 PN( 1T ) ( 1T( 1 e τ τ 0 ) )( 1 1+τ/ τ res 1 ( 1+( τ/ τ res ) ( ω 0 / ω Z ) 2 ) 1/2 )

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