Abstract

We present an implementation of fluorescence correlation spectroscopy with spectrally resolved detection based on a combined commercial confocal laser scanning/fluorescence correlation spectroscopy microscope. We have replaced the conventional detection scheme by a prism-based spectrometer and an electron-multiplying charge-coupled device camera used to record the photons. This allows us to read out more than 80,000 full spectra per second with a signal-to-noise ratio and a quantum efficiency high enough to allow single photon counting. We can identify up to four spectrally different quantum dots in vitro and demonstrate that spectrally resolved detection can be used to characterize photophysical properties of fluorophores by measuring the spectral dependence of quantum dot fluorescence emission intermittence. Moreover, we can confirm intracellular cross-correlation results as acquired with a conventional setup and show that spectral flexibility can help to optimize the choice of the detection windows.

©2010 Optical Society of America

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    [Crossref]
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    [Crossref]
  4. D. Magde, E. L. Elson, and W. W. Webb, “Fluorescence correlation spectroscopy. II. An experimental realization,” Biopolymers 13(1), 29–61 (1974).
    [Crossref] [PubMed]
  5. K. Bacia and P. Schwille, “A dynamic view of cellular processes by in vivo fluorescence auto- and cross-correlation spectroscopy,” Methods 29(1), 74–85 (2003).
    [Crossref] [PubMed]
  6. M. Gösch and R. Rigler, “Fluorescence correlation spectroscopy of molecular motions and kinetics,” Adv. Drug Deliv. Rev. 57(1), 169–190 (2005).
    [Crossref]
  7. J. Langowski, “Protein-protein interactions determined by fluorescence correlation spectroscopy,” Methods Cell Biol. 85, 471–484 (2008).
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  8. M. Wachsmuth, and K. Weisshart, “Fluorescence photobleaching and fluorescence correlation spectroscopy: two complementary technologies to study molecular dynamics in living cells,” in Imaging Cellular and Molecular Biological Functions (Springer Verlag, Heidelberg, 2007).
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    [Crossref]
  10. J. Rika and T. Binkert, “Direct measurement of a distinct correlation function by fluorescence cross correlation,” Phys. Rev. A 39(5), 2646–2652 (1989).
    [Crossref] [PubMed]
  11. P. Schwille, F. J. Meyer-Almes, and R. Rigler, “Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution,” Biophys. J. 72(4), 1878–1886 (1997).
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  12. M. Burkhardt, K. G. Heinze, and P. Schwille, “Four-color fluorescence correlation spectroscopy realized in a grating-based detection platform,” Opt. Lett. 30(17), 2266–2268 (2005).
    [Crossref] [PubMed]
  13. K. G. Heinze, M. Jahnz, and P. Schwille, “Triple-color coincidence analysis: one step further in following higher order molecular complex formation,” Biophys. J. 86(1), 506–516 (2004).
    [Crossref]
  14. L. C. Hwang, M. Leutenegger, M. Gösch, T. Lasser, P. Rigler, W. Meier, and T. Wohland, “Prism-based multicolor fluorescence correlation spectrometer,” Opt. Lett. 31(9), 1310–1312 (2006).
    [Crossref] [PubMed]
  15. M. J. R. Previte, S. Pelet, K. H. Kim, C. Buehler, and P. T. C. So, “Spectrally resolved fluorescence correlation spectroscopy based on global analysis,” Anal. Chem. 80(9), 3277–3284 (2008).
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  16. M. Burkhardt and P. Schwille, “Electron multiplying CCD based detection for spatially resolved fluorescence correlation spectroscopy,” Opt. Express 14(12), 5013–5020 (2006).
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  17. B. Kannan, J. Y. Har, P. Liu, I. Maruyama, J. L. Ding, and T. Wohland, “Electron multiplying charge-coupled device camera based fluorescence correlation spectroscopy,” Anal. Chem. 78(10), 3444–3451 (2006).
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  23. K. Schätzel, “Noise on photon correlation data. I. Autocorrelation functions,” Quantum Opt. 2(4), 287–305 (1990).
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  24. M. Wachsmuth, W. Waldeck, and J. Langowski, “Anomalous diffusion of fluorescent probes inside living cell nuclei investigated by spatially-resolved fluorescence correlation spectroscopy,” J. Mol. Biol. 298(4), 677–689 (2000).
    [Crossref] [PubMed]
  25. T. Weidemann, M. Wachsmuth, M. Tewes, K. Rippe, and J. Langowski, “Analysis of ligand binding by two-colour fluorescence cross-correlation spectroscopy,” Single Mol. 3(1), 49–61 (2002).
    [Crossref]
  26. K. Saito, I. Wada, M. Tamura, and M. Kinjo, “Direct detection of caspase-3 activation in single live cells by cross-correlation analysis,” Biochem. Biophys. Res. Commun. 324(2), 849–854 (2004).
    [Crossref] [PubMed]
  27. J. R. Unruh and 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]
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  29. X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir, and S. Weiss, “Quantum dots for live cells, in vivo imaging, and diagnostics,” Science 307(5709), 538–544 (2005).
    [Crossref] [PubMed]
  30. S. Doose, J. M. Tsay, F. Pinaud, and S. Weiss, “Comparison of photophysical and colloidal properties of biocompatible semiconductor nanocrystals using fluorescence correlation spectroscopy,” Anal. Chem. 77(7), 2235–2242 (2005).
    [Crossref] [PubMed]
  31. P. Frantsuzov, M. Kuno, B. Janko, and R. A. Marcus, “Universal emission intermittency in quantum dots, nanorods and nanowires,” Nat. Phys. 4(5), 519–522 (2008).
    [Crossref]
  32. R. Verberk and M. Orrit, “Photon statistics in the fluorescence of single molecules and nanocrystals: Correlation functions versus distributions of on- and off-times,” J. Chem. Phys. 119(4), 2214–2222 (2003).
    [Crossref]
  33. J. Yao, D. R. Larson, H. D. Vishwasrao, W. R. Zipfel, and W. W. Webb, “Blinking and nonradiant dark fraction of water-soluble quantum dots in aqueous solution,” Proc. Natl. Acad. Sci. U.S.A. 102(40), 14284–14289 (2005).
    [Crossref] [PubMed]
  34. A. N. Kapanidis, N. K. Lee, T. A. Laurence, S. Doose, E. Margeat, and S. Weiss, “Fluorescence-aided molecule sorting: analysis of structure and interactions by alternating-laser excitation of single molecules,” Proc. Natl. Acad. Sci. U.S.A. 101(24), 8936–8941 (2004).
    [Crossref] [PubMed]
  35. S. Rüttinger, R. Macdonald, B. Krämer, F. Koberling, M. Roos, and E. Hildt, “Accurate single-pair Förster resonant energy transfer through combination of pulsed interleaved excitation, time correlated single-photon counting, and fluorescence correlation spectroscopy,” J. Biomed. Opt. 11(2), 024012–024012 (2006).
    [Crossref] [PubMed]
  36. B. Kannan, L. Guo, T. Sudhaharan, S. Ahmed, I. Maruyama, and T. Wohland, “Spatially Resolved Total Internal Reflection Fluorescence Correlation Microscopy Using an Electron Multiplying Charge-Coupled Device Camera,” Analytical Chemistry (2007).

2010 (2)

2009 (2)

G. Heuvelman, F. Erdel, M. Wachsmuth, and K. Rippe, “Analysis of protein mobilities and interactions in living cells by multifocal fluorescence fluctuation microscopy,” Eur. Biophys. J. 38(6), 813–828 (2009).
[Crossref] [PubMed]

D. J. Needleman, Y. Xu, and T. J. Mitchison, “Pin-hole array correlation imaging: highly parallel fluorescence correlation spectroscopy,” Biophys. J. 96(12), 5050–5059 (2009).
[Crossref] [PubMed]

2008 (4)

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

J. Langowski, “Protein-protein interactions determined by fluorescence correlation spectroscopy,” Methods Cell Biol. 85, 471–484 (2008).
[Crossref]

J. R. Unruh and 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. Frantsuzov, M. Kuno, B. Janko, and R. A. Marcus, “Universal emission intermittency in quantum dots, nanorods and nanowires,” Nat. Phys. 4(5), 519–522 (2008).
[Crossref]

2006 (5)

S. Rüttinger, R. Macdonald, B. Krämer, F. Koberling, M. Roos, and E. Hildt, “Accurate single-pair Förster resonant energy transfer through combination of pulsed interleaved excitation, time correlated single-photon counting, and fluorescence correlation spectroscopy,” J. Biomed. Opt. 11(2), 024012–024012 (2006).
[Crossref] [PubMed]

L. C. Hwang, M. Leutenegger, M. Gösch, T. Lasser, P. Rigler, W. Meier, and T. Wohland, “Prism-based multicolor fluorescence correlation spectrometer,” Opt. Lett. 31(9), 1310–1312 (2006).
[Crossref] [PubMed]

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

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

D. R. Sisan, R. Arevalo, C. Graves, R. McAllister, and J. S. Urbach, “Spatially resolved fluorescence correlation spectroscopy using a spinning disk confocal microscope,” Biophys. J. 91(11), 4241–4252 (2006).
[Crossref] [PubMed]

2005 (5)

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

M. Gösch and R. Rigler, “Fluorescence correlation spectroscopy of molecular motions and kinetics,” Adv. Drug Deliv. Rev. 57(1), 169–190 (2005).
[Crossref]

X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir, and S. Weiss, “Quantum dots for live cells, in vivo imaging, and diagnostics,” Science 307(5709), 538–544 (2005).
[Crossref] [PubMed]

S. Doose, J. M. Tsay, F. Pinaud, and S. Weiss, “Comparison of photophysical and colloidal properties of biocompatible semiconductor nanocrystals using fluorescence correlation spectroscopy,” Anal. Chem. 77(7), 2235–2242 (2005).
[Crossref] [PubMed]

J. Yao, D. R. Larson, H. D. Vishwasrao, W. R. Zipfel, and W. W. Webb, “Blinking and nonradiant dark fraction of water-soluble quantum dots in aqueous solution,” Proc. Natl. Acad. Sci. U.S.A. 102(40), 14284–14289 (2005).
[Crossref] [PubMed]

2004 (3)

A. N. Kapanidis, N. K. Lee, T. A. Laurence, S. Doose, E. Margeat, and S. Weiss, “Fluorescence-aided molecule sorting: analysis of structure and interactions by alternating-laser excitation of single molecules,” Proc. Natl. Acad. Sci. U.S.A. 101(24), 8936–8941 (2004).
[Crossref] [PubMed]

K. Saito, I. Wada, M. Tamura, and M. Kinjo, “Direct detection of caspase-3 activation in single live cells by cross-correlation analysis,” Biochem. Biophys. Res. Commun. 324(2), 849–854 (2004).
[Crossref] [PubMed]

K. G. Heinze, M. Jahnz, and P. Schwille, “Triple-color coincidence analysis: one step further in following higher order molecular complex formation,” Biophys. J. 86(1), 506–516 (2004).
[Crossref]

2003 (2)

K. Bacia and P. Schwille, “A dynamic view of cellular processes by in vivo fluorescence auto- and cross-correlation spectroscopy,” Methods 29(1), 74–85 (2003).
[Crossref] [PubMed]

R. Verberk and M. Orrit, “Photon statistics in the fluorescence of single molecules and nanocrystals: Correlation functions versus distributions of on- and off-times,” J. Chem. Phys. 119(4), 2214–2222 (2003).
[Crossref]

2002 (1)

T. Weidemann, M. Wachsmuth, M. Tewes, K. Rippe, and J. Langowski, “Analysis of ligand binding by two-colour fluorescence cross-correlation spectroscopy,” Single Mol. 3(1), 49–61 (2002).
[Crossref]

2000 (1)

M. Wachsmuth, W. Waldeck, and J. Langowski, “Anomalous diffusion of fluorescent probes inside living cell nuclei investigated by spatially-resolved fluorescence correlation spectroscopy,” J. Mol. Biol. 298(4), 677–689 (2000).
[Crossref] [PubMed]

1997 (1)

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

1995 (1)

J. Widengren, Ü. Mets, and R. Rigler, “Fluorescence Correlation Spectroscopy of Triplet States in Solution: A Theoretical and Experimental Study,” J. Phys. Chem. 99(36), 13368–13379 (1995).
[Crossref]

1990 (1)

K. Schätzel, “Noise on photon correlation data. I. Autocorrelation functions,” Quantum Opt. 2(4), 287–305 (1990).
[Crossref]

1989 (1)

J. Rika and T. Binkert, “Direct measurement of a distinct correlation function by fluorescence cross correlation,” Phys. Rev. A 39(5), 2646–2652 (1989).
[Crossref] [PubMed]

1974 (3)

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

M. Ehrenberg and R. Rigler, “Rotational Brownian Motion and fluorescence intensity fluctuation,” Chem. Phys. 4(3), 390–401 (1974).
[Crossref]

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

1972 (1)

D. Magde, E. L. Elson, and W. W. Webb, “Thermodynamic fluctuations in a reacting system - measurement by fluorescence correlations spectroscopy,” Phys. Rev. Lett. 29(11), 705–708 (1972).
[Crossref]

Arevalo, R.

D. R. Sisan, R. Arevalo, C. Graves, R. McAllister, and J. S. Urbach, “Spatially resolved fluorescence correlation spectroscopy using a spinning disk confocal microscope,” Biophys. J. 91(11), 4241–4252 (2006).
[Crossref] [PubMed]

Bacia, K.

K. Bacia and P. Schwille, “A dynamic view of cellular processes by in vivo fluorescence auto- and cross-correlation spectroscopy,” Methods 29(1), 74–85 (2003).
[Crossref] [PubMed]

Bentolila, L. A.

X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir, and S. Weiss, “Quantum dots for live cells, in vivo imaging, and diagnostics,” Science 307(5709), 538–544 (2005).
[Crossref] [PubMed]

Binkert, T.

J. Rika and T. Binkert, “Direct measurement of a distinct correlation function by fluorescence cross correlation,” Phys. Rev. A 39(5), 2646–2652 (1989).
[Crossref] [PubMed]

Boening, D.

Buehler, C.

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

Burkhardt, M.

Ding, J. L.

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

Doose, S.

X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir, and S. Weiss, “Quantum dots for live cells, in vivo imaging, and diagnostics,” Science 307(5709), 538–544 (2005).
[Crossref] [PubMed]

S. Doose, J. M. Tsay, F. Pinaud, and S. Weiss, “Comparison of photophysical and colloidal properties of biocompatible semiconductor nanocrystals using fluorescence correlation spectroscopy,” Anal. Chem. 77(7), 2235–2242 (2005).
[Crossref] [PubMed]

A. N. Kapanidis, N. K. Lee, T. A. Laurence, S. Doose, E. Margeat, and S. Weiss, “Fluorescence-aided molecule sorting: analysis of structure and interactions by alternating-laser excitation of single molecules,” Proc. Natl. Acad. Sci. U.S.A. 101(24), 8936–8941 (2004).
[Crossref] [PubMed]

Ehrenberg, M.

M. Ehrenberg and R. Rigler, “Rotational Brownian Motion and fluorescence intensity fluctuation,” Chem. Phys. 4(3), 390–401 (1974).
[Crossref]

Elson, E. L.

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

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

D. Magde, E. L. Elson, and W. W. Webb, “Thermodynamic fluctuations in a reacting system - measurement by fluorescence correlations spectroscopy,” Phys. Rev. Lett. 29(11), 705–708 (1972).
[Crossref]

Erdel, F.

G. Heuvelman, F. Erdel, M. Wachsmuth, and K. Rippe, “Analysis of protein mobilities and interactions in living cells by multifocal fluorescence fluctuation microscopy,” Eur. Biophys. J. 38(6), 813–828 (2009).
[Crossref] [PubMed]

Frantsuzov, P.

P. Frantsuzov, M. Kuno, B. Janko, and R. A. Marcus, “Universal emission intermittency in quantum dots, nanorods and nanowires,” Nat. Phys. 4(5), 519–522 (2008).
[Crossref]

Gambhir, S. S.

X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir, and S. Weiss, “Quantum dots for live cells, in vivo imaging, and diagnostics,” Science 307(5709), 538–544 (2005).
[Crossref] [PubMed]

Gösch, M.

L. C. Hwang, M. Leutenegger, M. Gösch, T. Lasser, P. Rigler, W. Meier, and T. Wohland, “Prism-based multicolor fluorescence correlation spectrometer,” Opt. Lett. 31(9), 1310–1312 (2006).
[Crossref] [PubMed]

M. Gösch and R. Rigler, “Fluorescence correlation spectroscopy of molecular motions and kinetics,” Adv. Drug Deliv. Rev. 57(1), 169–190 (2005).
[Crossref]

Gratton, E.

J. R. Unruh and 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]

Graves, C.

D. R. Sisan, R. Arevalo, C. Graves, R. McAllister, and J. S. Urbach, “Spatially resolved fluorescence correlation spectroscopy using a spinning disk confocal microscope,” Biophys. J. 91(11), 4241–4252 (2006).
[Crossref] [PubMed]

Groemer, T. W.

Har, J. Y.

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

Heinze, K. G.

M. Burkhardt, K. G. Heinze, and 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, M. Jahnz, and P. Schwille, “Triple-color coincidence analysis: one step further in following higher order molecular complex formation,” Biophys. J. 86(1), 506–516 (2004).
[Crossref]

Heuvelman, G.

G. Heuvelman, F. Erdel, M. Wachsmuth, and K. Rippe, “Analysis of protein mobilities and interactions in living cells by multifocal fluorescence fluctuation microscopy,” Eur. Biophys. J. 38(6), 813–828 (2009).
[Crossref] [PubMed]

Hildt, E.

S. Rüttinger, R. Macdonald, B. Krämer, F. Koberling, M. Roos, and E. Hildt, “Accurate single-pair Förster resonant energy transfer through combination of pulsed interleaved excitation, time correlated single-photon counting, and fluorescence correlation spectroscopy,” J. Biomed. Opt. 11(2), 024012–024012 (2006).
[Crossref] [PubMed]

Hwang, L. C.

Jahnz, M.

K. G. Heinze, M. Jahnz, and P. Schwille, “Triple-color coincidence analysis: one step further in following higher order molecular complex formation,” Biophys. J. 86(1), 506–516 (2004).
[Crossref]

Janko, B.

P. Frantsuzov, M. Kuno, B. Janko, and R. A. Marcus, “Universal emission intermittency in quantum dots, nanorods and nanowires,” Nat. Phys. 4(5), 519–522 (2008).
[Crossref]

Kannan, B.

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

Kapanidis, A. N.

A. N. Kapanidis, N. K. Lee, T. A. Laurence, S. Doose, E. Margeat, and S. Weiss, “Fluorescence-aided molecule sorting: analysis of structure and interactions by alternating-laser excitation of single molecules,” Proc. Natl. Acad. Sci. U.S.A. 101(24), 8936–8941 (2004).
[Crossref] [PubMed]

Kim, K. H.

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

Kinjo, M.

K. Saito, I. Wada, M. Tamura, and M. Kinjo, “Direct detection of caspase-3 activation in single live cells by cross-correlation analysis,” Biochem. Biophys. Res. Commun. 324(2), 849–854 (2004).
[Crossref] [PubMed]

Klingauf, J.

Koberling, F.

S. Rüttinger, R. Macdonald, B. Krämer, F. Koberling, M. Roos, and E. Hildt, “Accurate single-pair Förster resonant energy transfer through combination of pulsed interleaved excitation, time correlated single-photon counting, and fluorescence correlation spectroscopy,” J. Biomed. Opt. 11(2), 024012–024012 (2006).
[Crossref] [PubMed]

Krämer, B.

S. Rüttinger, R. Macdonald, B. Krämer, F. Koberling, M. Roos, and E. Hildt, “Accurate single-pair Förster resonant energy transfer through combination of pulsed interleaved excitation, time correlated single-photon counting, and fluorescence correlation spectroscopy,” J. Biomed. Opt. 11(2), 024012–024012 (2006).
[Crossref] [PubMed]

Kuno, M.

P. Frantsuzov, M. Kuno, B. Janko, and R. A. Marcus, “Universal emission intermittency in quantum dots, nanorods and nanowires,” Nat. Phys. 4(5), 519–522 (2008).
[Crossref]

Langowski, J.

J. Langowski, “Protein-protein interactions determined by fluorescence correlation spectroscopy,” Methods Cell Biol. 85, 471–484 (2008).
[Crossref]

T. Weidemann, M. Wachsmuth, M. Tewes, K. Rippe, and J. Langowski, “Analysis of ligand binding by two-colour fluorescence cross-correlation spectroscopy,” Single Mol. 3(1), 49–61 (2002).
[Crossref]

M. Wachsmuth, W. Waldeck, and J. Langowski, “Anomalous diffusion of fluorescent probes inside living cell nuclei investigated by spatially-resolved fluorescence correlation spectroscopy,” J. Mol. Biol. 298(4), 677–689 (2000).
[Crossref] [PubMed]

Larson, D. R.

J. Yao, D. R. Larson, H. D. Vishwasrao, W. R. Zipfel, and W. W. Webb, “Blinking and nonradiant dark fraction of water-soluble quantum dots in aqueous solution,” Proc. Natl. Acad. Sci. U.S.A. 102(40), 14284–14289 (2005).
[Crossref] [PubMed]

Lasser, T.

Laurence, T. A.

A. N. Kapanidis, N. K. Lee, T. A. Laurence, S. Doose, E. Margeat, and S. Weiss, “Fluorescence-aided molecule sorting: analysis of structure and interactions by alternating-laser excitation of single molecules,” Proc. Natl. Acad. Sci. U.S.A. 101(24), 8936–8941 (2004).
[Crossref] [PubMed]

Lee, N. K.

A. N. Kapanidis, N. K. Lee, T. A. Laurence, S. Doose, E. Margeat, and S. Weiss, “Fluorescence-aided molecule sorting: analysis of structure and interactions by alternating-laser excitation of single molecules,” Proc. Natl. Acad. Sci. U.S.A. 101(24), 8936–8941 (2004).
[Crossref] [PubMed]

Leutenegger, M.

Li, J. J.

X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir, and S. Weiss, “Quantum dots for live cells, in vivo imaging, and diagnostics,” Science 307(5709), 538–544 (2005).
[Crossref] [PubMed]

Liu, P.

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

Macdonald, R.

S. Rüttinger, R. Macdonald, B. Krämer, F. Koberling, M. Roos, and E. Hildt, “Accurate single-pair Förster resonant energy transfer through combination of pulsed interleaved excitation, time correlated single-photon counting, and fluorescence correlation spectroscopy,” J. Biomed. Opt. 11(2), 024012–024012 (2006).
[Crossref] [PubMed]

Magde, D.

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

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

D. Magde, E. L. Elson, and W. W. Webb, “Thermodynamic fluctuations in a reacting system - measurement by fluorescence correlations spectroscopy,” Phys. Rev. Lett. 29(11), 705–708 (1972).
[Crossref]

Marcus, R. A.

P. Frantsuzov, M. Kuno, B. Janko, and R. A. Marcus, “Universal emission intermittency in quantum dots, nanorods and nanowires,” Nat. Phys. 4(5), 519–522 (2008).
[Crossref]

Margeat, E.

A. N. Kapanidis, N. K. Lee, T. A. Laurence, S. Doose, E. Margeat, and S. Weiss, “Fluorescence-aided molecule sorting: analysis of structure and interactions by alternating-laser excitation of single molecules,” Proc. Natl. Acad. Sci. U.S.A. 101(24), 8936–8941 (2004).
[Crossref] [PubMed]

Maruyama, I.

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

McAllister, R.

D. R. Sisan, R. Arevalo, C. Graves, R. McAllister, and J. S. Urbach, “Spatially resolved fluorescence correlation spectroscopy using a spinning disk confocal microscope,” Biophys. J. 91(11), 4241–4252 (2006).
[Crossref] [PubMed]

Meier, W.

Mets, Ü.

J. Widengren, Ü. Mets, and R. Rigler, “Fluorescence Correlation Spectroscopy of Triplet States in Solution: A Theoretical and Experimental Study,” J. Phys. Chem. 99(36), 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(4), 1878–1886 (1997).
[Crossref] [PubMed]

Michalet, X.

X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir, and S. Weiss, “Quantum dots for live cells, in vivo imaging, and diagnostics,” Science 307(5709), 538–544 (2005).
[Crossref] [PubMed]

Mitchison, T. J.

D. J. Needleman, Y. Xu, and T. J. Mitchison, “Pin-hole array correlation imaging: highly parallel fluorescence correlation spectroscopy,” Biophys. J. 96(12), 5050–5059 (2009).
[Crossref] [PubMed]

Needleman, D. J.

D. J. Needleman, Y. Xu, and T. J. Mitchison, “Pin-hole array correlation imaging: highly parallel fluorescence correlation spectroscopy,” Biophys. J. 96(12), 5050–5059 (2009).
[Crossref] [PubMed]

Orrit, M.

R. Verberk and M. Orrit, “Photon statistics in the fluorescence of single molecules and nanocrystals: Correlation functions versus distributions of on- and off-times,” J. Chem. Phys. 119(4), 2214–2222 (2003).
[Crossref]

Pelet, S.

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

Pinaud, F.

S. Doose, J. M. Tsay, F. Pinaud, and S. Weiss, “Comparison of photophysical and colloidal properties of biocompatible semiconductor nanocrystals using fluorescence correlation spectroscopy,” Anal. Chem. 77(7), 2235–2242 (2005).
[Crossref] [PubMed]

Pinaud, F. F.

X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir, and S. Weiss, “Quantum dots for live cells, in vivo imaging, and diagnostics,” Science 307(5709), 538–544 (2005).
[Crossref] [PubMed]

Previte, M. J. R.

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

Rigler, P.

Rigler, R.

M. Gösch and R. Rigler, “Fluorescence correlation spectroscopy of molecular motions and kinetics,” Adv. Drug Deliv. Rev. 57(1), 169–190 (2005).
[Crossref]

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

J. Widengren, Ü. Mets, and R. Rigler, “Fluorescence Correlation Spectroscopy of Triplet States in Solution: A Theoretical and Experimental Study,” J. Phys. Chem. 99(36), 13368–13379 (1995).
[Crossref]

M. Ehrenberg and R. Rigler, “Rotational Brownian Motion and fluorescence intensity fluctuation,” Chem. Phys. 4(3), 390–401 (1974).
[Crossref]

Rika, J.

J. Rika and T. Binkert, “Direct measurement of a distinct correlation function by fluorescence cross correlation,” Phys. Rev. A 39(5), 2646–2652 (1989).
[Crossref] [PubMed]

Rippe, K.

G. Heuvelman, F. Erdel, M. Wachsmuth, and K. Rippe, “Analysis of protein mobilities and interactions in living cells by multifocal fluorescence fluctuation microscopy,” Eur. Biophys. J. 38(6), 813–828 (2009).
[Crossref] [PubMed]

T. Weidemann, M. Wachsmuth, M. Tewes, K. Rippe, and J. Langowski, “Analysis of ligand binding by two-colour fluorescence cross-correlation spectroscopy,” Single Mol. 3(1), 49–61 (2002).
[Crossref]

Roos, M.

S. Rüttinger, R. Macdonald, B. Krämer, F. Koberling, M. Roos, and E. Hildt, “Accurate single-pair Förster resonant energy transfer through combination of pulsed interleaved excitation, time correlated single-photon counting, and fluorescence correlation spectroscopy,” J. Biomed. Opt. 11(2), 024012–024012 (2006).
[Crossref] [PubMed]

Rüttinger, S.

S. Rüttinger, R. Macdonald, B. Krämer, F. Koberling, M. Roos, and E. Hildt, “Accurate single-pair Förster resonant energy transfer through combination of pulsed interleaved excitation, time correlated single-photon counting, and fluorescence correlation spectroscopy,” J. Biomed. Opt. 11(2), 024012–024012 (2006).
[Crossref] [PubMed]

Saito, K.

K. Saito, I. Wada, M. Tamura, and M. Kinjo, “Direct detection of caspase-3 activation in single live cells by cross-correlation analysis,” Biochem. Biophys. Res. Commun. 324(2), 849–854 (2004).
[Crossref] [PubMed]

Sankaran, J.

Schätzel, K.

K. Schätzel, “Noise on photon correlation data. I. Autocorrelation functions,” Quantum Opt. 2(4), 287–305 (1990).
[Crossref]

Schwille, P.

M. Burkhardt and 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, and 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, M. Jahnz, and P. Schwille, “Triple-color coincidence analysis: one step further in following higher order molecular complex formation,” Biophys. J. 86(1), 506–516 (2004).
[Crossref]

K. Bacia and P. Schwille, “A dynamic view of cellular processes by in vivo fluorescence auto- and cross-correlation spectroscopy,” Methods 29(1), 74–85 (2003).
[Crossref] [PubMed]

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

Shi, X.

Sisan, D. R.

D. R. Sisan, R. Arevalo, C. Graves, R. McAllister, and J. S. Urbach, “Spatially resolved fluorescence correlation spectroscopy using a spinning disk confocal microscope,” Biophys. J. 91(11), 4241–4252 (2006).
[Crossref] [PubMed]

So, P. T. C.

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

Stelzer, E. H. K.

Sundaresan, G.

X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir, and S. Weiss, “Quantum dots for live cells, in vivo imaging, and diagnostics,” Science 307(5709), 538–544 (2005).
[Crossref] [PubMed]

Tamura, M.

K. Saito, I. Wada, M. Tamura, and M. Kinjo, “Direct detection of caspase-3 activation in single live cells by cross-correlation analysis,” Biochem. Biophys. Res. Commun. 324(2), 849–854 (2004).
[Crossref] [PubMed]

Tewes, M.

T. Weidemann, M. Wachsmuth, M. Tewes, K. Rippe, and J. Langowski, “Analysis of ligand binding by two-colour fluorescence cross-correlation spectroscopy,” Single Mol. 3(1), 49–61 (2002).
[Crossref]

Tsay, J. M.

S. Doose, J. M. Tsay, F. Pinaud, and S. Weiss, “Comparison of photophysical and colloidal properties of biocompatible semiconductor nanocrystals using fluorescence correlation spectroscopy,” Anal. Chem. 77(7), 2235–2242 (2005).
[Crossref] [PubMed]

X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir, and S. Weiss, “Quantum dots for live cells, in vivo imaging, and diagnostics,” Science 307(5709), 538–544 (2005).
[Crossref] [PubMed]

Unruh, J. R.

J. R. Unruh and 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]

Urbach, J. S.

D. R. Sisan, R. Arevalo, C. Graves, R. McAllister, and J. S. Urbach, “Spatially resolved fluorescence correlation spectroscopy using a spinning disk confocal microscope,” Biophys. J. 91(11), 4241–4252 (2006).
[Crossref] [PubMed]

Verberk, R.

R. Verberk and M. Orrit, “Photon statistics in the fluorescence of single molecules and nanocrystals: Correlation functions versus distributions of on- and off-times,” J. Chem. Phys. 119(4), 2214–2222 (2003).
[Crossref]

Vishwasrao, H. D.

J. Yao, D. R. Larson, H. D. Vishwasrao, W. R. Zipfel, and W. W. Webb, “Blinking and nonradiant dark fraction of water-soluble quantum dots in aqueous solution,” Proc. Natl. Acad. Sci. U.S.A. 102(40), 14284–14289 (2005).
[Crossref] [PubMed]

Wachsmuth, M.

G. Heuvelman, F. Erdel, M. Wachsmuth, and K. Rippe, “Analysis of protein mobilities and interactions in living cells by multifocal fluorescence fluctuation microscopy,” Eur. Biophys. J. 38(6), 813–828 (2009).
[Crossref] [PubMed]

T. Weidemann, M. Wachsmuth, M. Tewes, K. Rippe, and J. Langowski, “Analysis of ligand binding by two-colour fluorescence cross-correlation spectroscopy,” Single Mol. 3(1), 49–61 (2002).
[Crossref]

M. Wachsmuth, W. Waldeck, and J. Langowski, “Anomalous diffusion of fluorescent probes inside living cell nuclei investigated by spatially-resolved fluorescence correlation spectroscopy,” J. Mol. Biol. 298(4), 677–689 (2000).
[Crossref] [PubMed]

Wada, I.

K. Saito, I. Wada, M. Tamura, and M. Kinjo, “Direct detection of caspase-3 activation in single live cells by cross-correlation analysis,” Biochem. Biophys. Res. Commun. 324(2), 849–854 (2004).
[Crossref] [PubMed]

Waldeck, W.

M. Wachsmuth, W. Waldeck, and J. Langowski, “Anomalous diffusion of fluorescent probes inside living cell nuclei investigated by spatially-resolved fluorescence correlation spectroscopy,” J. Mol. Biol. 298(4), 677–689 (2000).
[Crossref] [PubMed]

Webb, W. W.

J. Yao, D. R. Larson, H. D. Vishwasrao, W. R. Zipfel, and W. W. Webb, “Blinking and nonradiant dark fraction of water-soluble quantum dots in aqueous solution,” Proc. Natl. Acad. Sci. U.S.A. 102(40), 14284–14289 (2005).
[Crossref] [PubMed]

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

D. Magde, E. L. Elson, and W. W. Webb, “Thermodynamic fluctuations in a reacting system - measurement by fluorescence correlations spectroscopy,” Phys. Rev. Lett. 29(11), 705–708 (1972).
[Crossref]

Weidemann, T.

T. Weidemann, M. Wachsmuth, M. Tewes, K. Rippe, and J. Langowski, “Analysis of ligand binding by two-colour fluorescence cross-correlation spectroscopy,” Single Mol. 3(1), 49–61 (2002).
[Crossref]

Weiss, S.

S. Doose, J. M. Tsay, F. Pinaud, and S. Weiss, “Comparison of photophysical and colloidal properties of biocompatible semiconductor nanocrystals using fluorescence correlation spectroscopy,” Anal. Chem. 77(7), 2235–2242 (2005).
[Crossref] [PubMed]

X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir, and S. Weiss, “Quantum dots for live cells, in vivo imaging, and diagnostics,” Science 307(5709), 538–544 (2005).
[Crossref] [PubMed]

A. N. Kapanidis, N. K. Lee, T. A. Laurence, S. Doose, E. Margeat, and S. Weiss, “Fluorescence-aided molecule sorting: analysis of structure and interactions by alternating-laser excitation of single molecules,” Proc. Natl. Acad. Sci. U.S.A. 101(24), 8936–8941 (2004).
[Crossref] [PubMed]

Widengren, J.

J. Widengren, Ü. Mets, and R. Rigler, “Fluorescence Correlation Spectroscopy of Triplet States in Solution: A Theoretical and Experimental Study,” J. Phys. Chem. 99(36), 13368–13379 (1995).
[Crossref]

Wohland, T.

Wu, A. M.

X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir, and S. Weiss, “Quantum dots for live cells, in vivo imaging, and diagnostics,” Science 307(5709), 538–544 (2005).
[Crossref] [PubMed]

Xu, Y.

D. J. Needleman, Y. Xu, and T. J. Mitchison, “Pin-hole array correlation imaging: highly parallel fluorescence correlation spectroscopy,” Biophys. J. 96(12), 5050–5059 (2009).
[Crossref] [PubMed]

Yao, J.

J. Yao, D. R. Larson, H. D. Vishwasrao, W. R. Zipfel, and W. W. Webb, “Blinking and nonradiant dark fraction of water-soluble quantum dots in aqueous solution,” Proc. Natl. Acad. Sci. U.S.A. 102(40), 14284–14289 (2005).
[Crossref] [PubMed]

Zipfel, W. R.

J. Yao, D. R. Larson, H. D. Vishwasrao, W. R. Zipfel, and W. W. Webb, “Blinking and nonradiant dark fraction of water-soluble quantum dots in aqueous solution,” Proc. Natl. Acad. Sci. U.S.A. 102(40), 14284–14289 (2005).
[Crossref] [PubMed]

Adv. Drug Deliv. Rev. (1)

M. Gösch and R. Rigler, “Fluorescence correlation spectroscopy of molecular motions and kinetics,” Adv. Drug Deliv. Rev. 57(1), 169–190 (2005).
[Crossref]

Anal. Chem. (3)

M. J. R. Previte, S. Pelet, K. H. Kim, C. Buehler, and P. T. C. 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, and T. Wohland, “Electron multiplying charge-coupled device camera based fluorescence correlation spectroscopy,” Anal. Chem. 78(10), 3444–3451 (2006).
[Crossref] [PubMed]

S. Doose, J. M. Tsay, F. Pinaud, and S. Weiss, “Comparison of photophysical and colloidal properties of biocompatible semiconductor nanocrystals using fluorescence correlation spectroscopy,” Anal. Chem. 77(7), 2235–2242 (2005).
[Crossref] [PubMed]

Biochem. Biophys. Res. Commun. (1)

K. Saito, I. Wada, M. Tamura, and M. Kinjo, “Direct detection of caspase-3 activation in single live cells by cross-correlation analysis,” Biochem. Biophys. Res. Commun. 324(2), 849–854 (2004).
[Crossref] [PubMed]

Biophys. J. (5)

J. R. Unruh and 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]

D. J. Needleman, Y. Xu, and T. J. Mitchison, “Pin-hole array correlation imaging: highly parallel fluorescence correlation spectroscopy,” Biophys. J. 96(12), 5050–5059 (2009).
[Crossref] [PubMed]

D. R. Sisan, R. Arevalo, C. Graves, R. McAllister, and J. S. Urbach, “Spatially resolved fluorescence correlation spectroscopy using a spinning disk confocal microscope,” Biophys. J. 91(11), 4241–4252 (2006).
[Crossref] [PubMed]

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

K. G. Heinze, M. Jahnz, and P. Schwille, “Triple-color coincidence analysis: one step further in following higher order molecular complex formation,” Biophys. J. 86(1), 506–516 (2004).
[Crossref]

Biopolymers (2)

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

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

Chem. Phys. (1)

M. Ehrenberg and R. Rigler, “Rotational Brownian Motion and fluorescence intensity fluctuation,” Chem. Phys. 4(3), 390–401 (1974).
[Crossref]

Eur. Biophys. J. (1)

G. Heuvelman, F. Erdel, M. Wachsmuth, and K. Rippe, “Analysis of protein mobilities and interactions in living cells by multifocal fluorescence fluctuation microscopy,” Eur. Biophys. J. 38(6), 813–828 (2009).
[Crossref] [PubMed]

J. Biomed. Opt. (1)

S. Rüttinger, R. Macdonald, B. Krämer, F. Koberling, M. Roos, and E. Hildt, “Accurate single-pair Förster resonant energy transfer through combination of pulsed interleaved excitation, time correlated single-photon counting, and fluorescence correlation spectroscopy,” J. Biomed. Opt. 11(2), 024012–024012 (2006).
[Crossref] [PubMed]

J. Chem. Phys. (1)

R. Verberk and M. Orrit, “Photon statistics in the fluorescence of single molecules and nanocrystals: Correlation functions versus distributions of on- and off-times,” J. Chem. Phys. 119(4), 2214–2222 (2003).
[Crossref]

J. Mol. Biol. (1)

M. Wachsmuth, W. Waldeck, and J. Langowski, “Anomalous diffusion of fluorescent probes inside living cell nuclei investigated by spatially-resolved fluorescence correlation spectroscopy,” J. Mol. Biol. 298(4), 677–689 (2000).
[Crossref] [PubMed]

J. Phys. Chem. (1)

J. Widengren, Ü. Mets, and R. Rigler, “Fluorescence Correlation Spectroscopy of Triplet States in Solution: A Theoretical and Experimental Study,” J. Phys. Chem. 99(36), 13368–13379 (1995).
[Crossref]

Methods (1)

K. Bacia and P. Schwille, “A dynamic view of cellular processes by in vivo fluorescence auto- and cross-correlation spectroscopy,” Methods 29(1), 74–85 (2003).
[Crossref] [PubMed]

Methods Cell Biol. (1)

J. Langowski, “Protein-protein interactions determined by fluorescence correlation spectroscopy,” Methods Cell Biol. 85, 471–484 (2008).
[Crossref]

Nat. Phys. (1)

P. Frantsuzov, M. Kuno, B. Janko, and R. A. Marcus, “Universal emission intermittency in quantum dots, nanorods and nanowires,” Nat. Phys. 4(5), 519–522 (2008).
[Crossref]

Opt. Express (3)

Opt. Lett. (2)

Phys. Rev. A (1)

J. Rika and T. Binkert, “Direct measurement of a distinct correlation function by fluorescence cross correlation,” Phys. Rev. A 39(5), 2646–2652 (1989).
[Crossref] [PubMed]

Phys. Rev. Lett. (1)

D. Magde, E. L. Elson, and W. W. Webb, “Thermodynamic fluctuations in a reacting system - measurement by fluorescence correlations spectroscopy,” Phys. Rev. Lett. 29(11), 705–708 (1972).
[Crossref]

Proc. Natl. Acad. Sci. U.S.A. (2)

J. Yao, D. R. Larson, H. D. Vishwasrao, W. R. Zipfel, and W. W. Webb, “Blinking and nonradiant dark fraction of water-soluble quantum dots in aqueous solution,” Proc. Natl. Acad. Sci. U.S.A. 102(40), 14284–14289 (2005).
[Crossref] [PubMed]

A. N. Kapanidis, N. K. Lee, T. A. Laurence, S. Doose, E. Margeat, and S. Weiss, “Fluorescence-aided molecule sorting: analysis of structure and interactions by alternating-laser excitation of single molecules,” Proc. Natl. Acad. Sci. U.S.A. 101(24), 8936–8941 (2004).
[Crossref] [PubMed]

Quantum Opt. (1)

K. Schätzel, “Noise on photon correlation data. I. Autocorrelation functions,” Quantum Opt. 2(4), 287–305 (1990).
[Crossref]

Science (1)

X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir, and S. Weiss, “Quantum dots for live cells, in vivo imaging, and diagnostics,” Science 307(5709), 538–544 (2005).
[Crossref] [PubMed]

Single Mol. (1)

T. Weidemann, M. Wachsmuth, M. Tewes, K. Rippe, and J. Langowski, “Analysis of ligand binding by two-colour fluorescence cross-correlation spectroscopy,” Single Mol. 3(1), 49–61 (2002).
[Crossref]

Other (3)

B. Kannan, L. Guo, T. Sudhaharan, S. Ahmed, I. Maruyama, and T. Wohland, “Spatially Resolved Total Internal Reflection Fluorescence Correlation Microscopy Using an Electron Multiplying Charge-Coupled Device Camera,” Analytical Chemistry (2007).

F. Christen, K. Kuijken, D. Baade, C. Cavadore, S. Deiries, and O. Iwert, “Fast Conversion Factor (Gain) Measurement of a CCD Using Images With Vertical Gradient,” in Scientific detectors for astronomy 2005 (Springer Netherlands, 2006).

M. Wachsmuth, and K. Weisshart, “Fluorescence photobleaching and fluorescence correlation spectroscopy: two complementary technologies to study molecular dynamics in living cells,” in Imaging Cellular and Molecular Biological Functions (Springer Verlag, Heidelberg, 2007).

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Figures (6)

Fig. 1
Fig. 1 (A) Experimental setup based on a commercial CLSM. (B) Readout speed in lines/second or Hz as a function of the number of pixels used. The spectrometer is operated with 80 pixels at up to 84 kHz. (C) A section of a typical raw data trace where a wavelength range for binning is indicated.

Download Full Size | PPT Slide | PDF

Fig. 2
Fig. 2 Spectral calibration. (A) Laser lines reflected at a coverglass in front of the objective lens. The nonlinear dispersion of the prism results in a distribution of data points that is not linear with the wavelength. (B) Measured time integrated intensity spectra of the mixed and separate QD solutions (open symbols) and fits of Gaussian curves (solid lines).

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Fig. 3
Fig. 3 Comparison with APD measurements. ACFs of Alexa 488 (A) and of QD 655 (B) in solution measured with the APD-based (blue) and with the EMCCD-based setup (red) normalized to the values at 12 µs lag time. The initial decrease at lag times <10 µs of the APD-based ACF of QD 655 was probably due to additional triplet state occupation and excluded for the fit. The diffusion correlation times from the fits are in good agreement. (C) Number of molecules in the focus as obtained from the fit of FCS data of QD 655 plotted against the concentration of the solution (black circles) and linear fit (red line).

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Fig. 4
Fig. 4 FCS of a mixture of QDs. (A) ACFs of QD 525 (blue), 565 (green), 605 (yellow), and 655 (red) based on time trace extracted from a full spectral intensity recording by binning over appropriate detection windows. (B) The resulting diffusion correlation times for the respective QDs as a function of the maximum emission wavelength (circles) and the spectral dependence as theoretically expected from size and chromatic effects (arrows).

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Fig. 5
Fig. 5 Spectrally resolved diffusion and fluorescence intermittence of QD 655. Spectral dependence of (A) the diffusion correlation time and (B) the number of molecules in the focal volume (circles) from fits with globally linked blinking parameters. The estimated chromatic dependence (solid lines) is based on the interpolated values at 655 nm. Spectral dependence of (C) the blinking correlation time and (D) the fraction in a non-fluorescent state from fits applying the estimated chromatic dependence from (A) and (B). Error bars are obtained from averaging over 4 experiments. (E) The measured ACFs (black) and the fits (red) resulting in (C) and (D) and the corresponding residuals for the data points 625, 633, 641, 649, 667 and 686 nm (top to bottom). The residuals were shifted by 0.002 each for better legibility. The qualitiy of the fits was similar for both approaches.

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Fig. 6
Fig. 6 Spectrally resolved FCCS in living cells. (A) Confocal images of a positive control MCF7 cell showing the EGFP (green) and the mRFP (red) fluorescence and an overlay image (yellow), scale bar 20µm. (B) ACFs of the EGFP (green) and the mRFP fluorescence signal (red) as well as their CCF acquired in positive (pos.) and negative control (neg.) MCF7 cells with the APD-based filter setup. (C) The same as in (B), but acquired with the EMCCD-based spectrometer setup. (D) ratioG values determined for 3×3 spectral windows from data acquired in positive (pos.) and negative (neg.) control cells and their difference (pos.-neg.).

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Equations (5)

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G k l ( τ ) = 〈 δ F k ( t ) δ F l ( t + τ ) 〉 〈 F k ( t ) 〉 〈 F l ( t ) 〉 ,   δ F k ( t ) = F k ( t ) − 〈 F k ( t ) 〉 .
G ( τ ) = 1 N ( f 1 [ 1 + ( τ τ diff , 1 ) α 1 ] − 1 [ 1 + 1 κ 2 ( τ τ diff , 1 ) α 1 ] − 1 / 2 + ( 1 − f 1 ) [ 1 + ( τ τ diff , 2 ) α 2 ] − 1 [ 1 + 1 κ 2 ( τ τ diff , 2 ) α 2 ] − 1 / 2 ) [ 1 + θ exp ( − τ τ blink ) ]
r a t i o G = G 12 ( 0 ) × [ G 11 ( 0 ) G 22 ( 0 ) ] − 1 / 2 .
σ ADU 2 = f conv − 1 μ ADU + f conv − 2 σ RO 2 .
w 0 2 ∝ λ ill 2 + λ det 2 so that τ diff ∝( 1+ λ det⁡ 2 λ ill 2 ) R i , N∝ ( 1+ λ det⁡ 2 λ ill 2 ) 3/2

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