Abstract

Pulsed Interleaved Excitation (PIE) improves fluorescence cross-correlation spectroscopy (FCCS) and single pair Förster Resonance Energy Transfer (spFRET) measurements, by correlating each detected photon to the excitation source that generated it. It relies on the interleaving of two picosecond laser sources and time correlated single photon counting (TCSPC) detection. Here, we present an optical configuration based on a commercial supercontinuum laser, which generates multicoulour interleaved picosecond pulses with arbitrary spacing and wavelengths within the visible spectrum. This simple, yet robust configuration can be used as a versatile source for PIE experiments, as an alternative to an array of picosecond lasers and drivers.

©2013 Optical Society of America

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    [Crossref] [PubMed]
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    [Crossref] [PubMed]
  20. H. N. Paulsen, K. M. Hilligsøe, J. Thøgersen, S. R. Keiding, and J. J. Larsen, “Coherent anti-Stokes Raman scattering microscopy with a photonic crystal fiber based light source,” Opt. Lett. 28(13), 1123–1125 (2003).
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  21. P. Blandin, S. Lévêque-Fort, S. Lécart, J. C. Cossec, M.-C. Potier, Z. Lenkei, F. Druon, and P. Georges, “Time-gated total internal reflection fluorescence microscopy with a supercontinuum excitation source,” Appl. Opt. 48(3), 553–559 (2009).
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  22. P. J. Rothwell, S. Berger, O. Kensch, S. Felekyan, M. Antonik, B. M. Wöhrl, T. Restle, R. S. Goody, and C. A. Seidel, “Multiparameter single-molecule fluorescence spectroscopy reveals heterogeneity of HIV-1 reverse transcriptase:primer/template complexes,” Proc. Natl. Acad. Sci. U.S.A. 100(4), 1655–1660 (2003).
    [Crossref] [PubMed]
  23. O. Krichevsky and G. Bonnet, “Fluorescence correlation spectroscopy: the technique and its applications,” Rep. Prog. Phys. 65(2), 251–297 (2002).
    [Crossref]
  24. J. R. Unruh, G. Gokulrangan, G. S. Wilson, and C. K. Johnson, “Fluorescence properties of fluorescein, tetramethylrhodamine and Texas Red linked to a DNA aptamer,” Photochem. Photobiol. 81(3), 682–690 (2005).
    [Crossref] [PubMed]
  25. 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]
  26. K. Bacia, S. A. Kim, and P. Schwille, “Fluorescence cross-correlation spectroscopy in living cells,” Nat. Methods 3, 83–89 (2006).
  27. M. Zhao, L. Jin, B. Chen, Y. Ding, H. Ma, and D. Chen, “Afterpulsing and its correction in fluorescence correlation spectroscopy experiments,” Appl. Opt. 42(19), 4031–4036 (2003).
    [Crossref] [PubMed]
  28. E. Sisamakis, A. Valeri, S. Kalinin, P. J. Rothwell, and C. A. Seidel, “Accurate single-molecule FRET studies using multiparameter fluorescence detection,” Methods Enzymol. 475, 455–514 (2010).
    [Crossref] [PubMed]
  29. J. Enderlein and I. Gregor, “Using fluorescence lifetime for discriminating detector afterpulsing in fluorescence-correlation spectroscopy,” Rev. Sci. Instrum. 76(3), 033102 (2005).
    [Crossref]
  30. S. Felekyan, S. Kalinin, H. Sanabria, A. Valeri, and C. A. Seidel, “Filtered FCS: species auto- and cross-correlation functions highlight binding and dynamics in biomolecules,” ChemPhysChem 13(4), 1036–1053 (2012).
    [Crossref] [PubMed]
  31. C. M. Pieper and J. Enderlein, “Fluorescence correlation spectroscopy as a tool for measuring the rotational diffusion of macromolecules,” Chem. Phys. Lett. 516(1-3), 1–11 (2011).
    [Crossref]

2012 (2)

V. Kudryavtsev, M. Sikor, S. Kalinin, D. Mokranjac, C. A. M. Seidel, and D. C. Lamb, “Combining MFD and PIE for accurate single-pair Förster resonance energy transfer measurements,” Chem Phys. Chem. 13, 1060–1078 (2012).

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

2011 (1)

C. M. Pieper and J. Enderlein, “Fluorescence correlation spectroscopy as a tool for measuring the rotational diffusion of macromolecules,” Chem. Phys. Lett. 516(1-3), 1–11 (2011).
[Crossref]

2010 (2)

E. Sisamakis, A. Valeri, S. Kalinin, P. J. Rothwell, and C. A. Seidel, “Accurate single-molecule FRET studies using multiparameter fluorescence detection,” Methods Enzymol. 475, 455–514 (2010).
[Crossref] [PubMed]

R. Mercatelli, S. Soria, G. Molesini, F. Bianco, G. Righini, and F. Quercioli, “Supercontinuum source tuned by an on-axis monochromator for fluorescence lifetime imaging,” Opt. Express 18(19), 20505–20511 (2010).
[Crossref] [PubMed]

2009 (1)

2008 (2)

2006 (4)

R. Fenske, D. Näther, M. Goossens, and S. D. Smith, “New light sources for time-correlated single-photon counting in commercially available spectrometers,” Proc. SPIE 6372, 63720H (2006).
[Crossref]

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

E. Margeat, A. N. Kapanidis, P. Tinnefeld, Y. Wang, J. Mukhopadhyay, R. H. Ebright, and S. Weiss, “Direct observation of abortive initiation and promoter escape within single immobilized transcription complexes,” Biophys. J. 90(4), 1419–1431 (2006).
[Crossref] [PubMed]

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

2005 (5)

J. R. Unruh, G. Gokulrangan, G. S. Wilson, and C. K. Johnson, “Fluorescence properties of fluorescein, tetramethylrhodamine and Texas Red linked to a DNA aptamer,” Photochem. Photobiol. 81(3), 682–690 (2005).
[Crossref] [PubMed]

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

T. A. Laurence, X. Kong, M. Jäger, and S. Weiss, “Probing structural heterogeneities and fluctuations of nucleic acids and denatured proteins,” Proc. Natl. Acad. Sci. U.S.A. 102(48), 17348–17353 (2005).
[Crossref] [PubMed]

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

E. Thews, M. Gerken, R. Eckert, J. Zäpfel, C. Tietz, and J. Wrachtrup, “Cross talk free fluorescence cross correlation spectroscopy in live cells,” Biophys. J. 89(3), 2069–2076 (2005).
[Crossref] [PubMed]

2004 (1)

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]

2003 (4)

E. Haustein and P. Schwille, “Ultrasensitive investigations of biological systems by fluorescence correlation spectroscopy,” Methods 29(2), 153–166 (2003).
[Crossref] [PubMed]

M. Zhao, L. Jin, B. Chen, Y. Ding, H. Ma, and D. Chen, “Afterpulsing and its correction in fluorescence correlation spectroscopy experiments,” Appl. Opt. 42(19), 4031–4036 (2003).
[Crossref] [PubMed]

P. J. Rothwell, S. Berger, O. Kensch, S. Felekyan, M. Antonik, B. M. Wöhrl, T. Restle, R. S. Goody, and C. A. Seidel, “Multiparameter single-molecule fluorescence spectroscopy reveals heterogeneity of HIV-1 reverse transcriptase:primer/template complexes,” Proc. Natl. Acad. Sci. U.S.A. 100(4), 1655–1660 (2003).
[Crossref] [PubMed]

H. N. Paulsen, K. M. Hilligsøe, J. Thøgersen, S. R. Keiding, and J. J. Larsen, “Coherent anti-Stokes Raman scattering microscopy with a photonic crystal fiber based light source,” Opt. Lett. 28(13), 1123–1125 (2003).
[Crossref] [PubMed]

2002 (2)

O. Krichevsky and G. Bonnet, “Fluorescence correlation spectroscopy: the technique and its applications,” Rep. Prog. Phys. 65(2), 251–297 (2002).
[Crossref]

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

1999 (1)

S. Weiss, “Fluorescence spectroscopy of single biomolecules,” Science 283(5408), 1676–1683 (1999).
[Crossref] [PubMed]

1998 (1)

X. S. Xie and J. K. Trautman, “Optical studies of single molecules at room temperature,” Annu. Rev. Phys. Chem. 49(1), 441–480 (1998).
[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]

1996 (1)

T. Ha, T. Enderle, D. F. Ogletree, D. S. Chemla, P. R. Selvin, and S. Weiss, “Probing the interaction between two single molecules: fluorescence resonance energy transfer between a single donor and a single acceptor,” Proc. Natl. Acad. Sci. U.S.A. 93(13), 6264–6268 (1996).
[Crossref] [PubMed]

1974 (1)

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 correlation spectroscopy,” Phys. Rev. Lett. 29(11), 705–708 (1972).
[Crossref]

Antonik, M.

P. J. Rothwell, S. Berger, O. Kensch, S. Felekyan, M. Antonik, B. M. Wöhrl, T. Restle, R. S. Goody, and C. A. Seidel, “Multiparameter single-molecule fluorescence spectroscopy reveals heterogeneity of HIV-1 reverse transcriptase:primer/template complexes,” Proc. Natl. Acad. Sci. U.S.A. 100(4), 1655–1660 (2003).
[Crossref] [PubMed]

Auksorius, E.

Bacia, K.

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

Benda, A.

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

Berger, S.

P. J. Rothwell, S. Berger, O. Kensch, S. Felekyan, M. Antonik, B. M. Wöhrl, T. Restle, R. S. Goody, and C. A. Seidel, “Multiparameter single-molecule fluorescence spectroscopy reveals heterogeneity of HIV-1 reverse transcriptase:primer/template complexes,” Proc. Natl. Acad. Sci. U.S.A. 100(4), 1655–1660 (2003).
[Crossref] [PubMed]

Bianco, F.

Blandin, P.

Böhmer, M.

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

Bonnet, G.

O. Krichevsky and G. Bonnet, “Fluorescence correlation spectroscopy: the technique and its applications,” Rep. Prog. Phys. 65(2), 251–297 (2002).
[Crossref]

Boruah, B. R.

Bräuchle, C.

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

Chemla, D. S.

T. Ha, T. Enderle, D. F. Ogletree, D. S. Chemla, P. R. Selvin, and S. Weiss, “Probing the interaction between two single molecules: fluorescence resonance energy transfer between a single donor and a single acceptor,” Proc. Natl. Acad. Sci. U.S.A. 93(13), 6264–6268 (1996).
[Crossref] [PubMed]

Chen, B.

Chen, D.

Cossec, J. C.

Ding, Y.

Doose, S.

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]

Druon, F.

Dunsby, C.

Ebright, R. H.

E. Margeat, A. N. Kapanidis, P. Tinnefeld, Y. Wang, J. Mukhopadhyay, R. H. Ebright, and S. Weiss, “Direct observation of abortive initiation and promoter escape within single immobilized transcription complexes,” Biophys. J. 90(4), 1419–1431 (2006).
[Crossref] [PubMed]

Eckert, R.

E. Thews, M. Gerken, R. Eckert, J. Zäpfel, C. Tietz, and J. Wrachtrup, “Cross talk free fluorescence cross correlation spectroscopy in live cells,” Biophys. J. 89(3), 2069–2076 (2005).
[Crossref] [PubMed]

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, “Thermodynamic fluctuations in a reacting system : measurement by fluorescence correlation spectroscopy,” Phys. Rev. Lett. 29(11), 705–708 (1972).
[Crossref]

Enderle, T.

T. Ha, T. Enderle, D. F. Ogletree, D. S. Chemla, P. R. Selvin, and S. Weiss, “Probing the interaction between two single molecules: fluorescence resonance energy transfer between a single donor and a single acceptor,” Proc. Natl. Acad. Sci. U.S.A. 93(13), 6264–6268 (1996).
[Crossref] [PubMed]

Enderlein, J.

C. M. Pieper and J. Enderlein, “Fluorescence correlation spectroscopy as a tool for measuring the rotational diffusion of macromolecules,” Chem. Phys. Lett. 516(1-3), 1–11 (2011).
[Crossref]

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

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

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

Erdmann, R.

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

Felekyan, S.

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

P. J. Rothwell, S. Berger, O. Kensch, S. Felekyan, M. Antonik, B. M. Wöhrl, T. Restle, R. S. Goody, and C. A. Seidel, “Multiparameter single-molecule fluorescence spectroscopy reveals heterogeneity of HIV-1 reverse transcriptase:primer/template complexes,” Proc. Natl. Acad. Sci. U.S.A. 100(4), 1655–1660 (2003).
[Crossref] [PubMed]

Fenske, R.

R. Fenske, D. Näther, M. Goossens, and S. D. Smith, “New light sources for time-correlated single-photon counting in commercially available spectrometers,” Proc. SPIE 6372, 63720H (2006).
[Crossref]

French, P. M.

Georges, P.

Gerken, M.

E. Thews, M. Gerken, R. Eckert, J. Zäpfel, C. Tietz, and J. Wrachtrup, “Cross talk free fluorescence cross correlation spectroscopy in live cells,” Biophys. J. 89(3), 2069–2076 (2005).
[Crossref] [PubMed]

Gokulrangan, G.

J. R. Unruh, G. Gokulrangan, G. S. Wilson, and C. K. Johnson, “Fluorescence properties of fluorescein, tetramethylrhodamine and Texas Red linked to a DNA aptamer,” Photochem. Photobiol. 81(3), 682–690 (2005).
[Crossref] [PubMed]

Goody, R. S.

P. J. Rothwell, S. Berger, O. Kensch, S. Felekyan, M. Antonik, B. M. Wöhrl, T. Restle, R. S. Goody, and C. A. Seidel, “Multiparameter single-molecule fluorescence spectroscopy reveals heterogeneity of HIV-1 reverse transcriptase:primer/template complexes,” Proc. Natl. Acad. Sci. U.S.A. 100(4), 1655–1660 (2003).
[Crossref] [PubMed]

Goossens, M.

R. Fenske, D. Näther, M. Goossens, and S. D. Smith, “New light sources for time-correlated single-photon counting in commercially available spectrometers,” Proc. SPIE 6372, 63720H (2006).
[Crossref]

Gregor, I.

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

Ha, T.

T. Ha, T. Enderle, D. F. Ogletree, D. S. Chemla, P. R. Selvin, and S. Weiss, “Probing the interaction between two single molecules: fluorescence resonance energy transfer between a single donor and a single acceptor,” Proc. Natl. Acad. Sci. U.S.A. 93(13), 6264–6268 (1996).
[Crossref] [PubMed]

Haustein, E.

E. Haustein and P. Schwille, “Ultrasensitive investigations of biological systems by fluorescence correlation spectroscopy,” Methods 29(2), 153–166 (2003).
[Crossref] [PubMed]

Hell, S. W.

Hilligsøe, K. M.

Hof, M.

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

Jäger, M.

T. A. Laurence, X. Kong, M. Jäger, and S. Weiss, “Probing structural heterogeneities and fluctuations of nucleic acids and denatured proteins,” Proc. Natl. Acad. Sci. U.S.A. 102(48), 17348–17353 (2005).
[Crossref] [PubMed]

Jin, L.

Johnson, C. K.

J. R. Unruh, G. Gokulrangan, G. S. Wilson, and C. K. Johnson, “Fluorescence properties of fluorescein, tetramethylrhodamine and Texas Red linked to a DNA aptamer,” Photochem. Photobiol. 81(3), 682–690 (2005).
[Crossref] [PubMed]

Kalinin, S.

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

V. Kudryavtsev, M. Sikor, S. Kalinin, D. Mokranjac, C. A. M. Seidel, and D. C. Lamb, “Combining MFD and PIE for accurate single-pair Förster resonance energy transfer measurements,” Chem Phys. Chem. 13, 1060–1078 (2012).

E. Sisamakis, A. Valeri, S. Kalinin, P. J. Rothwell, and C. A. Seidel, “Accurate single-molecule FRET studies using multiparameter fluorescence detection,” Methods Enzymol. 475, 455–514 (2010).
[Crossref] [PubMed]

Kapanidis, A. N.

E. Margeat, A. N. Kapanidis, P. Tinnefeld, Y. Wang, J. Mukhopadhyay, R. H. Ebright, and S. Weiss, “Direct observation of abortive initiation and promoter escape within single immobilized transcription complexes,” Biophys. J. 90(4), 1419–1431 (2006).
[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]

Kapusta, P.

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

Kastrup, L.

Keiding, S. R.

Kennedy, G.

Kensch, O.

P. J. Rothwell, S. Berger, O. Kensch, S. Felekyan, M. Antonik, B. M. Wöhrl, T. Restle, R. S. Goody, and C. A. Seidel, “Multiparameter single-molecule fluorescence spectroscopy reveals heterogeneity of HIV-1 reverse transcriptase:primer/template complexes,” Proc. Natl. Acad. Sci. U.S.A. 100(4), 1655–1660 (2003).
[Crossref] [PubMed]

Kim, S. A.

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

Kong, X.

T. A. Laurence, X. Kong, M. Jäger, and S. Weiss, “Probing structural heterogeneities and fluctuations of nucleic acids and denatured proteins,” Proc. Natl. Acad. Sci. U.S.A. 102(48), 17348–17353 (2005).
[Crossref] [PubMed]

Krichevsky, O.

O. Krichevsky and G. Bonnet, “Fluorescence correlation spectroscopy: the technique and its applications,” Rep. Prog. Phys. 65(2), 251–297 (2002).
[Crossref]

Kudryavtsev, V.

V. Kudryavtsev, M. Sikor, S. Kalinin, D. Mokranjac, C. A. M. Seidel, and D. C. Lamb, “Combining MFD and PIE for accurate single-pair Förster resonance energy transfer measurements,” Chem Phys. Chem. 13, 1060–1078 (2012).

Lamb, D. C.

V. Kudryavtsev, M. Sikor, S. Kalinin, D. Mokranjac, C. A. M. Seidel, and D. C. Lamb, “Combining MFD and PIE for accurate single-pair Förster resonance energy transfer measurements,” Chem Phys. Chem. 13, 1060–1078 (2012).

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

Lanigan, P. M.

Larsen, J. J.

Laurence, T. A.

T. A. Laurence, X. Kong, M. Jäger, and S. Weiss, “Probing structural heterogeneities and fluctuations of nucleic acids and denatured proteins,” Proc. Natl. Acad. Sci. U.S.A. 102(48), 17348–17353 (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]

Lécart, S.

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]

Lenkei, Z.

Lévêque-Fort, S.

Ma, H.

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, “Thermodynamic fluctuations in a reacting system : measurement by fluorescence correlation spectroscopy,” Phys. Rev. Lett. 29(11), 705–708 (1972).
[Crossref]

Margeat, E.

E. Margeat, A. N. Kapanidis, P. Tinnefeld, Y. Wang, J. Mukhopadhyay, R. H. Ebright, and S. Weiss, “Direct observation of abortive initiation and promoter escape within single immobilized transcription complexes,” Biophys. J. 90(4), 1419–1431 (2006).
[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]

Mercatelli, R.

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]

Mokranjac, D.

V. Kudryavtsev, M. Sikor, S. Kalinin, D. Mokranjac, C. A. M. Seidel, and D. C. Lamb, “Combining MFD and PIE for accurate single-pair Förster resonance energy transfer measurements,” Chem Phys. Chem. 13, 1060–1078 (2012).

Molesini, G.

Mukhopadhyay, J.

E. Margeat, A. N. Kapanidis, P. Tinnefeld, Y. Wang, J. Mukhopadhyay, R. H. Ebright, and S. Weiss, “Direct observation of abortive initiation and promoter escape within single immobilized transcription complexes,” Biophys. J. 90(4), 1419–1431 (2006).
[Crossref] [PubMed]

Müller, B. K.

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

Näther, D.

R. Fenske, D. Näther, M. Goossens, and S. D. Smith, “New light sources for time-correlated single-photon counting in commercially available spectrometers,” Proc. SPIE 6372, 63720H (2006).
[Crossref]

Neil, M. A.

Ogletree, D. F.

T. Ha, T. Enderle, D. F. Ogletree, D. S. Chemla, P. R. Selvin, and S. Weiss, “Probing the interaction between two single molecules: fluorescence resonance energy transfer between a single donor and a single acceptor,” Proc. Natl. Acad. Sci. U.S.A. 93(13), 6264–6268 (1996).
[Crossref] [PubMed]

Paulsen, H. N.

Pieper, C. M.

C. M. Pieper and J. Enderlein, “Fluorescence correlation spectroscopy as a tool for measuring the rotational diffusion of macromolecules,” Chem. Phys. Lett. 516(1-3), 1–11 (2011).
[Crossref]

Potier, M.-C.

Quercioli, F.

Rahn, H.

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

Restle, T.

P. J. Rothwell, S. Berger, O. Kensch, S. Felekyan, M. Antonik, B. M. Wöhrl, T. Restle, R. S. Goody, and C. A. Seidel, “Multiparameter single-molecule fluorescence spectroscopy reveals heterogeneity of HIV-1 reverse transcriptase:primer/template complexes,” Proc. Natl. Acad. Sci. U.S.A. 100(4), 1655–1660 (2003).
[Crossref] [PubMed]

Righini, G.

Rigler, R.

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

Rittweger, E.

Rothwell, P. J.

E. Sisamakis, A. Valeri, S. Kalinin, P. J. Rothwell, and C. A. Seidel, “Accurate single-molecule FRET studies using multiparameter fluorescence detection,” Methods Enzymol. 475, 455–514 (2010).
[Crossref] [PubMed]

P. J. Rothwell, S. Berger, O. Kensch, S. Felekyan, M. Antonik, B. M. Wöhrl, T. Restle, R. S. Goody, and C. A. Seidel, “Multiparameter single-molecule fluorescence spectroscopy reveals heterogeneity of HIV-1 reverse transcriptase:primer/template complexes,” Proc. Natl. Acad. Sci. U.S.A. 100(4), 1655–1660 (2003).
[Crossref] [PubMed]

Sanabria, H.

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

Schwille, P.

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

E. Haustein and P. Schwille, “Ultrasensitive investigations of biological systems by fluorescence correlation spectroscopy,” Methods 29(2), 153–166 (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]

Seidel, C. A.

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

E. Sisamakis, A. Valeri, S. Kalinin, P. J. Rothwell, and C. A. Seidel, “Accurate single-molecule FRET studies using multiparameter fluorescence detection,” Methods Enzymol. 475, 455–514 (2010).
[Crossref] [PubMed]

P. J. Rothwell, S. Berger, O. Kensch, S. Felekyan, M. Antonik, B. M. Wöhrl, T. Restle, R. S. Goody, and C. A. Seidel, “Multiparameter single-molecule fluorescence spectroscopy reveals heterogeneity of HIV-1 reverse transcriptase:primer/template complexes,” Proc. Natl. Acad. Sci. U.S.A. 100(4), 1655–1660 (2003).
[Crossref] [PubMed]

Seidel, C. A. M.

V. Kudryavtsev, M. Sikor, S. Kalinin, D. Mokranjac, C. A. M. Seidel, and D. C. Lamb, “Combining MFD and PIE for accurate single-pair Förster resonance energy transfer measurements,” Chem Phys. Chem. 13, 1060–1078 (2012).

Selvin, P. R.

T. Ha, T. Enderle, D. F. Ogletree, D. S. Chemla, P. R. Selvin, and S. Weiss, “Probing the interaction between two single molecules: fluorescence resonance energy transfer between a single donor and a single acceptor,” Proc. Natl. Acad. Sci. U.S.A. 93(13), 6264–6268 (1996).
[Crossref] [PubMed]

Sikor, M.

V. Kudryavtsev, M. Sikor, S. Kalinin, D. Mokranjac, C. A. M. Seidel, and D. C. Lamb, “Combining MFD and PIE for accurate single-pair Förster resonance energy transfer measurements,” Chem Phys. Chem. 13, 1060–1078 (2012).

Sisamakis, E.

E. Sisamakis, A. Valeri, S. Kalinin, P. J. Rothwell, and C. A. Seidel, “Accurate single-molecule FRET studies using multiparameter fluorescence detection,” Methods Enzymol. 475, 455–514 (2010).
[Crossref] [PubMed]

Smith, S. D.

R. Fenske, D. Näther, M. Goossens, and S. D. Smith, “New light sources for time-correlated single-photon counting in commercially available spectrometers,” Proc. SPIE 6372, 63720H (2006).
[Crossref]

Soria, S.

Thews, E.

E. Thews, M. Gerken, R. Eckert, J. Zäpfel, C. Tietz, and J. Wrachtrup, “Cross talk free fluorescence cross correlation spectroscopy in live cells,” Biophys. J. 89(3), 2069–2076 (2005).
[Crossref] [PubMed]

Thøgersen, J.

Tietz, C.

E. Thews, M. Gerken, R. Eckert, J. Zäpfel, C. Tietz, and J. Wrachtrup, “Cross talk free fluorescence cross correlation spectroscopy in live cells,” Biophys. J. 89(3), 2069–2076 (2005).
[Crossref] [PubMed]

Tinnefeld, P.

E. Margeat, A. N. Kapanidis, P. Tinnefeld, Y. Wang, J. Mukhopadhyay, R. H. Ebright, and S. Weiss, “Direct observation of abortive initiation and promoter escape within single immobilized transcription complexes,” Biophys. J. 90(4), 1419–1431 (2006).
[Crossref] [PubMed]

Trautman, J. K.

X. S. Xie and J. K. Trautman, “Optical studies of single molecules at room temperature,” Annu. Rev. Phys. Chem. 49(1), 441–480 (1998).
[Crossref] [PubMed]

Unruh, J. R.

J. R. Unruh, G. Gokulrangan, G. S. Wilson, and C. K. Johnson, “Fluorescence properties of fluorescein, tetramethylrhodamine and Texas Red linked to a DNA aptamer,” Photochem. Photobiol. 81(3), 682–690 (2005).
[Crossref] [PubMed]

Valeri, A.

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

E. Sisamakis, A. Valeri, S. Kalinin, P. J. Rothwell, and C. A. Seidel, “Accurate single-molecule FRET studies using multiparameter fluorescence detection,” Methods Enzymol. 475, 455–514 (2010).
[Crossref] [PubMed]

Wahl, M.

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

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

Wang, Y.

E. Margeat, A. N. Kapanidis, P. Tinnefeld, Y. Wang, J. Mukhopadhyay, R. H. Ebright, and S. Weiss, “Direct observation of abortive initiation and promoter escape within single immobilized transcription complexes,” Biophys. J. 90(4), 1419–1431 (2006).
[Crossref] [PubMed]

Webb, W. W.

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

Weiss, S.

E. Margeat, A. N. Kapanidis, P. Tinnefeld, Y. Wang, J. Mukhopadhyay, R. H. Ebright, and S. Weiss, “Direct observation of abortive initiation and promoter escape within single immobilized transcription complexes,” Biophys. J. 90(4), 1419–1431 (2006).
[Crossref] [PubMed]

T. A. Laurence, X. Kong, M. Jäger, and S. Weiss, “Probing structural heterogeneities and fluctuations of nucleic acids and denatured proteins,” Proc. Natl. Acad. Sci. U.S.A. 102(48), 17348–17353 (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]

S. Weiss, “Fluorescence spectroscopy of single biomolecules,” Science 283(5408), 1676–1683 (1999).
[Crossref] [PubMed]

T. Ha, T. Enderle, D. F. Ogletree, D. S. Chemla, P. R. Selvin, and S. Weiss, “Probing the interaction between two single molecules: fluorescence resonance energy transfer between a single donor and a single acceptor,” Proc. Natl. Acad. Sci. U.S.A. 93(13), 6264–6268 (1996).
[Crossref] [PubMed]

Wildanger, D.

Wilson, G. S.

J. R. Unruh, G. Gokulrangan, G. S. Wilson, and C. K. Johnson, “Fluorescence properties of fluorescein, tetramethylrhodamine and Texas Red linked to a DNA aptamer,” Photochem. Photobiol. 81(3), 682–690 (2005).
[Crossref] [PubMed]

Wöhrl, B. M.

P. J. Rothwell, S. Berger, O. Kensch, S. Felekyan, M. Antonik, B. M. Wöhrl, T. Restle, R. S. Goody, and C. A. Seidel, “Multiparameter single-molecule fluorescence spectroscopy reveals heterogeneity of HIV-1 reverse transcriptase:primer/template complexes,” Proc. Natl. Acad. Sci. U.S.A. 100(4), 1655–1660 (2003).
[Crossref] [PubMed]

Wrachtrup, J.

E. Thews, M. Gerken, R. Eckert, J. Zäpfel, C. Tietz, and J. Wrachtrup, “Cross talk free fluorescence cross correlation spectroscopy in live cells,” Biophys. J. 89(3), 2069–2076 (2005).
[Crossref] [PubMed]

Xie, X. S.

X. S. Xie and J. K. Trautman, “Optical studies of single molecules at room temperature,” Annu. Rev. Phys. Chem. 49(1), 441–480 (1998).
[Crossref] [PubMed]

Zäpfel, J.

E. Thews, M. Gerken, R. Eckert, J. Zäpfel, C. Tietz, and J. Wrachtrup, “Cross talk free fluorescence cross correlation spectroscopy in live cells,” Biophys. J. 89(3), 2069–2076 (2005).
[Crossref] [PubMed]

Zaychikov, E.

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

Zhao, M.

Annu. Rev. Phys. Chem. (1)

X. S. Xie and J. K. Trautman, “Optical studies of single molecules at room temperature,” Annu. Rev. Phys. Chem. 49(1), 441–480 (1998).
[Crossref] [PubMed]

Appl. Opt. (2)

Biophys. J. (4)

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]

E. Margeat, A. N. Kapanidis, P. Tinnefeld, Y. Wang, J. Mukhopadhyay, R. H. Ebright, and S. Weiss, “Direct observation of abortive initiation and promoter escape within single immobilized transcription complexes,” Biophys. J. 90(4), 1419–1431 (2006).
[Crossref] [PubMed]

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

E. Thews, M. Gerken, R. Eckert, J. Zäpfel, C. Tietz, and J. Wrachtrup, “Cross talk free fluorescence cross correlation spectroscopy in live cells,” Biophys. J. 89(3), 2069–2076 (2005).
[Crossref] [PubMed]

Biopolymers (1)

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

Chem Phys. Chem. (1)

V. Kudryavtsev, M. Sikor, S. Kalinin, D. Mokranjac, C. A. M. Seidel, and D. C. Lamb, “Combining MFD and PIE for accurate single-pair Förster resonance energy transfer measurements,” Chem Phys. Chem. 13, 1060–1078 (2012).

Chem. Phys. (1)

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

Chem. Phys. Lett. (1)

C. M. Pieper and J. Enderlein, “Fluorescence correlation spectroscopy as a tool for measuring the rotational diffusion of macromolecules,” Chem. Phys. Lett. 516(1-3), 1–11 (2011).
[Crossref]

ChemPhysChem (1)

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

J. Fluoresc. (1)

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

Methods (1)

E. Haustein and P. Schwille, “Ultrasensitive investigations of biological systems by fluorescence correlation spectroscopy,” Methods 29(2), 153–166 (2003).
[Crossref] [PubMed]

Methods Enzymol. (1)

E. Sisamakis, A. Valeri, S. Kalinin, P. J. Rothwell, and C. A. Seidel, “Accurate single-molecule FRET studies using multiparameter fluorescence detection,” Methods Enzymol. 475, 455–514 (2010).
[Crossref] [PubMed]

Nat. Methods (1)

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

Opt. Express (2)

Opt. Lett. (2)

Photochem. Photobiol. (1)

J. R. Unruh, G. Gokulrangan, G. S. Wilson, and C. K. Johnson, “Fluorescence properties of fluorescein, tetramethylrhodamine and Texas Red linked to a DNA aptamer,” Photochem. Photobiol. 81(3), 682–690 (2005).
[Crossref] [PubMed]

Phys. Rev. Lett. (1)

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

Proc. Natl. Acad. Sci. U.S.A. (4)

T. Ha, T. Enderle, D. F. Ogletree, D. S. Chemla, P. R. Selvin, and S. Weiss, “Probing the interaction between two single molecules: fluorescence resonance energy transfer between a single donor and a single acceptor,” Proc. Natl. Acad. Sci. U.S.A. 93(13), 6264–6268 (1996).
[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]

T. A. Laurence, X. Kong, M. Jäger, and S. Weiss, “Probing structural heterogeneities and fluctuations of nucleic acids and denatured proteins,” Proc. Natl. Acad. Sci. U.S.A. 102(48), 17348–17353 (2005).
[Crossref] [PubMed]

P. J. Rothwell, S. Berger, O. Kensch, S. Felekyan, M. Antonik, B. M. Wöhrl, T. Restle, R. S. Goody, and C. A. Seidel, “Multiparameter single-molecule fluorescence spectroscopy reveals heterogeneity of HIV-1 reverse transcriptase:primer/template complexes,” Proc. Natl. Acad. Sci. U.S.A. 100(4), 1655–1660 (2003).
[Crossref] [PubMed]

Proc. SPIE (1)

R. Fenske, D. Näther, M. Goossens, and S. D. Smith, “New light sources for time-correlated single-photon counting in commercially available spectrometers,” Proc. SPIE 6372, 63720H (2006).
[Crossref]

Rep. Prog. Phys. (1)

O. Krichevsky and G. Bonnet, “Fluorescence correlation spectroscopy: the technique and its applications,” Rep. Prog. Phys. 65(2), 251–297 (2002).
[Crossref]

Rev. Sci. Instrum. (1)

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

Science (1)

S. Weiss, “Fluorescence spectroscopy of single biomolecules,” Science 283(5408), 1676–1683 (1999).
[Crossref] [PubMed]

Other (1)

P. Schwille and E. Haustein, “Fluorescence correlation spectroscopy. An introduction to its concepts and applications,” Spectroscopy 1–33 (2009).

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Figures (4)

Fig. 1
Fig. 1 Experimental setup (see text). Briefly, the output of the supercontinuum source is separated into two paths (the prompt and the delayed, the latter corresponding to a 8 meters long delay line) using a beamsplitter cube (BS1). The beams are recombined, thus interleaving the pulses, and coupled into a single mode fiber (SMF). The output of the fiber is then directed into a home-built confocal microscope, equipped with a 4-channels Single Photon Avalanche Diodes detection, and a Single Photon Counting Module (SPCM). L: lens; DM: dichroic mirror; TL: tube lens; BP: bandpass filter.

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Fig. 2
Fig. 2 Fluorescence decays obtained for a mixture of TMR and Atto655. Photons detected in the green channels appear in green, and are only observed upon excitation by the prompt pulses (at 532nm). Photons detected in the red channels appear in red. They are detected upon excitation by the delayed pulses (at 635nm) as expected, but also upon excitation by the prompt pulses, due to the leakage of the emission of TMR in the red channels, and due to direct excitation of Atto655 by the prompt pulses at 532nm. Insets represent the same decays on a logarithmic scale. The observed offset in the decays from the green channel arises from higher dark count rates in the green detectors than in the red detectors.

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Fig. 3
Fig. 3 A - Fluorescence autocorrelation (green and red) and cross-correlation (black) curves for a TMR solution. The cross-correlation amplitude represents 100% of the autocorrelation amplitudes of both channels, indicating a perfect overlap of the green and the red detection channels. B - Fluorescence autocorrelation and cross correlation (black) curves for an Atto655 solution upon excitation by the prompt and the delayed beams (green and red, respectively). The difference in correlation amplitudes indicate that the excitation volume generated by the delayed (red) beam is slightly larger than the other. However, the cross-correlation curves lie between the autocorrelation curves for both excitation beams, which indicates an excellent overlap between the two excitation volumes. Data were fit with a model taking into account one diffusion component and triplet blinking.

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Fig. 4
Fig. 4 Fluorescence autocorrelation (green and red) and cross correlation (black) curves for a 1:1 mixture of cy3 (green)- and Atto655 (red)- labeled DNA fragments either non complementary ((A) and (B)) or complementary and hybridized ((C) and (D)). A and C represent the correlation curves obtained for all photons, while B and D are obtained by selecting only the green photons generated by the prompt pulse and the red photons generated by the delayed pulse. For the non-complementary strands, the apparent cross-correlation amplitude that represents 14% of the green cross-correlation (A) is due to spectral cross-talk, and is completely removed by applying the PIE algorithm (B). For the complementary strands, the apparent cross-correlation amplitude represents 57% of the green cross-correlation (C). After removal of the cross-talk (D), this cross-correlation decreases to 46%, that represents the fraction of DNA molecules labeled with cy3 that are hybridized to DNA molecules labeled with Atto655. Data were fit with a model taking into account one diffusion component and triplet blinking (except for the cross correlation curves, where there is no triplet blinking).

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