Abstract

There is currently great interest in determining physical parameters, e.g. fluorescence lifetime, of individual molecules that inform on environmental conditions, whilst avoiding the artefacts of ensemble averaging. Protein interactions, molecular dynamics and sub-species can all be studied. In a burst integrated fluorescence lifetime (BIFL) experiment, identification of fluorescent bursts from single molecules above background detection is a problem. This paper presents a Bayesian method for burst identification based on model selection and demonstrates the detection of bursts consisting of 10% signal amplitude. The method also estimates the fluorescence lifetime (and its error) from the burst data.

© 2010 OSA

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  1. M. Böhmer and J. Enderlein, “Fluorescence spectroscopy of single molecules under ambient conditions: methodology and technology,” ChemPhysChem 4(8), 792–808 (2003).
    [CrossRef] [PubMed]
  2. C. Eggeling, J. R. Fries, L. Brand, R. Günther, and C. A. Seidel, “Monitoring conformational dynamics of a single molecule by selective fluorescence spectroscopy,” Proc. Natl. Acad. Sci. U.S.A. 95(4), 1556–1561 (1998).
    [CrossRef] [PubMed]
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    [CrossRef]
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    [CrossRef] [PubMed]
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    [CrossRef]
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    [CrossRef] [PubMed]
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    [CrossRef]
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    [CrossRef]
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    [CrossRef]

2009

P. R. Barber, S. M. Ameer-Beg, J. Gilbey, L. M. Carlin, M. Keppler, T. C. Ng, and B. Vojnovic, “Multiphoton time-domain fluorescence lifetime imaging microscopy: practical application to protein–protein interactions using global analysis,” J. R. Soc. Interface 6, S93–S105 (2009).
[CrossRef]

2008

W. Grange, P. Haas, A. Wild, M. A. Lieb, M. Calame, M. Hegner, and B. Hecht, “Detection of transient events in the presence of background noise,” J. Phys. Chem. B 112(23), 7140–7144 (2008).
[CrossRef] [PubMed]

2007

S. H. Lee, M. Jae, and R. P. Gardner, “Non-Poisson counting statistics of a hybrid G-M counter dead time model,” Nucl. Instrum. Methods Phys. Res. B 263, 46–49 (2007).

2006

W. Becker, A. Bergmann, E. Haustein, Z. Petrasek, P. Schwille, C. Biskup, L. Kelbauskas, K. Benndorf, N. Klöcker, T. Anhut, I. Riemann, and K. König, “Fluorescence lifetime images and correlation spectra obtained by multidimensional time-correlated single photon counting,” Microsc. Res. Tech. 69(3), 186–195 (2006).
[CrossRef] [PubMed]

2005

T. C. Voss, I. A. Demarco, and R. N. Day, “Quantitative imaging of protein interactions in the cell nucleus,” Biotechniques 38(3), 413–424 (2005).
[CrossRef] [PubMed]

H. Wallrabe and A. Periasamy, “Imaging protein molecules using FRET and FLIM microscopy,” Curr. Opin. Biotechnol. 16(1), 19–27 (2005).
[CrossRef] [PubMed]

M. Parsons, J. Monypenny, S. M. Ameer-Beg, T. H. Millard, L. M. Machesky, M. Peter, M. D. Keppler, G. Schiavo, R. Watson, J. Chernoff, D. Zicha, B. Vojnovic, and T. Ng, “Spatially distinct binding of Cdc42 to PAK1 and N-WASP in breast carcinoma cells,” Mol. Cell. Biol. 25(5), 1680–1695 (2005).
[CrossRef] [PubMed]

M. Peter, S. M. Ameer-Beg, M. K. Y. Hughes, M. D. Keppler, S. Prag, M. Marsh, B. Vojnovic, and T. Ng, “Multiphoton-FLIM quantification of the EGFP-mRFP1 FRET pair for localization of membrane receptor-kinase interactions,” Biophys. J. 88(2), 1224–1237 (2005).
[CrossRef] [PubMed]

2004

M. Prummer, B. Sick, A. Renn, and U. P. Wild, “Multiparameter microscopy and spectroscopy for single-molecule analytics,” Anal. Chem. 76(6), 1633–1640 (2004).
[CrossRef] [PubMed]

2003

M. Böhmer and J. Enderlein, “Fluorescence spectroscopy of single molecules under ambient conditions: methodology and technology,” ChemPhysChem 4(8), 792–808 (2003).
[CrossRef] [PubMed]

2001

R. Kühnemuth and C. A. M. Seidel, “Principles of Single Molecule Multiparameter Fluorescence Spectroscopy,” Single Mol. 2(4), 251–254 (2001).

2000

M. Prummer, C. G. Hubner, B. Sick, B. Hecht, A. Renn, and U. P. Wild, “Single-molecule identification by spectrally and time-resolved fluorescence detection,” Anal. Chem. 72(3), 443–447 (2000).
[CrossRef] [PubMed]

1998

C. Eggeling, J. R. Fries, L. Brand, R. Günther, and C. A. Seidel, “Monitoring conformational dynamics of a single molecule by selective fluorescence spectroscopy,” Proc. Natl. Acad. Sci. U.S.A. 95(4), 1556–1561 (1998).
[CrossRef] [PubMed]

J. R. Fries, L. Brand, C. Eggeling, M. Kollner, and C. A. M. Seidel, “Quantitative Identification of Different Single Molecules by Selective Time-Resolved Confocal Fluorescence Spectroscopy,” J. Phys. Chem. A 102(33), 6601–6613 (1998).
[CrossRef]

J. Enderlein, D. L. Robbins, W. P. Ambrose, and R. A. Keller, “Molecular Shot Noise, Burst Size Distribution, and Single-Molecule Detection in Fluid Flow: Effects of Multiple Occupancy,” J. Phys. Chem. A 102(30), 6089–6094 (1998).
[CrossRef]

1997

J. Enderlein, P. M. Goodwin, A. Van Orden, W. Patrick Ambrose, R. Erdmann, and R. A. Keller, “A maximum likelihood estimator to distinguish single molecules by their fluorescence decays,” Chem. Phys. Lett. 270(5-6), 464–470 (1997).
[CrossRef]

1990

E. Brooks Shera, N. K. Seitzinger, L. M. Davis, R. A. Keller, and S. A. Soper, “Detection of single fluorescent molecules,” Chem. Phys. Lett. 174(6), 553–557 (1990).
[CrossRef]

Ambrose, W. P.

J. Enderlein, D. L. Robbins, W. P. Ambrose, and R. A. Keller, “Molecular Shot Noise, Burst Size Distribution, and Single-Molecule Detection in Fluid Flow: Effects of Multiple Occupancy,” J. Phys. Chem. A 102(30), 6089–6094 (1998).
[CrossRef]

Ameer-Beg, S. M.

P. R. Barber, S. M. Ameer-Beg, J. Gilbey, L. M. Carlin, M. Keppler, T. C. Ng, and B. Vojnovic, “Multiphoton time-domain fluorescence lifetime imaging microscopy: practical application to protein–protein interactions using global analysis,” J. R. Soc. Interface 6, S93–S105 (2009).
[CrossRef]

M. Parsons, J. Monypenny, S. M. Ameer-Beg, T. H. Millard, L. M. Machesky, M. Peter, M. D. Keppler, G. Schiavo, R. Watson, J. Chernoff, D. Zicha, B. Vojnovic, and T. Ng, “Spatially distinct binding of Cdc42 to PAK1 and N-WASP in breast carcinoma cells,” Mol. Cell. Biol. 25(5), 1680–1695 (2005).
[CrossRef] [PubMed]

M. Peter, S. M. Ameer-Beg, M. K. Y. Hughes, M. D. Keppler, S. Prag, M. Marsh, B. Vojnovic, and T. Ng, “Multiphoton-FLIM quantification of the EGFP-mRFP1 FRET pair for localization of membrane receptor-kinase interactions,” Biophys. J. 88(2), 1224–1237 (2005).
[CrossRef] [PubMed]

Anhut, T.

W. Becker, A. Bergmann, E. Haustein, Z. Petrasek, P. Schwille, C. Biskup, L. Kelbauskas, K. Benndorf, N. Klöcker, T. Anhut, I. Riemann, and K. König, “Fluorescence lifetime images and correlation spectra obtained by multidimensional time-correlated single photon counting,” Microsc. Res. Tech. 69(3), 186–195 (2006).
[CrossRef] [PubMed]

Barber, P. R.

P. R. Barber, S. M. Ameer-Beg, J. Gilbey, L. M. Carlin, M. Keppler, T. C. Ng, and B. Vojnovic, “Multiphoton time-domain fluorescence lifetime imaging microscopy: practical application to protein–protein interactions using global analysis,” J. R. Soc. Interface 6, S93–S105 (2009).
[CrossRef]

Becker, W.

W. Becker, A. Bergmann, E. Haustein, Z. Petrasek, P. Schwille, C. Biskup, L. Kelbauskas, K. Benndorf, N. Klöcker, T. Anhut, I. Riemann, and K. König, “Fluorescence lifetime images and correlation spectra obtained by multidimensional time-correlated single photon counting,” Microsc. Res. Tech. 69(3), 186–195 (2006).
[CrossRef] [PubMed]

Benndorf, K.

W. Becker, A. Bergmann, E. Haustein, Z. Petrasek, P. Schwille, C. Biskup, L. Kelbauskas, K. Benndorf, N. Klöcker, T. Anhut, I. Riemann, and K. König, “Fluorescence lifetime images and correlation spectra obtained by multidimensional time-correlated single photon counting,” Microsc. Res. Tech. 69(3), 186–195 (2006).
[CrossRef] [PubMed]

Bergmann, A.

W. Becker, A. Bergmann, E. Haustein, Z. Petrasek, P. Schwille, C. Biskup, L. Kelbauskas, K. Benndorf, N. Klöcker, T. Anhut, I. Riemann, and K. König, “Fluorescence lifetime images and correlation spectra obtained by multidimensional time-correlated single photon counting,” Microsc. Res. Tech. 69(3), 186–195 (2006).
[CrossRef] [PubMed]

Biskup, C.

W. Becker, A. Bergmann, E. Haustein, Z. Petrasek, P. Schwille, C. Biskup, L. Kelbauskas, K. Benndorf, N. Klöcker, T. Anhut, I. Riemann, and K. König, “Fluorescence lifetime images and correlation spectra obtained by multidimensional time-correlated single photon counting,” Microsc. Res. Tech. 69(3), 186–195 (2006).
[CrossRef] [PubMed]

Böhmer, M.

M. Böhmer and J. Enderlein, “Fluorescence spectroscopy of single molecules under ambient conditions: methodology and technology,” ChemPhysChem 4(8), 792–808 (2003).
[CrossRef] [PubMed]

Brand, L.

C. Eggeling, J. R. Fries, L. Brand, R. Günther, and C. A. Seidel, “Monitoring conformational dynamics of a single molecule by selective fluorescence spectroscopy,” Proc. Natl. Acad. Sci. U.S.A. 95(4), 1556–1561 (1998).
[CrossRef] [PubMed]

J. R. Fries, L. Brand, C. Eggeling, M. Kollner, and C. A. M. Seidel, “Quantitative Identification of Different Single Molecules by Selective Time-Resolved Confocal Fluorescence Spectroscopy,” J. Phys. Chem. A 102(33), 6601–6613 (1998).
[CrossRef]

Brooks Shera, E.

E. Brooks Shera, N. K. Seitzinger, L. M. Davis, R. A. Keller, and S. A. Soper, “Detection of single fluorescent molecules,” Chem. Phys. Lett. 174(6), 553–557 (1990).
[CrossRef]

Calame, M.

W. Grange, P. Haas, A. Wild, M. A. Lieb, M. Calame, M. Hegner, and B. Hecht, “Detection of transient events in the presence of background noise,” J. Phys. Chem. B 112(23), 7140–7144 (2008).
[CrossRef] [PubMed]

Carlin, L. M.

P. R. Barber, S. M. Ameer-Beg, J. Gilbey, L. M. Carlin, M. Keppler, T. C. Ng, and B. Vojnovic, “Multiphoton time-domain fluorescence lifetime imaging microscopy: practical application to protein–protein interactions using global analysis,” J. R. Soc. Interface 6, S93–S105 (2009).
[CrossRef]

Chernoff, J.

M. Parsons, J. Monypenny, S. M. Ameer-Beg, T. H. Millard, L. M. Machesky, M. Peter, M. D. Keppler, G. Schiavo, R. Watson, J. Chernoff, D. Zicha, B. Vojnovic, and T. Ng, “Spatially distinct binding of Cdc42 to PAK1 and N-WASP in breast carcinoma cells,” Mol. Cell. Biol. 25(5), 1680–1695 (2005).
[CrossRef] [PubMed]

Davis, L. M.

E. Brooks Shera, N. K. Seitzinger, L. M. Davis, R. A. Keller, and S. A. Soper, “Detection of single fluorescent molecules,” Chem. Phys. Lett. 174(6), 553–557 (1990).
[CrossRef]

Day, R. N.

T. C. Voss, I. A. Demarco, and R. N. Day, “Quantitative imaging of protein interactions in the cell nucleus,” Biotechniques 38(3), 413–424 (2005).
[CrossRef] [PubMed]

Demarco, I. A.

T. C. Voss, I. A. Demarco, and R. N. Day, “Quantitative imaging of protein interactions in the cell nucleus,” Biotechniques 38(3), 413–424 (2005).
[CrossRef] [PubMed]

Eggeling, C.

J. R. Fries, L. Brand, C. Eggeling, M. Kollner, and C. A. M. Seidel, “Quantitative Identification of Different Single Molecules by Selective Time-Resolved Confocal Fluorescence Spectroscopy,” J. Phys. Chem. A 102(33), 6601–6613 (1998).
[CrossRef]

C. Eggeling, J. R. Fries, L. Brand, R. Günther, and C. A. Seidel, “Monitoring conformational dynamics of a single molecule by selective fluorescence spectroscopy,” Proc. Natl. Acad. Sci. U.S.A. 95(4), 1556–1561 (1998).
[CrossRef] [PubMed]

Enderlein, J.

M. Böhmer and J. Enderlein, “Fluorescence spectroscopy of single molecules under ambient conditions: methodology and technology,” ChemPhysChem 4(8), 792–808 (2003).
[CrossRef] [PubMed]

J. Enderlein, D. L. Robbins, W. P. Ambrose, and R. A. Keller, “Molecular Shot Noise, Burst Size Distribution, and Single-Molecule Detection in Fluid Flow: Effects of Multiple Occupancy,” J. Phys. Chem. A 102(30), 6089–6094 (1998).
[CrossRef]

J. Enderlein, P. M. Goodwin, A. Van Orden, W. Patrick Ambrose, R. Erdmann, and R. A. Keller, “A maximum likelihood estimator to distinguish single molecules by their fluorescence decays,” Chem. Phys. Lett. 270(5-6), 464–470 (1997).
[CrossRef]

Erdmann, R.

J. Enderlein, P. M. Goodwin, A. Van Orden, W. Patrick Ambrose, R. Erdmann, and R. A. Keller, “A maximum likelihood estimator to distinguish single molecules by their fluorescence decays,” Chem. Phys. Lett. 270(5-6), 464–470 (1997).
[CrossRef]

Fries, J. R.

C. Eggeling, J. R. Fries, L. Brand, R. Günther, and C. A. Seidel, “Monitoring conformational dynamics of a single molecule by selective fluorescence spectroscopy,” Proc. Natl. Acad. Sci. U.S.A. 95(4), 1556–1561 (1998).
[CrossRef] [PubMed]

J. R. Fries, L. Brand, C. Eggeling, M. Kollner, and C. A. M. Seidel, “Quantitative Identification of Different Single Molecules by Selective Time-Resolved Confocal Fluorescence Spectroscopy,” J. Phys. Chem. A 102(33), 6601–6613 (1998).
[CrossRef]

Gardner, R. P.

S. H. Lee, M. Jae, and R. P. Gardner, “Non-Poisson counting statistics of a hybrid G-M counter dead time model,” Nucl. Instrum. Methods Phys. Res. B 263, 46–49 (2007).

Gilbey, J.

P. R. Barber, S. M. Ameer-Beg, J. Gilbey, L. M. Carlin, M. Keppler, T. C. Ng, and B. Vojnovic, “Multiphoton time-domain fluorescence lifetime imaging microscopy: practical application to protein–protein interactions using global analysis,” J. R. Soc. Interface 6, S93–S105 (2009).
[CrossRef]

Goodwin, P. M.

J. Enderlein, P. M. Goodwin, A. Van Orden, W. Patrick Ambrose, R. Erdmann, and R. A. Keller, “A maximum likelihood estimator to distinguish single molecules by their fluorescence decays,” Chem. Phys. Lett. 270(5-6), 464–470 (1997).
[CrossRef]

Grange, W.

W. Grange, P. Haas, A. Wild, M. A. Lieb, M. Calame, M. Hegner, and B. Hecht, “Detection of transient events in the presence of background noise,” J. Phys. Chem. B 112(23), 7140–7144 (2008).
[CrossRef] [PubMed]

Günther, R.

C. Eggeling, J. R. Fries, L. Brand, R. Günther, and C. A. Seidel, “Monitoring conformational dynamics of a single molecule by selective fluorescence spectroscopy,” Proc. Natl. Acad. Sci. U.S.A. 95(4), 1556–1561 (1998).
[CrossRef] [PubMed]

Haas, P.

W. Grange, P. Haas, A. Wild, M. A. Lieb, M. Calame, M. Hegner, and B. Hecht, “Detection of transient events in the presence of background noise,” J. Phys. Chem. B 112(23), 7140–7144 (2008).
[CrossRef] [PubMed]

Haustein, E.

W. Becker, A. Bergmann, E. Haustein, Z. Petrasek, P. Schwille, C. Biskup, L. Kelbauskas, K. Benndorf, N. Klöcker, T. Anhut, I. Riemann, and K. König, “Fluorescence lifetime images and correlation spectra obtained by multidimensional time-correlated single photon counting,” Microsc. Res. Tech. 69(3), 186–195 (2006).
[CrossRef] [PubMed]

Hecht, B.

W. Grange, P. Haas, A. Wild, M. A. Lieb, M. Calame, M. Hegner, and B. Hecht, “Detection of transient events in the presence of background noise,” J. Phys. Chem. B 112(23), 7140–7144 (2008).
[CrossRef] [PubMed]

M. Prummer, C. G. Hubner, B. Sick, B. Hecht, A. Renn, and U. P. Wild, “Single-molecule identification by spectrally and time-resolved fluorescence detection,” Anal. Chem. 72(3), 443–447 (2000).
[CrossRef] [PubMed]

Hegner, M.

W. Grange, P. Haas, A. Wild, M. A. Lieb, M. Calame, M. Hegner, and B. Hecht, “Detection of transient events in the presence of background noise,” J. Phys. Chem. B 112(23), 7140–7144 (2008).
[CrossRef] [PubMed]

Hubner, C. G.

M. Prummer, C. G. Hubner, B. Sick, B. Hecht, A. Renn, and U. P. Wild, “Single-molecule identification by spectrally and time-resolved fluorescence detection,” Anal. Chem. 72(3), 443–447 (2000).
[CrossRef] [PubMed]

Hughes, M. K. Y.

M. Peter, S. M. Ameer-Beg, M. K. Y. Hughes, M. D. Keppler, S. Prag, M. Marsh, B. Vojnovic, and T. Ng, “Multiphoton-FLIM quantification of the EGFP-mRFP1 FRET pair for localization of membrane receptor-kinase interactions,” Biophys. J. 88(2), 1224–1237 (2005).
[CrossRef] [PubMed]

Jae, M.

S. H. Lee, M. Jae, and R. P. Gardner, “Non-Poisson counting statistics of a hybrid G-M counter dead time model,” Nucl. Instrum. Methods Phys. Res. B 263, 46–49 (2007).

Kelbauskas, L.

W. Becker, A. Bergmann, E. Haustein, Z. Petrasek, P. Schwille, C. Biskup, L. Kelbauskas, K. Benndorf, N. Klöcker, T. Anhut, I. Riemann, and K. König, “Fluorescence lifetime images and correlation spectra obtained by multidimensional time-correlated single photon counting,” Microsc. Res. Tech. 69(3), 186–195 (2006).
[CrossRef] [PubMed]

Keller, R. A.

J. Enderlein, D. L. Robbins, W. P. Ambrose, and R. A. Keller, “Molecular Shot Noise, Burst Size Distribution, and Single-Molecule Detection in Fluid Flow: Effects of Multiple Occupancy,” J. Phys. Chem. A 102(30), 6089–6094 (1998).
[CrossRef]

J. Enderlein, P. M. Goodwin, A. Van Orden, W. Patrick Ambrose, R. Erdmann, and R. A. Keller, “A maximum likelihood estimator to distinguish single molecules by their fluorescence decays,” Chem. Phys. Lett. 270(5-6), 464–470 (1997).
[CrossRef]

E. Brooks Shera, N. K. Seitzinger, L. M. Davis, R. A. Keller, and S. A. Soper, “Detection of single fluorescent molecules,” Chem. Phys. Lett. 174(6), 553–557 (1990).
[CrossRef]

Keppler, M.

P. R. Barber, S. M. Ameer-Beg, J. Gilbey, L. M. Carlin, M. Keppler, T. C. Ng, and B. Vojnovic, “Multiphoton time-domain fluorescence lifetime imaging microscopy: practical application to protein–protein interactions using global analysis,” J. R. Soc. Interface 6, S93–S105 (2009).
[CrossRef]

Keppler, M. D.

M. Peter, S. M. Ameer-Beg, M. K. Y. Hughes, M. D. Keppler, S. Prag, M. Marsh, B. Vojnovic, and T. Ng, “Multiphoton-FLIM quantification of the EGFP-mRFP1 FRET pair for localization of membrane receptor-kinase interactions,” Biophys. J. 88(2), 1224–1237 (2005).
[CrossRef] [PubMed]

M. Parsons, J. Monypenny, S. M. Ameer-Beg, T. H. Millard, L. M. Machesky, M. Peter, M. D. Keppler, G. Schiavo, R. Watson, J. Chernoff, D. Zicha, B. Vojnovic, and T. Ng, “Spatially distinct binding of Cdc42 to PAK1 and N-WASP in breast carcinoma cells,” Mol. Cell. Biol. 25(5), 1680–1695 (2005).
[CrossRef] [PubMed]

Klöcker, N.

W. Becker, A. Bergmann, E. Haustein, Z. Petrasek, P. Schwille, C. Biskup, L. Kelbauskas, K. Benndorf, N. Klöcker, T. Anhut, I. Riemann, and K. König, “Fluorescence lifetime images and correlation spectra obtained by multidimensional time-correlated single photon counting,” Microsc. Res. Tech. 69(3), 186–195 (2006).
[CrossRef] [PubMed]

Kollner, M.

J. R. Fries, L. Brand, C. Eggeling, M. Kollner, and C. A. M. Seidel, “Quantitative Identification of Different Single Molecules by Selective Time-Resolved Confocal Fluorescence Spectroscopy,” J. Phys. Chem. A 102(33), 6601–6613 (1998).
[CrossRef]

König, K.

W. Becker, A. Bergmann, E. Haustein, Z. Petrasek, P. Schwille, C. Biskup, L. Kelbauskas, K. Benndorf, N. Klöcker, T. Anhut, I. Riemann, and K. König, “Fluorescence lifetime images and correlation spectra obtained by multidimensional time-correlated single photon counting,” Microsc. Res. Tech. 69(3), 186–195 (2006).
[CrossRef] [PubMed]

Kühnemuth, R.

R. Kühnemuth and C. A. M. Seidel, “Principles of Single Molecule Multiparameter Fluorescence Spectroscopy,” Single Mol. 2(4), 251–254 (2001).

Lee, S. H.

S. H. Lee, M. Jae, and R. P. Gardner, “Non-Poisson counting statistics of a hybrid G-M counter dead time model,” Nucl. Instrum. Methods Phys. Res. B 263, 46–49 (2007).

Lieb, M. A.

W. Grange, P. Haas, A. Wild, M. A. Lieb, M. Calame, M. Hegner, and B. Hecht, “Detection of transient events in the presence of background noise,” J. Phys. Chem. B 112(23), 7140–7144 (2008).
[CrossRef] [PubMed]

Machesky, L. M.

M. Parsons, J. Monypenny, S. M. Ameer-Beg, T. H. Millard, L. M. Machesky, M. Peter, M. D. Keppler, G. Schiavo, R. Watson, J. Chernoff, D. Zicha, B. Vojnovic, and T. Ng, “Spatially distinct binding of Cdc42 to PAK1 and N-WASP in breast carcinoma cells,” Mol. Cell. Biol. 25(5), 1680–1695 (2005).
[CrossRef] [PubMed]

Marsh, M.

M. Peter, S. M. Ameer-Beg, M. K. Y. Hughes, M. D. Keppler, S. Prag, M. Marsh, B. Vojnovic, and T. Ng, “Multiphoton-FLIM quantification of the EGFP-mRFP1 FRET pair for localization of membrane receptor-kinase interactions,” Biophys. J. 88(2), 1224–1237 (2005).
[CrossRef] [PubMed]

Millard, T. H.

M. Parsons, J. Monypenny, S. M. Ameer-Beg, T. H. Millard, L. M. Machesky, M. Peter, M. D. Keppler, G. Schiavo, R. Watson, J. Chernoff, D. Zicha, B. Vojnovic, and T. Ng, “Spatially distinct binding of Cdc42 to PAK1 and N-WASP in breast carcinoma cells,” Mol. Cell. Biol. 25(5), 1680–1695 (2005).
[CrossRef] [PubMed]

Monypenny, J.

M. Parsons, J. Monypenny, S. M. Ameer-Beg, T. H. Millard, L. M. Machesky, M. Peter, M. D. Keppler, G. Schiavo, R. Watson, J. Chernoff, D. Zicha, B. Vojnovic, and T. Ng, “Spatially distinct binding of Cdc42 to PAK1 and N-WASP in breast carcinoma cells,” Mol. Cell. Biol. 25(5), 1680–1695 (2005).
[CrossRef] [PubMed]

Ng, T.

M. Parsons, J. Monypenny, S. M. Ameer-Beg, T. H. Millard, L. M. Machesky, M. Peter, M. D. Keppler, G. Schiavo, R. Watson, J. Chernoff, D. Zicha, B. Vojnovic, and T. Ng, “Spatially distinct binding of Cdc42 to PAK1 and N-WASP in breast carcinoma cells,” Mol. Cell. Biol. 25(5), 1680–1695 (2005).
[CrossRef] [PubMed]

M. Peter, S. M. Ameer-Beg, M. K. Y. Hughes, M. D. Keppler, S. Prag, M. Marsh, B. Vojnovic, and T. Ng, “Multiphoton-FLIM quantification of the EGFP-mRFP1 FRET pair for localization of membrane receptor-kinase interactions,” Biophys. J. 88(2), 1224–1237 (2005).
[CrossRef] [PubMed]

Ng, T. C.

P. R. Barber, S. M. Ameer-Beg, J. Gilbey, L. M. Carlin, M. Keppler, T. C. Ng, and B. Vojnovic, “Multiphoton time-domain fluorescence lifetime imaging microscopy: practical application to protein–protein interactions using global analysis,” J. R. Soc. Interface 6, S93–S105 (2009).
[CrossRef]

Parsons, M.

M. Parsons, J. Monypenny, S. M. Ameer-Beg, T. H. Millard, L. M. Machesky, M. Peter, M. D. Keppler, G. Schiavo, R. Watson, J. Chernoff, D. Zicha, B. Vojnovic, and T. Ng, “Spatially distinct binding of Cdc42 to PAK1 and N-WASP in breast carcinoma cells,” Mol. Cell. Biol. 25(5), 1680–1695 (2005).
[CrossRef] [PubMed]

Patrick Ambrose, W.

J. Enderlein, P. M. Goodwin, A. Van Orden, W. Patrick Ambrose, R. Erdmann, and R. A. Keller, “A maximum likelihood estimator to distinguish single molecules by their fluorescence decays,” Chem. Phys. Lett. 270(5-6), 464–470 (1997).
[CrossRef]

Periasamy, A.

H. Wallrabe and A. Periasamy, “Imaging protein molecules using FRET and FLIM microscopy,” Curr. Opin. Biotechnol. 16(1), 19–27 (2005).
[CrossRef] [PubMed]

Peter, M.

M. Parsons, J. Monypenny, S. M. Ameer-Beg, T. H. Millard, L. M. Machesky, M. Peter, M. D. Keppler, G. Schiavo, R. Watson, J. Chernoff, D. Zicha, B. Vojnovic, and T. Ng, “Spatially distinct binding of Cdc42 to PAK1 and N-WASP in breast carcinoma cells,” Mol. Cell. Biol. 25(5), 1680–1695 (2005).
[CrossRef] [PubMed]

M. Peter, S. M. Ameer-Beg, M. K. Y. Hughes, M. D. Keppler, S. Prag, M. Marsh, B. Vojnovic, and T. Ng, “Multiphoton-FLIM quantification of the EGFP-mRFP1 FRET pair for localization of membrane receptor-kinase interactions,” Biophys. J. 88(2), 1224–1237 (2005).
[CrossRef] [PubMed]

Petrasek, Z.

W. Becker, A. Bergmann, E. Haustein, Z. Petrasek, P. Schwille, C. Biskup, L. Kelbauskas, K. Benndorf, N. Klöcker, T. Anhut, I. Riemann, and K. König, “Fluorescence lifetime images and correlation spectra obtained by multidimensional time-correlated single photon counting,” Microsc. Res. Tech. 69(3), 186–195 (2006).
[CrossRef] [PubMed]

Prag, S.

M. Peter, S. M. Ameer-Beg, M. K. Y. Hughes, M. D. Keppler, S. Prag, M. Marsh, B. Vojnovic, and T. Ng, “Multiphoton-FLIM quantification of the EGFP-mRFP1 FRET pair for localization of membrane receptor-kinase interactions,” Biophys. J. 88(2), 1224–1237 (2005).
[CrossRef] [PubMed]

Prummer, M.

M. Prummer, B. Sick, A. Renn, and U. P. Wild, “Multiparameter microscopy and spectroscopy for single-molecule analytics,” Anal. Chem. 76(6), 1633–1640 (2004).
[CrossRef] [PubMed]

M. Prummer, C. G. Hubner, B. Sick, B. Hecht, A. Renn, and U. P. Wild, “Single-molecule identification by spectrally and time-resolved fluorescence detection,” Anal. Chem. 72(3), 443–447 (2000).
[CrossRef] [PubMed]

Renn, A.

M. Prummer, B. Sick, A. Renn, and U. P. Wild, “Multiparameter microscopy and spectroscopy for single-molecule analytics,” Anal. Chem. 76(6), 1633–1640 (2004).
[CrossRef] [PubMed]

M. Prummer, C. G. Hubner, B. Sick, B. Hecht, A. Renn, and U. P. Wild, “Single-molecule identification by spectrally and time-resolved fluorescence detection,” Anal. Chem. 72(3), 443–447 (2000).
[CrossRef] [PubMed]

Riemann, I.

W. Becker, A. Bergmann, E. Haustein, Z. Petrasek, P. Schwille, C. Biskup, L. Kelbauskas, K. Benndorf, N. Klöcker, T. Anhut, I. Riemann, and K. König, “Fluorescence lifetime images and correlation spectra obtained by multidimensional time-correlated single photon counting,” Microsc. Res. Tech. 69(3), 186–195 (2006).
[CrossRef] [PubMed]

Robbins, D. L.

J. Enderlein, D. L. Robbins, W. P. Ambrose, and R. A. Keller, “Molecular Shot Noise, Burst Size Distribution, and Single-Molecule Detection in Fluid Flow: Effects of Multiple Occupancy,” J. Phys. Chem. A 102(30), 6089–6094 (1998).
[CrossRef]

Schiavo, G.

M. Parsons, J. Monypenny, S. M. Ameer-Beg, T. H. Millard, L. M. Machesky, M. Peter, M. D. Keppler, G. Schiavo, R. Watson, J. Chernoff, D. Zicha, B. Vojnovic, and T. Ng, “Spatially distinct binding of Cdc42 to PAK1 and N-WASP in breast carcinoma cells,” Mol. Cell. Biol. 25(5), 1680–1695 (2005).
[CrossRef] [PubMed]

Schwille, P.

W. Becker, A. Bergmann, E. Haustein, Z. Petrasek, P. Schwille, C. Biskup, L. Kelbauskas, K. Benndorf, N. Klöcker, T. Anhut, I. Riemann, and K. König, “Fluorescence lifetime images and correlation spectra obtained by multidimensional time-correlated single photon counting,” Microsc. Res. Tech. 69(3), 186–195 (2006).
[CrossRef] [PubMed]

Seidel, C. A.

C. Eggeling, J. R. Fries, L. Brand, R. Günther, and C. A. Seidel, “Monitoring conformational dynamics of a single molecule by selective fluorescence spectroscopy,” Proc. Natl. Acad. Sci. U.S.A. 95(4), 1556–1561 (1998).
[CrossRef] [PubMed]

Seidel, C. A. M.

R. Kühnemuth and C. A. M. Seidel, “Principles of Single Molecule Multiparameter Fluorescence Spectroscopy,” Single Mol. 2(4), 251–254 (2001).

J. R. Fries, L. Brand, C. Eggeling, M. Kollner, and C. A. M. Seidel, “Quantitative Identification of Different Single Molecules by Selective Time-Resolved Confocal Fluorescence Spectroscopy,” J. Phys. Chem. A 102(33), 6601–6613 (1998).
[CrossRef]

Seitzinger, N. K.

E. Brooks Shera, N. K. Seitzinger, L. M. Davis, R. A. Keller, and S. A. Soper, “Detection of single fluorescent molecules,” Chem. Phys. Lett. 174(6), 553–557 (1990).
[CrossRef]

Sick, B.

M. Prummer, B. Sick, A. Renn, and U. P. Wild, “Multiparameter microscopy and spectroscopy for single-molecule analytics,” Anal. Chem. 76(6), 1633–1640 (2004).
[CrossRef] [PubMed]

M. Prummer, C. G. Hubner, B. Sick, B. Hecht, A. Renn, and U. P. Wild, “Single-molecule identification by spectrally and time-resolved fluorescence detection,” Anal. Chem. 72(3), 443–447 (2000).
[CrossRef] [PubMed]

Soper, S. A.

E. Brooks Shera, N. K. Seitzinger, L. M. Davis, R. A. Keller, and S. A. Soper, “Detection of single fluorescent molecules,” Chem. Phys. Lett. 174(6), 553–557 (1990).
[CrossRef]

Van Orden, A.

J. Enderlein, P. M. Goodwin, A. Van Orden, W. Patrick Ambrose, R. Erdmann, and R. A. Keller, “A maximum likelihood estimator to distinguish single molecules by their fluorescence decays,” Chem. Phys. Lett. 270(5-6), 464–470 (1997).
[CrossRef]

Vojnovic, B.

P. R. Barber, S. M. Ameer-Beg, J. Gilbey, L. M. Carlin, M. Keppler, T. C. Ng, and B. Vojnovic, “Multiphoton time-domain fluorescence lifetime imaging microscopy: practical application to protein–protein interactions using global analysis,” J. R. Soc. Interface 6, S93–S105 (2009).
[CrossRef]

M. Peter, S. M. Ameer-Beg, M. K. Y. Hughes, M. D. Keppler, S. Prag, M. Marsh, B. Vojnovic, and T. Ng, “Multiphoton-FLIM quantification of the EGFP-mRFP1 FRET pair for localization of membrane receptor-kinase interactions,” Biophys. J. 88(2), 1224–1237 (2005).
[CrossRef] [PubMed]

M. Parsons, J. Monypenny, S. M. Ameer-Beg, T. H. Millard, L. M. Machesky, M. Peter, M. D. Keppler, G. Schiavo, R. Watson, J. Chernoff, D. Zicha, B. Vojnovic, and T. Ng, “Spatially distinct binding of Cdc42 to PAK1 and N-WASP in breast carcinoma cells,” Mol. Cell. Biol. 25(5), 1680–1695 (2005).
[CrossRef] [PubMed]

Voss, T. C.

T. C. Voss, I. A. Demarco, and R. N. Day, “Quantitative imaging of protein interactions in the cell nucleus,” Biotechniques 38(3), 413–424 (2005).
[CrossRef] [PubMed]

Wallrabe, H.

H. Wallrabe and A. Periasamy, “Imaging protein molecules using FRET and FLIM microscopy,” Curr. Opin. Biotechnol. 16(1), 19–27 (2005).
[CrossRef] [PubMed]

Watson, R.

M. Parsons, J. Monypenny, S. M. Ameer-Beg, T. H. Millard, L. M. Machesky, M. Peter, M. D. Keppler, G. Schiavo, R. Watson, J. Chernoff, D. Zicha, B. Vojnovic, and T. Ng, “Spatially distinct binding of Cdc42 to PAK1 and N-WASP in breast carcinoma cells,” Mol. Cell. Biol. 25(5), 1680–1695 (2005).
[CrossRef] [PubMed]

Wild, A.

W. Grange, P. Haas, A. Wild, M. A. Lieb, M. Calame, M. Hegner, and B. Hecht, “Detection of transient events in the presence of background noise,” J. Phys. Chem. B 112(23), 7140–7144 (2008).
[CrossRef] [PubMed]

Wild, U. P.

M. Prummer, B. Sick, A. Renn, and U. P. Wild, “Multiparameter microscopy and spectroscopy for single-molecule analytics,” Anal. Chem. 76(6), 1633–1640 (2004).
[CrossRef] [PubMed]

M. Prummer, C. G. Hubner, B. Sick, B. Hecht, A. Renn, and U. P. Wild, “Single-molecule identification by spectrally and time-resolved fluorescence detection,” Anal. Chem. 72(3), 443–447 (2000).
[CrossRef] [PubMed]

Zicha, D.

M. Parsons, J. Monypenny, S. M. Ameer-Beg, T. H. Millard, L. M. Machesky, M. Peter, M. D. Keppler, G. Schiavo, R. Watson, J. Chernoff, D. Zicha, B. Vojnovic, and T. Ng, “Spatially distinct binding of Cdc42 to PAK1 and N-WASP in breast carcinoma cells,” Mol. Cell. Biol. 25(5), 1680–1695 (2005).
[CrossRef] [PubMed]

Nucl. Instrum. Methods Phys. Res. B

S. H. Lee, M. Jae, and R. P. Gardner, “Non-Poisson counting statistics of a hybrid G-M counter dead time model,” Nucl. Instrum. Methods Phys. Res. B 263, 46–49 (2007).

Anal. Chem.

M. Prummer, C. G. Hubner, B. Sick, B. Hecht, A. Renn, and U. P. Wild, “Single-molecule identification by spectrally and time-resolved fluorescence detection,” Anal. Chem. 72(3), 443–447 (2000).
[CrossRef] [PubMed]

M. Prummer, B. Sick, A. Renn, and U. P. Wild, “Multiparameter microscopy and spectroscopy for single-molecule analytics,” Anal. Chem. 76(6), 1633–1640 (2004).
[CrossRef] [PubMed]

Biophys. J.

M. Peter, S. M. Ameer-Beg, M. K. Y. Hughes, M. D. Keppler, S. Prag, M. Marsh, B. Vojnovic, and T. Ng, “Multiphoton-FLIM quantification of the EGFP-mRFP1 FRET pair for localization of membrane receptor-kinase interactions,” Biophys. J. 88(2), 1224–1237 (2005).
[CrossRef] [PubMed]

Biotechniques

T. C. Voss, I. A. Demarco, and R. N. Day, “Quantitative imaging of protein interactions in the cell nucleus,” Biotechniques 38(3), 413–424 (2005).
[CrossRef] [PubMed]

Chem. Phys. Lett.

J. Enderlein, P. M. Goodwin, A. Van Orden, W. Patrick Ambrose, R. Erdmann, and R. A. Keller, “A maximum likelihood estimator to distinguish single molecules by their fluorescence decays,” Chem. Phys. Lett. 270(5-6), 464–470 (1997).
[CrossRef]

E. Brooks Shera, N. K. Seitzinger, L. M. Davis, R. A. Keller, and S. A. Soper, “Detection of single fluorescent molecules,” Chem. Phys. Lett. 174(6), 553–557 (1990).
[CrossRef]

ChemPhysChem

M. Böhmer and J. Enderlein, “Fluorescence spectroscopy of single molecules under ambient conditions: methodology and technology,” ChemPhysChem 4(8), 792–808 (2003).
[CrossRef] [PubMed]

Curr. Opin. Biotechnol.

H. Wallrabe and A. Periasamy, “Imaging protein molecules using FRET and FLIM microscopy,” Curr. Opin. Biotechnol. 16(1), 19–27 (2005).
[CrossRef] [PubMed]

J. Phys. Chem. A

J. R. Fries, L. Brand, C. Eggeling, M. Kollner, and C. A. M. Seidel, “Quantitative Identification of Different Single Molecules by Selective Time-Resolved Confocal Fluorescence Spectroscopy,” J. Phys. Chem. A 102(33), 6601–6613 (1998).
[CrossRef]

J. Enderlein, D. L. Robbins, W. P. Ambrose, and R. A. Keller, “Molecular Shot Noise, Burst Size Distribution, and Single-Molecule Detection in Fluid Flow: Effects of Multiple Occupancy,” J. Phys. Chem. A 102(30), 6089–6094 (1998).
[CrossRef]

J. Phys. Chem. B

W. Grange, P. Haas, A. Wild, M. A. Lieb, M. Calame, M. Hegner, and B. Hecht, “Detection of transient events in the presence of background noise,” J. Phys. Chem. B 112(23), 7140–7144 (2008).
[CrossRef] [PubMed]

J. R. Soc. Interface

P. R. Barber, S. M. Ameer-Beg, J. Gilbey, L. M. Carlin, M. Keppler, T. C. Ng, and B. Vojnovic, “Multiphoton time-domain fluorescence lifetime imaging microscopy: practical application to protein–protein interactions using global analysis,” J. R. Soc. Interface 6, S93–S105 (2009).
[CrossRef]

Microsc. Res. Tech.

W. Becker, A. Bergmann, E. Haustein, Z. Petrasek, P. Schwille, C. Biskup, L. Kelbauskas, K. Benndorf, N. Klöcker, T. Anhut, I. Riemann, and K. König, “Fluorescence lifetime images and correlation spectra obtained by multidimensional time-correlated single photon counting,” Microsc. Res. Tech. 69(3), 186–195 (2006).
[CrossRef] [PubMed]

Mol. Cell. Biol.

M. Parsons, J. Monypenny, S. M. Ameer-Beg, T. H. Millard, L. M. Machesky, M. Peter, M. D. Keppler, G. Schiavo, R. Watson, J. Chernoff, D. Zicha, B. Vojnovic, and T. Ng, “Spatially distinct binding of Cdc42 to PAK1 and N-WASP in breast carcinoma cells,” Mol. Cell. Biol. 25(5), 1680–1695 (2005).
[CrossRef] [PubMed]

Proc. Natl. Acad. Sci. U.S.A.

C. Eggeling, J. R. Fries, L. Brand, R. Günther, and C. A. Seidel, “Monitoring conformational dynamics of a single molecule by selective fluorescence spectroscopy,” Proc. Natl. Acad. Sci. U.S.A. 95(4), 1556–1561 (1998).
[CrossRef] [PubMed]

Other

R. Kühnemuth and C. A. M. Seidel, “Principles of Single Molecule Multiparameter Fluorescence Spectroscopy,” Single Mol. 2(4), 251–254 (2001).

C. M. Bishop, Neural Networks for Pattern Recognition (Oxford University Press, Oxford, 1995).

W. H. Press, B. P. Flannery, S. A. Teukolsky, and W. T. Vetterling, Numerical Recipes in C: The Art of Scientific Computing (Cambridge University Press, 1992).

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Figures (3)

Fig. 1
Fig. 1

A simplified schematic illustrating the mathematical process of forming the posterior probability density function (PDF, blue lines) versus lifetime, from which the most likely lifetime and an associated error interval can be calculated. Starting from the prior probability density function, successive photon arrival times (red bars, shown at the appropriate microtime) contribute some evidence under this Bayesian framework, as calculated by the function F (see text). As more photons are measured the posterior PDF may become a single well-defined peak indicating the most likely lifetime value, w 1.

Fig. 2
Fig. 2

Bayesian burst detection from simulated data. Signal fluorescence lifetime is set to 2 ns in all cases. (a) Bursts where the signal proportion of 1/3 (bg: 2 x 104 s−1 sig: 1 x 104 s−1) are easily detected (and would equally be by simple thresholding of the macrotime trace). (b) Bursts with a signal proportion of 1/10 (bg: 2 x 104 s−1 sig: 2 x 103 s−1) cannot be detected from the macrotime trace alone. The Bayesian technique detects these bursts as shown by the resulting probability of a signal, P(signal) = P(H sig|D) (red line). (c) The microtime trace with 1/3 signal, extracted from the photon train near 0.8 seconds (see dotted line labelled ‘c’ in (a)). (d and e) Microtime traces representing 1/10 signal and pure background noise (see dotted lines in (b)). Microtime traces were formed from data re-binned into 100 bins for plotting clarity. Where bursts were detected, lifetime fitting was performed by the full continuous Bayes algorithm on the re-binned data. The fitted curves are shown (solid line) and on the right hand side (f and g) are plots of the 2D PDF around the most likely value, where white indicates high values, decreasing through a spectrum to black which indicate low values. (h) The percentage of true detected bursts (blue line) and false positive detections (red line) as a function of signal proportion. (‘counts’ = number of photon counts in trace, ‘lifetime’ = determined lifetime).

Fig. 3
Fig. 3

Bayesian burst detection from experimental data of a dilute solution of quantum dots. (a) Macrotime intensity trace and corresponding signal probability. The obvious high-intensity burst is detected along with several low intensity bursts that are not apparent through analysis of the macrotime trace alone. (b, c and d) Microtime traces extracted from the photon train at the positions indicated by the dotted lines in (a). Lifetime fitting was performed on the microtime data and the fitted curve is shown (solid line, re-binned into 32 bins for clarity) and inset is a plot of the PDF around the most likely value. Data in graph (c) was rejected as background since P(signal) is below 0.5. Lifetimes of 6.20 ns and 4.88 ns were extracted by a full, continuous, Bayesian analysis of the high- and low-intensity bursts, respectively.

Tables (2)

Tables Icon

Table 1 Values used by the discrete algorithm when analysing the simulated experiments

Tables Icon

Table 2 Values used by the discrete algorithm when analysing the experimental data

Equations (12)

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

P ( H s i g | D ) = P ( D | H s i g ) P ( H s i g ) P ( H s i g ) P ( D | H s i g ) + P ( H b g ) P ( D | H b g ) ,
p ( Δ t | H b g ) = 1 T ,
p ( Δ t | H s i g , w ) = ( 1 w 0 ) F ( Δ t | w 1 ) + w 0 T ,
F ( Δ t | w 1 ) = e Δ t / w 1 w 1 ( 1 e T / w 1 ) ,
p ( w ) = α e α w 1 ,
P ( D | H s i g ) = d w p ( w ) . μ = 1 p [ p ( Δ t μ | H s i g , w ) ] ,
P ( D | H s i g ) = d w α e α w 1 μ = 1 p [ ( 1 w 0 )    F ( Δ t μ | w 1 ) + w 0 T ] ,
P ( D | H b g ) = μ = 1 p [ 1 T ] = 1 T p .
P ( H s i g | D ) = P ( H s i g ) . d w α e α w 1 μ = 1 p [ ( 1 w 0 )    T F ( Δ t μ | w 1 ) + w 0 ] [ 1 P ( H s i g ) ] + P ( H s i g ) . d w α e α w 1 μ = 1 p [ ( 1 w 0 )    T F ( Δ t μ | w 1 ) + w 0 ] ,
p ( w | D , H s i g ) = e α w 1 μ = 1 p [ ( 1 w 0 ) T F ( Δ t μ | w 1 ) + w 0 ] d w e α w 0 μ = 1 p [ ( 1 w 0 ) T F ( Δ t μ | w 1 ) + w 0 ] .
F ¯ ( Δ t | w 1 ) = d s Γ ( Δ t s ) F ( s | w 1 ) 0 T d t d s Γ ( t s ) F ( s | w 1 ) ,
Γ ( u ) = e 1 2 ( u u c ) 2 / σ 2 σ 2 π 2 θ [ u ] 1 + e r f ( u c / σ 2 ) .

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