Abstract

The ability to quantify the fluorescence signals from multiply labeled biological samples is highly desirable in the life sciences but often difficult, because of spectral overlap between fluorescent species and the presence of autofluorescence. Several so called unmixing algorithms have been developed to address this problem. Here, we present a novel algorithm that combines measurements of lifetime and spectrum to achieve unmixing without a priori information on the spectral properties of the fluorophore labels. The only assumption made is that the lifetimes of the fluorophores differ. Our method combines global analysis for a measurement of lifetime distributions with singular value decomposition to recover individual fluorescence spectra. We demonstrate the technique on simulated datasets and subsequently by an experiment on a biological sample. The method is computationally efficient and straightforward to implement. Applications range from histopathology of complex and multiply labelled samples to functional imaging in live cells.

© 2009 Optical Society of America

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  1. J. H. Frank, A. D. Elder, J. Swartling, A. R. Venkitaraman, A. D. Jeyasekharan, and C. F. Kaminski, "A white light confocal microscope for spectrally resolved multidimensional imaging," J Microsc. 227, 203-215 (2007).
    [CrossRef] [PubMed]
  2. C. F. Kaminski, R. S. Watt, A. D. Elder, J. H. Frank, and J. Hult, "Supercontinuum radiation for applications in chemical sensing and microscopy," Appl. Phys. B: Lasers and Optics 92(3), 367-378 (2008).
    [CrossRef]
  3. S. Schlachter, A. D. Elder, A. Esposito, G. S. Kaminski, J. H. Frank, L. K. van Geest, and C. F. Kaminski, "mhFLIM: Resolution of heterogeneous fluorescence decays in widefield lifetime microscopy," Opt. Express 17(3), 1557-1570 (2009).
    [CrossRef]
  4. A. Esposito, T. Tiffert, J. M. A. Mauritz, S. Schlachter, L. H. Bannister, C. F. Kaminski, and V. L. Lew, "FRET Imaging of Hemoglobin Concentration in Plasmodium falciparum-Infected Red Cells," PLoS ONE 3(11), e3780 (2008).
    [CrossRef]
  5. T. Zimmermann, J. Rietdorf, and R. Pepperkok, "Spectral imaging and its applications in live cell microscopy," FEBS Lett. 546(1), 87-92 (2003).
    [CrossRef]
  6. A. D. Elder, A. Domin, G. S. Kaminski Schierle, C. Lindon, J. Pines, A. Esposito, and C. F. Kaminski, "A quantitative protocol for dynamic measurements of protein interactions by Frster resonance energy transfersensitized fluorescence emission," J. Roy. Soc. Interface 6(1), S59-S81 (2009).
    [CrossRef]
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    [CrossRef]
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    [CrossRef] [PubMed]
  12. G.-J. Kremers, E. B. van Munster, J. Goedhart, and T. W. J. Gadella, "Quantitative Lifetime Unmixing of Multiexponentially Decaying Fluorophores Using Single-Frequency Fluorescence Lifetime Imaging Microscopy," Biophys. J. 95(1), 378-389 (2008).
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    [CrossRef]
  15. F. S. Wouters and A. Esposito, "Quantitative analysis of fluorescence lifetime imaging made easy," HFSP J. 2(1), 7-11 (2008).
    [CrossRef]
  16. M. A. Digman, V. R. Caiolfa, M. Zamai, and E. Gratton, "The phasor approach to fluorescence lifetime imaging analysis," Biophys. J. 94, L14-L16 (2007).
    [CrossRef] [PubMed]
  17. A. D. Elder, S. C. Schlachter, and C. F. Kaminski, "Theoretical investigation of the photon efficiency in frequency-domain FLIM," J. Opt. Soc. Am. A 25, 452-462 (2008).
    [CrossRef]
  18. D. M. Owen, E. Auksorius, H. B. Manning, C. B. Talbot, P. A. A. de Beule, C. Dunsby, M. A. A. Neil, and P. M. W. French, "Excitation-resolved hyperspectral fluorescence lifetime imaging using a UV-extended supercontinuum source," Opt. Lett. 32(23), 3408-3410 (2007).
    [CrossRef]
  19. P. Serge, J. R. P. Michael, K. Daekeun, K. Ki Hean, J. S. Tsu-Te, and T. C. S. Peter, "Frequency domain lifetime and spectral imaging microscopy," Microsc. Res. Tech. 69(11), 861-874 (2006).
  20. Q. S. Hanley, D. J. Arndt-Jovin, and T. M. Jovin, "Spectrally Resolved Fluorescence Lifetime Imaging Microscopy," Appl. Spectrosc. 56, 155-166 (2002).
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  21. J. A. Levitt, D. R. Matthews, S. M. Ameer-Beg, and K. Suhling, "Fluorescence lifetime and polarization-resolved imaging in cell biology," Curr. Opin. Biotechnol. 20(1), 28-36 (2009).
    [CrossRef]
  22. N. P. Galletly, J. McGinty, C. Dunsby, F. Teixeira, J. Requejo-Isidro, I. Munro, D. S. Elson, M. A. A. Neil, A. C. Chu, P. M. French, and G. W. Stamp, "Fluorescence lifetime imaging distinguishes basal cell carcinoma from surrounding uninvolved skin." British Journal of Dermatology 159, 152-161 (2008).
    [CrossRef] [PubMed]
  23. A. D. Elder, S.M. Matthews, J. Swartling, K. Yunus, J. H. Frank, C. M. Brennan, A. C. Fisher, and C. F. Kaminski, "The application of frequency-domain fluorescence lifetime imaging microscopy as a quantitative analytical tool for microfluidic devices," Opt. Express 14(12), 5456-5467 (2006).
    [CrossRef]
  24. S. M. Matthews, A. D. Elder, K. Yunus, C. F. Kaminski, C. M. Brennan, and A. C. Fisher, "Quantitative kinetic analysis in a microfluidic device using frequency-domain fluorescence lifetime imaging," Anal. Chem. 79(11), 4101-4109 (2007).
    [CrossRef]
  25. X. Dai, Z. Yue, M. E. Eccleston, J. Swartling, N. K. H. Slater, and C. F. Kaminski, "Fluorescence intensity and lifetime imaging of free and micellar-encapsulated doxorubicin in living cells," Nanomed. Nanotechnol. Bio. Med. 4(1), 49-56 (2008).
    [CrossRef]
  26. X. W. Dai, M. E. Eccleston, Z. L. Yue, N. K. H. Slater, and C. F. Kaminski, "A spectroscopic study of the self-association and inter-molecular aggregation behaviour of pH-responsive poly(L-lysine iso-phthalamide)," Polymer 47(8), 2689-2698 (2006).
    [CrossRef]

2009 (4)

A. D. Elder, A. Domin, G. S. Kaminski Schierle, C. Lindon, J. Pines, A. Esposito, and C. F. Kaminski, "A quantitative protocol for dynamic measurements of protein interactions by Frster resonance energy transfersensitized fluorescence emission," J. Roy. Soc. Interface 6(1), S59-S81 (2009).
[CrossRef]

R. A. Neher,M. Mitkovski, F. Kirchhoff, E. Neher, F. J. Theis, and A. Zeug, "Blind Source Separation Techniques for the Decomposition of Multiply Labeled Fluorescence Images," Biophys. J. 96(9), 3791-3800 (2009).
[CrossRef]

J. A. Levitt, D. R. Matthews, S. M. Ameer-Beg, and K. Suhling, "Fluorescence lifetime and polarization-resolved imaging in cell biology," Curr. Opin. Biotechnol. 20(1), 28-36 (2009).
[CrossRef]

S. Schlachter, A. D. Elder, A. Esposito, G. S. Kaminski, J. H. Frank, L. K. van Geest, and C. F. Kaminski, "mhFLIM: Resolution of heterogeneous fluorescence decays in widefield lifetime microscopy," Opt. Express 17(3), 1557-1570 (2009).
[CrossRef]

2008 (7)

A. D. Elder, S. C. Schlachter, and C. F. Kaminski, "Theoretical investigation of the photon efficiency in frequency-domain FLIM," J. Opt. Soc. Am. A 25, 452-462 (2008).
[CrossRef]

N. P. Galletly, J. McGinty, C. Dunsby, F. Teixeira, J. Requejo-Isidro, I. Munro, D. S. Elson, M. A. A. Neil, A. C. Chu, P. M. French, and G. W. Stamp, "Fluorescence lifetime imaging distinguishes basal cell carcinoma from surrounding uninvolved skin." British Journal of Dermatology 159, 152-161 (2008).
[CrossRef] [PubMed]

G.-J. Kremers, E. B. van Munster, J. Goedhart, and T. W. J. Gadella, "Quantitative Lifetime Unmixing of Multiexponentially Decaying Fluorophores Using Single-Frequency Fluorescence Lifetime Imaging Microscopy," Biophys. J. 95(1), 378-389 (2008).
[CrossRef]

A. Esposito, T. Tiffert, J. M. A. Mauritz, S. Schlachter, L. H. Bannister, C. F. Kaminski, and V. L. Lew, "FRET Imaging of Hemoglobin Concentration in Plasmodium falciparum-Infected Red Cells," PLoS ONE 3(11), e3780 (2008).
[CrossRef]

F. S. Wouters and A. Esposito, "Quantitative analysis of fluorescence lifetime imaging made easy," HFSP J. 2(1), 7-11 (2008).
[CrossRef]

C. F. Kaminski, R. S. Watt, A. D. Elder, J. H. Frank, and J. Hult, "Supercontinuum radiation for applications in chemical sensing and microscopy," Appl. Phys. B: Lasers and Optics 92(3), 367-378 (2008).
[CrossRef]

X. Dai, Z. Yue, M. E. Eccleston, J. Swartling, N. K. H. Slater, and C. F. Kaminski, "Fluorescence intensity and lifetime imaging of free and micellar-encapsulated doxorubicin in living cells," Nanomed. Nanotechnol. Bio. Med. 4(1), 49-56 (2008).
[CrossRef]

2007 (4)

M. A. Digman, V. R. Caiolfa, M. Zamai, and E. Gratton, "The phasor approach to fluorescence lifetime imaging analysis," Biophys. J. 94, L14-L16 (2007).
[CrossRef] [PubMed]

J. H. Frank, A. D. Elder, J. Swartling, A. R. Venkitaraman, A. D. Jeyasekharan, and C. F. Kaminski, "A white light confocal microscope for spectrally resolved multidimensional imaging," J Microsc. 227, 203-215 (2007).
[CrossRef] [PubMed]

S. M. Matthews, A. D. Elder, K. Yunus, C. F. Kaminski, C. M. Brennan, and A. C. Fisher, "Quantitative kinetic analysis in a microfluidic device using frequency-domain fluorescence lifetime imaging," Anal. Chem. 79(11), 4101-4109 (2007).
[CrossRef]

D. M. Owen, E. Auksorius, H. B. Manning, C. B. Talbot, P. A. A. de Beule, C. Dunsby, M. A. A. Neil, and P. M. W. French, "Excitation-resolved hyperspectral fluorescence lifetime imaging using a UV-extended supercontinuum source," Opt. Lett. 32(23), 3408-3410 (2007).
[CrossRef]

2006 (3)

P. Serge, J. R. P. Michael, K. Daekeun, K. Ki Hean, J. S. Tsu-Te, and T. C. S. Peter, "Frequency domain lifetime and spectral imaging microscopy," Microsc. Res. Tech. 69(11), 861-874 (2006).

X. W. Dai, M. E. Eccleston, Z. L. Yue, N. K. H. Slater, and C. F. Kaminski, "A spectroscopic study of the self-association and inter-molecular aggregation behaviour of pH-responsive poly(L-lysine iso-phthalamide)," Polymer 47(8), 2689-2698 (2006).
[CrossRef]

A. D. Elder, S.M. Matthews, J. Swartling, K. Yunus, J. H. Frank, C. M. Brennan, A. C. Fisher, and C. F. Kaminski, "The application of frequency-domain fluorescence lifetime imaging microscopy as a quantitative analytical tool for microfluidic devices," Opt. Express 14(12), 5456-5467 (2006).
[CrossRef]

2005 (1)

D. Chorvat, J. Kirchnerova, M. Cagalinec, J. Smolka, A. Mateasik, and A. Chorvatova, "Spectral Unmixing of Flavin Autofluorescence Components in Cardiac Myocytes," Biophys. J. 89(6), L55-L57 (2005).
[CrossRef]

2004 (2)

H. Shirakawa and S. Miyazaki, "Blind Spectral Decomposition of Single-Cell Fluorescence by Parallel Factor Analysis," Biophys. J. 86(3), 1739-1752 (2004).
[CrossRef]

A. H. A. Clayton, Q. S. Hanley, and P. J. Verveer, "Graphical representation and multicomponent analysis of single-frequency fluorescence lifetime imaging microscopy data," J. Microscopy 213(1), 1-5 (2004).
[CrossRef]

2003 (1)

T. Zimmermann, J. Rietdorf, and R. Pepperkok, "Spectral imaging and its applications in live cell microscopy," FEBS Lett. 546(1), 87-92 (2003).
[CrossRef]

2002 (1)

2000 (1)

P. Verveer, A. Squire, and P. Bastiaens, "Global Analysis of fluorescence lifetime imaging microscopy data," Biophys. J. 78, 2127-2137 (2000).
[CrossRef] [PubMed]

Ameer-Beg, S. M.

J. A. Levitt, D. R. Matthews, S. M. Ameer-Beg, and K. Suhling, "Fluorescence lifetime and polarization-resolved imaging in cell biology," Curr. Opin. Biotechnol. 20(1), 28-36 (2009).
[CrossRef]

Arndt-Jovin, D. J.

Auksorius, E.

Bannister, L. H.

A. Esposito, T. Tiffert, J. M. A. Mauritz, S. Schlachter, L. H. Bannister, C. F. Kaminski, and V. L. Lew, "FRET Imaging of Hemoglobin Concentration in Plasmodium falciparum-Infected Red Cells," PLoS ONE 3(11), e3780 (2008).
[CrossRef]

Bastiaens, P.

P. Verveer, A. Squire, and P. Bastiaens, "Global Analysis of fluorescence lifetime imaging microscopy data," Biophys. J. 78, 2127-2137 (2000).
[CrossRef] [PubMed]

Brennan, C. M.

S. M. Matthews, A. D. Elder, K. Yunus, C. F. Kaminski, C. M. Brennan, and A. C. Fisher, "Quantitative kinetic analysis in a microfluidic device using frequency-domain fluorescence lifetime imaging," Anal. Chem. 79(11), 4101-4109 (2007).
[CrossRef]

A. D. Elder, S.M. Matthews, J. Swartling, K. Yunus, J. H. Frank, C. M. Brennan, A. C. Fisher, and C. F. Kaminski, "The application of frequency-domain fluorescence lifetime imaging microscopy as a quantitative analytical tool for microfluidic devices," Opt. Express 14(12), 5456-5467 (2006).
[CrossRef]

Cagalinec, M.

D. Chorvat, J. Kirchnerova, M. Cagalinec, J. Smolka, A. Mateasik, and A. Chorvatova, "Spectral Unmixing of Flavin Autofluorescence Components in Cardiac Myocytes," Biophys. J. 89(6), L55-L57 (2005).
[CrossRef]

Caiolfa, V. R.

M. A. Digman, V. R. Caiolfa, M. Zamai, and E. Gratton, "The phasor approach to fluorescence lifetime imaging analysis," Biophys. J. 94, L14-L16 (2007).
[CrossRef] [PubMed]

Chorvat, D.

D. Chorvat, J. Kirchnerova, M. Cagalinec, J. Smolka, A. Mateasik, and A. Chorvatova, "Spectral Unmixing of Flavin Autofluorescence Components in Cardiac Myocytes," Biophys. J. 89(6), L55-L57 (2005).
[CrossRef]

Chorvatova, A.

D. Chorvat, J. Kirchnerova, M. Cagalinec, J. Smolka, A. Mateasik, and A. Chorvatova, "Spectral Unmixing of Flavin Autofluorescence Components in Cardiac Myocytes," Biophys. J. 89(6), L55-L57 (2005).
[CrossRef]

Chu, A. C.

N. P. Galletly, J. McGinty, C. Dunsby, F. Teixeira, J. Requejo-Isidro, I. Munro, D. S. Elson, M. A. A. Neil, A. C. Chu, P. M. French, and G. W. Stamp, "Fluorescence lifetime imaging distinguishes basal cell carcinoma from surrounding uninvolved skin." British Journal of Dermatology 159, 152-161 (2008).
[CrossRef] [PubMed]

Clayton, A. H. A.

A. H. A. Clayton, Q. S. Hanley, and P. J. Verveer, "Graphical representation and multicomponent analysis of single-frequency fluorescence lifetime imaging microscopy data," J. Microscopy 213(1), 1-5 (2004).
[CrossRef]

Daekeun, K.

P. Serge, J. R. P. Michael, K. Daekeun, K. Ki Hean, J. S. Tsu-Te, and T. C. S. Peter, "Frequency domain lifetime and spectral imaging microscopy," Microsc. Res. Tech. 69(11), 861-874 (2006).

Dai, X.

X. Dai, Z. Yue, M. E. Eccleston, J. Swartling, N. K. H. Slater, and C. F. Kaminski, "Fluorescence intensity and lifetime imaging of free and micellar-encapsulated doxorubicin in living cells," Nanomed. Nanotechnol. Bio. Med. 4(1), 49-56 (2008).
[CrossRef]

Dai, X. W.

X. W. Dai, M. E. Eccleston, Z. L. Yue, N. K. H. Slater, and C. F. Kaminski, "A spectroscopic study of the self-association and inter-molecular aggregation behaviour of pH-responsive poly(L-lysine iso-phthalamide)," Polymer 47(8), 2689-2698 (2006).
[CrossRef]

de Beule, P. A. A.

Digman, M. A.

M. A. Digman, V. R. Caiolfa, M. Zamai, and E. Gratton, "The phasor approach to fluorescence lifetime imaging analysis," Biophys. J. 94, L14-L16 (2007).
[CrossRef] [PubMed]

Domin, A.

A. D. Elder, A. Domin, G. S. Kaminski Schierle, C. Lindon, J. Pines, A. Esposito, and C. F. Kaminski, "A quantitative protocol for dynamic measurements of protein interactions by Frster resonance energy transfersensitized fluorescence emission," J. Roy. Soc. Interface 6(1), S59-S81 (2009).
[CrossRef]

Dunsby, C.

N. P. Galletly, J. McGinty, C. Dunsby, F. Teixeira, J. Requejo-Isidro, I. Munro, D. S. Elson, M. A. A. Neil, A. C. Chu, P. M. French, and G. W. Stamp, "Fluorescence lifetime imaging distinguishes basal cell carcinoma from surrounding uninvolved skin." British Journal of Dermatology 159, 152-161 (2008).
[CrossRef] [PubMed]

D. M. Owen, E. Auksorius, H. B. Manning, C. B. Talbot, P. A. A. de Beule, C. Dunsby, M. A. A. Neil, and P. M. W. French, "Excitation-resolved hyperspectral fluorescence lifetime imaging using a UV-extended supercontinuum source," Opt. Lett. 32(23), 3408-3410 (2007).
[CrossRef]

Eccleston, M. E.

X. Dai, Z. Yue, M. E. Eccleston, J. Swartling, N. K. H. Slater, and C. F. Kaminski, "Fluorescence intensity and lifetime imaging of free and micellar-encapsulated doxorubicin in living cells," Nanomed. Nanotechnol. Bio. Med. 4(1), 49-56 (2008).
[CrossRef]

X. W. Dai, M. E. Eccleston, Z. L. Yue, N. K. H. Slater, and C. F. Kaminski, "A spectroscopic study of the self-association and inter-molecular aggregation behaviour of pH-responsive poly(L-lysine iso-phthalamide)," Polymer 47(8), 2689-2698 (2006).
[CrossRef]

Elder, A. D.

S. Schlachter, A. D. Elder, A. Esposito, G. S. Kaminski, J. H. Frank, L. K. van Geest, and C. F. Kaminski, "mhFLIM: Resolution of heterogeneous fluorescence decays in widefield lifetime microscopy," Opt. Express 17(3), 1557-1570 (2009).
[CrossRef]

A. D. Elder, A. Domin, G. S. Kaminski Schierle, C. Lindon, J. Pines, A. Esposito, and C. F. Kaminski, "A quantitative protocol for dynamic measurements of protein interactions by Frster resonance energy transfersensitized fluorescence emission," J. Roy. Soc. Interface 6(1), S59-S81 (2009).
[CrossRef]

A. D. Elder, S. C. Schlachter, and C. F. Kaminski, "Theoretical investigation of the photon efficiency in frequency-domain FLIM," J. Opt. Soc. Am. A 25, 452-462 (2008).
[CrossRef]

C. F. Kaminski, R. S. Watt, A. D. Elder, J. H. Frank, and J. Hult, "Supercontinuum radiation for applications in chemical sensing and microscopy," Appl. Phys. B: Lasers and Optics 92(3), 367-378 (2008).
[CrossRef]

J. H. Frank, A. D. Elder, J. Swartling, A. R. Venkitaraman, A. D. Jeyasekharan, and C. F. Kaminski, "A white light confocal microscope for spectrally resolved multidimensional imaging," J Microsc. 227, 203-215 (2007).
[CrossRef] [PubMed]

S. M. Matthews, A. D. Elder, K. Yunus, C. F. Kaminski, C. M. Brennan, and A. C. Fisher, "Quantitative kinetic analysis in a microfluidic device using frequency-domain fluorescence lifetime imaging," Anal. Chem. 79(11), 4101-4109 (2007).
[CrossRef]

A. D. Elder, S.M. Matthews, J. Swartling, K. Yunus, J. H. Frank, C. M. Brennan, A. C. Fisher, and C. F. Kaminski, "The application of frequency-domain fluorescence lifetime imaging microscopy as a quantitative analytical tool for microfluidic devices," Opt. Express 14(12), 5456-5467 (2006).
[CrossRef]

Elson, D. S.

N. P. Galletly, J. McGinty, C. Dunsby, F. Teixeira, J. Requejo-Isidro, I. Munro, D. S. Elson, M. A. A. Neil, A. C. Chu, P. M. French, and G. W. Stamp, "Fluorescence lifetime imaging distinguishes basal cell carcinoma from surrounding uninvolved skin." British Journal of Dermatology 159, 152-161 (2008).
[CrossRef] [PubMed]

Esposito, A.

A. D. Elder, A. Domin, G. S. Kaminski Schierle, C. Lindon, J. Pines, A. Esposito, and C. F. Kaminski, "A quantitative protocol for dynamic measurements of protein interactions by Frster resonance energy transfersensitized fluorescence emission," J. Roy. Soc. Interface 6(1), S59-S81 (2009).
[CrossRef]

S. Schlachter, A. D. Elder, A. Esposito, G. S. Kaminski, J. H. Frank, L. K. van Geest, and C. F. Kaminski, "mhFLIM: Resolution of heterogeneous fluorescence decays in widefield lifetime microscopy," Opt. Express 17(3), 1557-1570 (2009).
[CrossRef]

A. Esposito, T. Tiffert, J. M. A. Mauritz, S. Schlachter, L. H. Bannister, C. F. Kaminski, and V. L. Lew, "FRET Imaging of Hemoglobin Concentration in Plasmodium falciparum-Infected Red Cells," PLoS ONE 3(11), e3780 (2008).
[CrossRef]

F. S. Wouters and A. Esposito, "Quantitative analysis of fluorescence lifetime imaging made easy," HFSP J. 2(1), 7-11 (2008).
[CrossRef]

Fisher, A. C.

S. M. Matthews, A. D. Elder, K. Yunus, C. F. Kaminski, C. M. Brennan, and A. C. Fisher, "Quantitative kinetic analysis in a microfluidic device using frequency-domain fluorescence lifetime imaging," Anal. Chem. 79(11), 4101-4109 (2007).
[CrossRef]

A. D. Elder, S.M. Matthews, J. Swartling, K. Yunus, J. H. Frank, C. M. Brennan, A. C. Fisher, and C. F. Kaminski, "The application of frequency-domain fluorescence lifetime imaging microscopy as a quantitative analytical tool for microfluidic devices," Opt. Express 14(12), 5456-5467 (2006).
[CrossRef]

Frank, J. H.

S. Schlachter, A. D. Elder, A. Esposito, G. S. Kaminski, J. H. Frank, L. K. van Geest, and C. F. Kaminski, "mhFLIM: Resolution of heterogeneous fluorescence decays in widefield lifetime microscopy," Opt. Express 17(3), 1557-1570 (2009).
[CrossRef]

C. F. Kaminski, R. S. Watt, A. D. Elder, J. H. Frank, and J. Hult, "Supercontinuum radiation for applications in chemical sensing and microscopy," Appl. Phys. B: Lasers and Optics 92(3), 367-378 (2008).
[CrossRef]

J. H. Frank, A. D. Elder, J. Swartling, A. R. Venkitaraman, A. D. Jeyasekharan, and C. F. Kaminski, "A white light confocal microscope for spectrally resolved multidimensional imaging," J Microsc. 227, 203-215 (2007).
[CrossRef] [PubMed]

A. D. Elder, S.M. Matthews, J. Swartling, K. Yunus, J. H. Frank, C. M. Brennan, A. C. Fisher, and C. F. Kaminski, "The application of frequency-domain fluorescence lifetime imaging microscopy as a quantitative analytical tool for microfluidic devices," Opt. Express 14(12), 5456-5467 (2006).
[CrossRef]

French, P. M.

N. P. Galletly, J. McGinty, C. Dunsby, F. Teixeira, J. Requejo-Isidro, I. Munro, D. S. Elson, M. A. A. Neil, A. C. Chu, P. M. French, and G. W. Stamp, "Fluorescence lifetime imaging distinguishes basal cell carcinoma from surrounding uninvolved skin." British Journal of Dermatology 159, 152-161 (2008).
[CrossRef] [PubMed]

French, P. M. W.

Gadella, T. W. J.

G.-J. Kremers, E. B. van Munster, J. Goedhart, and T. W. J. Gadella, "Quantitative Lifetime Unmixing of Multiexponentially Decaying Fluorophores Using Single-Frequency Fluorescence Lifetime Imaging Microscopy," Biophys. J. 95(1), 378-389 (2008).
[CrossRef]

Galletly, N. P.

N. P. Galletly, J. McGinty, C. Dunsby, F. Teixeira, J. Requejo-Isidro, I. Munro, D. S. Elson, M. A. A. Neil, A. C. Chu, P. M. French, and G. W. Stamp, "Fluorescence lifetime imaging distinguishes basal cell carcinoma from surrounding uninvolved skin." British Journal of Dermatology 159, 152-161 (2008).
[CrossRef] [PubMed]

Goedhart, J.

G.-J. Kremers, E. B. van Munster, J. Goedhart, and T. W. J. Gadella, "Quantitative Lifetime Unmixing of Multiexponentially Decaying Fluorophores Using Single-Frequency Fluorescence Lifetime Imaging Microscopy," Biophys. J. 95(1), 378-389 (2008).
[CrossRef]

Gratton, E.

M. A. Digman, V. R. Caiolfa, M. Zamai, and E. Gratton, "The phasor approach to fluorescence lifetime imaging analysis," Biophys. J. 94, L14-L16 (2007).
[CrossRef] [PubMed]

Hanley, Q. S.

A. H. A. Clayton, Q. S. Hanley, and P. J. Verveer, "Graphical representation and multicomponent analysis of single-frequency fluorescence lifetime imaging microscopy data," J. Microscopy 213(1), 1-5 (2004).
[CrossRef]

Q. S. Hanley, D. J. Arndt-Jovin, and T. M. Jovin, "Spectrally Resolved Fluorescence Lifetime Imaging Microscopy," Appl. Spectrosc. 56, 155-166 (2002).
[CrossRef]

Hult, J.

C. F. Kaminski, R. S. Watt, A. D. Elder, J. H. Frank, and J. Hult, "Supercontinuum radiation for applications in chemical sensing and microscopy," Appl. Phys. B: Lasers and Optics 92(3), 367-378 (2008).
[CrossRef]

Jeyasekharan, A. D.

J. H. Frank, A. D. Elder, J. Swartling, A. R. Venkitaraman, A. D. Jeyasekharan, and C. F. Kaminski, "A white light confocal microscope for spectrally resolved multidimensional imaging," J Microsc. 227, 203-215 (2007).
[CrossRef] [PubMed]

Jovin, T. M.

Kaminski, C. F.

S. Schlachter, A. D. Elder, A. Esposito, G. S. Kaminski, J. H. Frank, L. K. van Geest, and C. F. Kaminski, "mhFLIM: Resolution of heterogeneous fluorescence decays in widefield lifetime microscopy," Opt. Express 17(3), 1557-1570 (2009).
[CrossRef]

A. D. Elder, A. Domin, G. S. Kaminski Schierle, C. Lindon, J. Pines, A. Esposito, and C. F. Kaminski, "A quantitative protocol for dynamic measurements of protein interactions by Frster resonance energy transfersensitized fluorescence emission," J. Roy. Soc. Interface 6(1), S59-S81 (2009).
[CrossRef]

A. Esposito, T. Tiffert, J. M. A. Mauritz, S. Schlachter, L. H. Bannister, C. F. Kaminski, and V. L. Lew, "FRET Imaging of Hemoglobin Concentration in Plasmodium falciparum-Infected Red Cells," PLoS ONE 3(11), e3780 (2008).
[CrossRef]

X. Dai, Z. Yue, M. E. Eccleston, J. Swartling, N. K. H. Slater, and C. F. Kaminski, "Fluorescence intensity and lifetime imaging of free and micellar-encapsulated doxorubicin in living cells," Nanomed. Nanotechnol. Bio. Med. 4(1), 49-56 (2008).
[CrossRef]

C. F. Kaminski, R. S. Watt, A. D. Elder, J. H. Frank, and J. Hult, "Supercontinuum radiation for applications in chemical sensing and microscopy," Appl. Phys. B: Lasers and Optics 92(3), 367-378 (2008).
[CrossRef]

A. D. Elder, S. C. Schlachter, and C. F. Kaminski, "Theoretical investigation of the photon efficiency in frequency-domain FLIM," J. Opt. Soc. Am. A 25, 452-462 (2008).
[CrossRef]

S. M. Matthews, A. D. Elder, K. Yunus, C. F. Kaminski, C. M. Brennan, and A. C. Fisher, "Quantitative kinetic analysis in a microfluidic device using frequency-domain fluorescence lifetime imaging," Anal. Chem. 79(11), 4101-4109 (2007).
[CrossRef]

J. H. Frank, A. D. Elder, J. Swartling, A. R. Venkitaraman, A. D. Jeyasekharan, and C. F. Kaminski, "A white light confocal microscope for spectrally resolved multidimensional imaging," J Microsc. 227, 203-215 (2007).
[CrossRef] [PubMed]

A. D. Elder, S.M. Matthews, J. Swartling, K. Yunus, J. H. Frank, C. M. Brennan, A. C. Fisher, and C. F. Kaminski, "The application of frequency-domain fluorescence lifetime imaging microscopy as a quantitative analytical tool for microfluidic devices," Opt. Express 14(12), 5456-5467 (2006).
[CrossRef]

X. W. Dai, M. E. Eccleston, Z. L. Yue, N. K. H. Slater, and C. F. Kaminski, "A spectroscopic study of the self-association and inter-molecular aggregation behaviour of pH-responsive poly(L-lysine iso-phthalamide)," Polymer 47(8), 2689-2698 (2006).
[CrossRef]

Kaminski, G. S.

Kaminski Schierle, G. S.

A. D. Elder, A. Domin, G. S. Kaminski Schierle, C. Lindon, J. Pines, A. Esposito, and C. F. Kaminski, "A quantitative protocol for dynamic measurements of protein interactions by Frster resonance energy transfersensitized fluorescence emission," J. Roy. Soc. Interface 6(1), S59-S81 (2009).
[CrossRef]

Ki Hean, K.

P. Serge, J. R. P. Michael, K. Daekeun, K. Ki Hean, J. S. Tsu-Te, and T. C. S. Peter, "Frequency domain lifetime and spectral imaging microscopy," Microsc. Res. Tech. 69(11), 861-874 (2006).

Kirchhoff, F.

R. A. Neher,M. Mitkovski, F. Kirchhoff, E. Neher, F. J. Theis, and A. Zeug, "Blind Source Separation Techniques for the Decomposition of Multiply Labeled Fluorescence Images," Biophys. J. 96(9), 3791-3800 (2009).
[CrossRef]

Kirchnerova, J.

D. Chorvat, J. Kirchnerova, M. Cagalinec, J. Smolka, A. Mateasik, and A. Chorvatova, "Spectral Unmixing of Flavin Autofluorescence Components in Cardiac Myocytes," Biophys. J. 89(6), L55-L57 (2005).
[CrossRef]

Kremers, G.-J.

G.-J. Kremers, E. B. van Munster, J. Goedhart, and T. W. J. Gadella, "Quantitative Lifetime Unmixing of Multiexponentially Decaying Fluorophores Using Single-Frequency Fluorescence Lifetime Imaging Microscopy," Biophys. J. 95(1), 378-389 (2008).
[CrossRef]

Levitt, J. A.

J. A. Levitt, D. R. Matthews, S. M. Ameer-Beg, and K. Suhling, "Fluorescence lifetime and polarization-resolved imaging in cell biology," Curr. Opin. Biotechnol. 20(1), 28-36 (2009).
[CrossRef]

Lew, V. L.

A. Esposito, T. Tiffert, J. M. A. Mauritz, S. Schlachter, L. H. Bannister, C. F. Kaminski, and V. L. Lew, "FRET Imaging of Hemoglobin Concentration in Plasmodium falciparum-Infected Red Cells," PLoS ONE 3(11), e3780 (2008).
[CrossRef]

Lindon, C.

A. D. Elder, A. Domin, G. S. Kaminski Schierle, C. Lindon, J. Pines, A. Esposito, and C. F. Kaminski, "A quantitative protocol for dynamic measurements of protein interactions by Frster resonance energy transfersensitized fluorescence emission," J. Roy. Soc. Interface 6(1), S59-S81 (2009).
[CrossRef]

Manning, H. B.

Mateasik, A.

D. Chorvat, J. Kirchnerova, M. Cagalinec, J. Smolka, A. Mateasik, and A. Chorvatova, "Spectral Unmixing of Flavin Autofluorescence Components in Cardiac Myocytes," Biophys. J. 89(6), L55-L57 (2005).
[CrossRef]

Matthews, D. R.

J. A. Levitt, D. R. Matthews, S. M. Ameer-Beg, and K. Suhling, "Fluorescence lifetime and polarization-resolved imaging in cell biology," Curr. Opin. Biotechnol. 20(1), 28-36 (2009).
[CrossRef]

Matthews, S. M.

S. M. Matthews, A. D. Elder, K. Yunus, C. F. Kaminski, C. M. Brennan, and A. C. Fisher, "Quantitative kinetic analysis in a microfluidic device using frequency-domain fluorescence lifetime imaging," Anal. Chem. 79(11), 4101-4109 (2007).
[CrossRef]

Matthews, S.M.

Mauritz, J. M. A.

A. Esposito, T. Tiffert, J. M. A. Mauritz, S. Schlachter, L. H. Bannister, C. F. Kaminski, and V. L. Lew, "FRET Imaging of Hemoglobin Concentration in Plasmodium falciparum-Infected Red Cells," PLoS ONE 3(11), e3780 (2008).
[CrossRef]

McGinty, J.

N. P. Galletly, J. McGinty, C. Dunsby, F. Teixeira, J. Requejo-Isidro, I. Munro, D. S. Elson, M. A. A. Neil, A. C. Chu, P. M. French, and G. W. Stamp, "Fluorescence lifetime imaging distinguishes basal cell carcinoma from surrounding uninvolved skin." British Journal of Dermatology 159, 152-161 (2008).
[CrossRef] [PubMed]

Michael, J. R. P.

P. Serge, J. R. P. Michael, K. Daekeun, K. Ki Hean, J. S. Tsu-Te, and T. C. S. Peter, "Frequency domain lifetime and spectral imaging microscopy," Microsc. Res. Tech. 69(11), 861-874 (2006).

Mitkovski, M.

R. A. Neher,M. Mitkovski, F. Kirchhoff, E. Neher, F. J. Theis, and A. Zeug, "Blind Source Separation Techniques for the Decomposition of Multiply Labeled Fluorescence Images," Biophys. J. 96(9), 3791-3800 (2009).
[CrossRef]

Miyazaki, S.

H. Shirakawa and S. Miyazaki, "Blind Spectral Decomposition of Single-Cell Fluorescence by Parallel Factor Analysis," Biophys. J. 86(3), 1739-1752 (2004).
[CrossRef]

Munro, I.

N. P. Galletly, J. McGinty, C. Dunsby, F. Teixeira, J. Requejo-Isidro, I. Munro, D. S. Elson, M. A. A. Neil, A. C. Chu, P. M. French, and G. W. Stamp, "Fluorescence lifetime imaging distinguishes basal cell carcinoma from surrounding uninvolved skin." British Journal of Dermatology 159, 152-161 (2008).
[CrossRef] [PubMed]

Neher, E.

R. A. Neher,M. Mitkovski, F. Kirchhoff, E. Neher, F. J. Theis, and A. Zeug, "Blind Source Separation Techniques for the Decomposition of Multiply Labeled Fluorescence Images," Biophys. J. 96(9), 3791-3800 (2009).
[CrossRef]

Neher, R. A.

R. A. Neher,M. Mitkovski, F. Kirchhoff, E. Neher, F. J. Theis, and A. Zeug, "Blind Source Separation Techniques for the Decomposition of Multiply Labeled Fluorescence Images," Biophys. J. 96(9), 3791-3800 (2009).
[CrossRef]

Neil, M. A. A.

N. P. Galletly, J. McGinty, C. Dunsby, F. Teixeira, J. Requejo-Isidro, I. Munro, D. S. Elson, M. A. A. Neil, A. C. Chu, P. M. French, and G. W. Stamp, "Fluorescence lifetime imaging distinguishes basal cell carcinoma from surrounding uninvolved skin." British Journal of Dermatology 159, 152-161 (2008).
[CrossRef] [PubMed]

D. M. Owen, E. Auksorius, H. B. Manning, C. B. Talbot, P. A. A. de Beule, C. Dunsby, M. A. A. Neil, and P. M. W. French, "Excitation-resolved hyperspectral fluorescence lifetime imaging using a UV-extended supercontinuum source," Opt. Lett. 32(23), 3408-3410 (2007).
[CrossRef]

Owen, D. M.

Pepperkok, R.

T. Zimmermann, J. Rietdorf, and R. Pepperkok, "Spectral imaging and its applications in live cell microscopy," FEBS Lett. 546(1), 87-92 (2003).
[CrossRef]

Peter, T. C. S.

P. Serge, J. R. P. Michael, K. Daekeun, K. Ki Hean, J. S. Tsu-Te, and T. C. S. Peter, "Frequency domain lifetime and spectral imaging microscopy," Microsc. Res. Tech. 69(11), 861-874 (2006).

Pines, J.

A. D. Elder, A. Domin, G. S. Kaminski Schierle, C. Lindon, J. Pines, A. Esposito, and C. F. Kaminski, "A quantitative protocol for dynamic measurements of protein interactions by Frster resonance energy transfersensitized fluorescence emission," J. Roy. Soc. Interface 6(1), S59-S81 (2009).
[CrossRef]

Requejo-Isidro, J.

N. P. Galletly, J. McGinty, C. Dunsby, F. Teixeira, J. Requejo-Isidro, I. Munro, D. S. Elson, M. A. A. Neil, A. C. Chu, P. M. French, and G. W. Stamp, "Fluorescence lifetime imaging distinguishes basal cell carcinoma from surrounding uninvolved skin." British Journal of Dermatology 159, 152-161 (2008).
[CrossRef] [PubMed]

Rietdorf, J.

T. Zimmermann, J. Rietdorf, and R. Pepperkok, "Spectral imaging and its applications in live cell microscopy," FEBS Lett. 546(1), 87-92 (2003).
[CrossRef]

Schlachter, S.

S. Schlachter, A. D. Elder, A. Esposito, G. S. Kaminski, J. H. Frank, L. K. van Geest, and C. F. Kaminski, "mhFLIM: Resolution of heterogeneous fluorescence decays in widefield lifetime microscopy," Opt. Express 17(3), 1557-1570 (2009).
[CrossRef]

A. Esposito, T. Tiffert, J. M. A. Mauritz, S. Schlachter, L. H. Bannister, C. F. Kaminski, and V. L. Lew, "FRET Imaging of Hemoglobin Concentration in Plasmodium falciparum-Infected Red Cells," PLoS ONE 3(11), e3780 (2008).
[CrossRef]

Schlachter, S. C.

Serge, P.

P. Serge, J. R. P. Michael, K. Daekeun, K. Ki Hean, J. S. Tsu-Te, and T. C. S. Peter, "Frequency domain lifetime and spectral imaging microscopy," Microsc. Res. Tech. 69(11), 861-874 (2006).

Shirakawa, H.

H. Shirakawa and S. Miyazaki, "Blind Spectral Decomposition of Single-Cell Fluorescence by Parallel Factor Analysis," Biophys. J. 86(3), 1739-1752 (2004).
[CrossRef]

Slater, N. K. H.

X. Dai, Z. Yue, M. E. Eccleston, J. Swartling, N. K. H. Slater, and C. F. Kaminski, "Fluorescence intensity and lifetime imaging of free and micellar-encapsulated doxorubicin in living cells," Nanomed. Nanotechnol. Bio. Med. 4(1), 49-56 (2008).
[CrossRef]

X. W. Dai, M. E. Eccleston, Z. L. Yue, N. K. H. Slater, and C. F. Kaminski, "A spectroscopic study of the self-association and inter-molecular aggregation behaviour of pH-responsive poly(L-lysine iso-phthalamide)," Polymer 47(8), 2689-2698 (2006).
[CrossRef]

Smolka, J.

D. Chorvat, J. Kirchnerova, M. Cagalinec, J. Smolka, A. Mateasik, and A. Chorvatova, "Spectral Unmixing of Flavin Autofluorescence Components in Cardiac Myocytes," Biophys. J. 89(6), L55-L57 (2005).
[CrossRef]

Squire, A.

P. Verveer, A. Squire, and P. Bastiaens, "Global Analysis of fluorescence lifetime imaging microscopy data," Biophys. J. 78, 2127-2137 (2000).
[CrossRef] [PubMed]

Stamp, G. W.

N. P. Galletly, J. McGinty, C. Dunsby, F. Teixeira, J. Requejo-Isidro, I. Munro, D. S. Elson, M. A. A. Neil, A. C. Chu, P. M. French, and G. W. Stamp, "Fluorescence lifetime imaging distinguishes basal cell carcinoma from surrounding uninvolved skin." British Journal of Dermatology 159, 152-161 (2008).
[CrossRef] [PubMed]

Suhling, K.

J. A. Levitt, D. R. Matthews, S. M. Ameer-Beg, and K. Suhling, "Fluorescence lifetime and polarization-resolved imaging in cell biology," Curr. Opin. Biotechnol. 20(1), 28-36 (2009).
[CrossRef]

Swartling, J.

X. Dai, Z. Yue, M. E. Eccleston, J. Swartling, N. K. H. Slater, and C. F. Kaminski, "Fluorescence intensity and lifetime imaging of free and micellar-encapsulated doxorubicin in living cells," Nanomed. Nanotechnol. Bio. Med. 4(1), 49-56 (2008).
[CrossRef]

J. H. Frank, A. D. Elder, J. Swartling, A. R. Venkitaraman, A. D. Jeyasekharan, and C. F. Kaminski, "A white light confocal microscope for spectrally resolved multidimensional imaging," J Microsc. 227, 203-215 (2007).
[CrossRef] [PubMed]

A. D. Elder, S.M. Matthews, J. Swartling, K. Yunus, J. H. Frank, C. M. Brennan, A. C. Fisher, and C. F. Kaminski, "The application of frequency-domain fluorescence lifetime imaging microscopy as a quantitative analytical tool for microfluidic devices," Opt. Express 14(12), 5456-5467 (2006).
[CrossRef]

Talbot, C. B.

Teixeira, F.

N. P. Galletly, J. McGinty, C. Dunsby, F. Teixeira, J. Requejo-Isidro, I. Munro, D. S. Elson, M. A. A. Neil, A. C. Chu, P. M. French, and G. W. Stamp, "Fluorescence lifetime imaging distinguishes basal cell carcinoma from surrounding uninvolved skin." British Journal of Dermatology 159, 152-161 (2008).
[CrossRef] [PubMed]

Theis, F. J.

R. A. Neher,M. Mitkovski, F. Kirchhoff, E. Neher, F. J. Theis, and A. Zeug, "Blind Source Separation Techniques for the Decomposition of Multiply Labeled Fluorescence Images," Biophys. J. 96(9), 3791-3800 (2009).
[CrossRef]

Tiffert, T.

A. Esposito, T. Tiffert, J. M. A. Mauritz, S. Schlachter, L. H. Bannister, C. F. Kaminski, and V. L. Lew, "FRET Imaging of Hemoglobin Concentration in Plasmodium falciparum-Infected Red Cells," PLoS ONE 3(11), e3780 (2008).
[CrossRef]

Tsu-Te, J. S.

P. Serge, J. R. P. Michael, K. Daekeun, K. Ki Hean, J. S. Tsu-Te, and T. C. S. Peter, "Frequency domain lifetime and spectral imaging microscopy," Microsc. Res. Tech. 69(11), 861-874 (2006).

van Geest, L. K.

van Munster, E. B.

G.-J. Kremers, E. B. van Munster, J. Goedhart, and T. W. J. Gadella, "Quantitative Lifetime Unmixing of Multiexponentially Decaying Fluorophores Using Single-Frequency Fluorescence Lifetime Imaging Microscopy," Biophys. J. 95(1), 378-389 (2008).
[CrossRef]

Venkitaraman, A. R.

J. H. Frank, A. D. Elder, J. Swartling, A. R. Venkitaraman, A. D. Jeyasekharan, and C. F. Kaminski, "A white light confocal microscope for spectrally resolved multidimensional imaging," J Microsc. 227, 203-215 (2007).
[CrossRef] [PubMed]

Verveer, P.

P. Verveer, A. Squire, and P. Bastiaens, "Global Analysis of fluorescence lifetime imaging microscopy data," Biophys. J. 78, 2127-2137 (2000).
[CrossRef] [PubMed]

Verveer, P. J.

A. H. A. Clayton, Q. S. Hanley, and P. J. Verveer, "Graphical representation and multicomponent analysis of single-frequency fluorescence lifetime imaging microscopy data," J. Microscopy 213(1), 1-5 (2004).
[CrossRef]

Watt, R. S.

C. F. Kaminski, R. S. Watt, A. D. Elder, J. H. Frank, and J. Hult, "Supercontinuum radiation for applications in chemical sensing and microscopy," Appl. Phys. B: Lasers and Optics 92(3), 367-378 (2008).
[CrossRef]

Wouters, F. S.

F. S. Wouters and A. Esposito, "Quantitative analysis of fluorescence lifetime imaging made easy," HFSP J. 2(1), 7-11 (2008).
[CrossRef]

Yue, Z.

X. Dai, Z. Yue, M. E. Eccleston, J. Swartling, N. K. H. Slater, and C. F. Kaminski, "Fluorescence intensity and lifetime imaging of free and micellar-encapsulated doxorubicin in living cells," Nanomed. Nanotechnol. Bio. Med. 4(1), 49-56 (2008).
[CrossRef]

Yue, Z. L.

X. W. Dai, M. E. Eccleston, Z. L. Yue, N. K. H. Slater, and C. F. Kaminski, "A spectroscopic study of the self-association and inter-molecular aggregation behaviour of pH-responsive poly(L-lysine iso-phthalamide)," Polymer 47(8), 2689-2698 (2006).
[CrossRef]

Yunus, K.

S. M. Matthews, A. D. Elder, K. Yunus, C. F. Kaminski, C. M. Brennan, and A. C. Fisher, "Quantitative kinetic analysis in a microfluidic device using frequency-domain fluorescence lifetime imaging," Anal. Chem. 79(11), 4101-4109 (2007).
[CrossRef]

A. D. Elder, S.M. Matthews, J. Swartling, K. Yunus, J. H. Frank, C. M. Brennan, A. C. Fisher, and C. F. Kaminski, "The application of frequency-domain fluorescence lifetime imaging microscopy as a quantitative analytical tool for microfluidic devices," Opt. Express 14(12), 5456-5467 (2006).
[CrossRef]

Zamai, M.

M. A. Digman, V. R. Caiolfa, M. Zamai, and E. Gratton, "The phasor approach to fluorescence lifetime imaging analysis," Biophys. J. 94, L14-L16 (2007).
[CrossRef] [PubMed]

Zeug, A.

R. A. Neher,M. Mitkovski, F. Kirchhoff, E. Neher, F. J. Theis, and A. Zeug, "Blind Source Separation Techniques for the Decomposition of Multiply Labeled Fluorescence Images," Biophys. J. 96(9), 3791-3800 (2009).
[CrossRef]

Zimmermann, T.

T. Zimmermann, J. Rietdorf, and R. Pepperkok, "Spectral imaging and its applications in live cell microscopy," FEBS Lett. 546(1), 87-92 (2003).
[CrossRef]

Anal. Chem. (1)

S. M. Matthews, A. D. Elder, K. Yunus, C. F. Kaminski, C. M. Brennan, and A. C. Fisher, "Quantitative kinetic analysis in a microfluidic device using frequency-domain fluorescence lifetime imaging," Anal. Chem. 79(11), 4101-4109 (2007).
[CrossRef]

Appl. Phys. B: Lasers and Optics (1)

C. F. Kaminski, R. S. Watt, A. D. Elder, J. H. Frank, and J. Hult, "Supercontinuum radiation for applications in chemical sensing and microscopy," Appl. Phys. B: Lasers and Optics 92(3), 367-378 (2008).
[CrossRef]

Appl. Spectrosc. (1)

Biophys. J. (6)

M. A. Digman, V. R. Caiolfa, M. Zamai, and E. Gratton, "The phasor approach to fluorescence lifetime imaging analysis," Biophys. J. 94, L14-L16 (2007).
[CrossRef] [PubMed]

H. Shirakawa and S. Miyazaki, "Blind Spectral Decomposition of Single-Cell Fluorescence by Parallel Factor Analysis," Biophys. J. 86(3), 1739-1752 (2004).
[CrossRef]

D. Chorvat, J. Kirchnerova, M. Cagalinec, J. Smolka, A. Mateasik, and A. Chorvatova, "Spectral Unmixing of Flavin Autofluorescence Components in Cardiac Myocytes," Biophys. J. 89(6), L55-L57 (2005).
[CrossRef]

R. A. Neher,M. Mitkovski, F. Kirchhoff, E. Neher, F. J. Theis, and A. Zeug, "Blind Source Separation Techniques for the Decomposition of Multiply Labeled Fluorescence Images," Biophys. J. 96(9), 3791-3800 (2009).
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P. Verveer, A. Squire, and P. Bastiaens, "Global Analysis of fluorescence lifetime imaging microscopy data," Biophys. J. 78, 2127-2137 (2000).
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G.-J. Kremers, E. B. van Munster, J. Goedhart, and T. W. J. Gadella, "Quantitative Lifetime Unmixing of Multiexponentially Decaying Fluorophores Using Single-Frequency Fluorescence Lifetime Imaging Microscopy," Biophys. J. 95(1), 378-389 (2008).
[CrossRef]

British Journal of Dermatology (1)

N. P. Galletly, J. McGinty, C. Dunsby, F. Teixeira, J. Requejo-Isidro, I. Munro, D. S. Elson, M. A. A. Neil, A. C. Chu, P. M. French, and G. W. Stamp, "Fluorescence lifetime imaging distinguishes basal cell carcinoma from surrounding uninvolved skin." British Journal of Dermatology 159, 152-161 (2008).
[CrossRef] [PubMed]

Curr. Opin. Biotechnol. (1)

J. A. Levitt, D. R. Matthews, S. M. Ameer-Beg, and K. Suhling, "Fluorescence lifetime and polarization-resolved imaging in cell biology," Curr. Opin. Biotechnol. 20(1), 28-36 (2009).
[CrossRef]

FEBS Lett. (1)

T. Zimmermann, J. Rietdorf, and R. Pepperkok, "Spectral imaging and its applications in live cell microscopy," FEBS Lett. 546(1), 87-92 (2003).
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HFSP J. (1)

F. S. Wouters and A. Esposito, "Quantitative analysis of fluorescence lifetime imaging made easy," HFSP J. 2(1), 7-11 (2008).
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J Microsc. (1)

J. H. Frank, A. D. Elder, J. Swartling, A. R. Venkitaraman, A. D. Jeyasekharan, and C. F. Kaminski, "A white light confocal microscope for spectrally resolved multidimensional imaging," J Microsc. 227, 203-215 (2007).
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J. Microscopy (1)

A. H. A. Clayton, Q. S. Hanley, and P. J. Verveer, "Graphical representation and multicomponent analysis of single-frequency fluorescence lifetime imaging microscopy data," J. Microscopy 213(1), 1-5 (2004).
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J. Opt. Soc. Am. A (1)

J. Roy. Soc. Interface (1)

A. D. Elder, A. Domin, G. S. Kaminski Schierle, C. Lindon, J. Pines, A. Esposito, and C. F. Kaminski, "A quantitative protocol for dynamic measurements of protein interactions by Frster resonance energy transfersensitized fluorescence emission," J. Roy. Soc. Interface 6(1), S59-S81 (2009).
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Microsc. Res. Tech. (1)

P. Serge, J. R. P. Michael, K. Daekeun, K. Ki Hean, J. S. Tsu-Te, and T. C. S. Peter, "Frequency domain lifetime and spectral imaging microscopy," Microsc. Res. Tech. 69(11), 861-874 (2006).

Nanomed. Nanotechnol. Bio. Med. (1)

X. Dai, Z. Yue, M. E. Eccleston, J. Swartling, N. K. H. Slater, and C. F. Kaminski, "Fluorescence intensity and lifetime imaging of free and micellar-encapsulated doxorubicin in living cells," Nanomed. Nanotechnol. Bio. Med. 4(1), 49-56 (2008).
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Opt. Express (2)

Opt. Lett. (1)

PLoS ONE (1)

A. Esposito, T. Tiffert, J. M. A. Mauritz, S. Schlachter, L. H. Bannister, C. F. Kaminski, and V. L. Lew, "FRET Imaging of Hemoglobin Concentration in Plasmodium falciparum-Infected Red Cells," PLoS ONE 3(11), e3780 (2008).
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Polymer (1)

X. W. Dai, M. E. Eccleston, Z. L. Yue, N. K. H. Slater, and C. F. Kaminski, "A spectroscopic study of the self-association and inter-molecular aggregation behaviour of pH-responsive poly(L-lysine iso-phthalamide)," Polymer 47(8), 2689-2698 (2006).
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G. Strang, Linear algebra and its applications (Thomson, 2006).

J. R. Lakowicz, Principles of Fluorescence Spectroscopy (Springer, 2006).

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Figures (7)

Fig. 1.
Fig. 1.

Simulated dataset. Distribution of fluorophore species 1 (A) and 2 (B). (C) Molecular Fraction of species 1. (D) Distribution of excitation spectra. The excitation spectra for fluorescent species 1 and 2 are shown in blue and red, respectively. Each fluorophore is modeled as having a slightly different excitation spectrum, hence there are thousands of individual spectra plotted and overlaid. (E) Distribution of fluorophore lifetimes in a given pixel. This histogram displays the distribution of lifetimes assigned to each fluorescent species in a given pixel. Fluorescent species 1 and 2 are modelled as having a mean lifetime of 2 ns and 1 ns, respectively. (F) Exponential decay curve with Poisson noise from pixel denoted by white square in (C) at 510 nm excitation wavelength. (G) Intensity images at each excitation wavelength.

Fig. 2.
Fig. 2.

(A) AB plot showing simulated dataset. AB clouds of different colour represent data obtained at different excitation wavelengths. Cloud centroids are indicated by yellow dots. (B) Graphical global analysis showing linear fit to signals from all pixels and excitation wavelengths. The global lifetimes are described by the intersection of the linear fit with the red monoexponential circle. (C) Fractional intensity images calculated using Eq. (9) and global lifetime estimates.

Fig. 3.
Fig. 3.

(A) Original (noise free) fluorophore distributions and (B) estimates derived from SVD. (C) Retrieved fluorophore excitation spectra for fluorescent species 1 and 2, in yellow and green, respectively (peak normalized and plotted against true spectra)

Fig. 4.
Fig. 4.

Molecular fraction for simulated dataset. (A) Noise free molecular fraction. (B) Molecular fraction estimated by SVD unmixing algorithm. (C) Estimated molecular fraction corrected using molecular fraction known at one pixel. (D) Mean squared error between corrected molecular fraction and true molecular fraction

Fig. 5.
Fig. 5.

Figure-of-Merit (FoM) for unmixing algorithm at a range of spectral - lifetime separations. The lifetime of Fluorophore 1 is held at 2 ns and peak excitation at 500 nm. The lifetime of Fluorophore 2 is then varied from 1.2 ns to 2.2 ns and the spectral peak is varied from 500 nm to 540 nm. The insets show the estimators for the unmixed images (N̂1 and N̂2), ratios to the true images (n 1 and n 2) and the associated excitation spectra and lifetime distributions. See text for details.

Fig. 6.
Fig. 6.

AB plot showing biological sample FLIM images taken at several excitation wavelengths (mean of each AB cloud is shown by a yellow dot) and (B) Intensity images at each excitation wavelength. Images are approximately 150µm×150µm

Fig. 7.
Fig. 7.

(A) Estimated fluorophore excitation spectra and literature values for Alexa 546 and 555 dyes (peak normalized). (B) Estimated distribution of Fluorophore 1 (N̂ 1 ) and (C) Fluorophore 2 (N̂ 1 ). (D) Estimated molecular fraction f̂ 1 and (E) intensity image overlaid with estimated molecular fraction. All images are approximately 150µm×150µm

Equations (19)

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I ( p ) = a ( p ) e t τ 1 ( p ) + b ( p ) e t τ 2 ( p )
α ( p ) = τ 1 a ( p ) τ 1 a ( p ) + τ 2 b ( p )
I λ p t = Σ i = 1 F B i ( λ ) N i ( p ) e ( t τ i )
I λ p = 0 Σ i = 1 F B i ( λ ) N i ( p ) e ( t τ i ) dt
= Σ i = 1 F B i ( λ ) N i ( p ) τ i
α i λ p = τ i B i ( λ ) N i ( p ) I λ p
f i ( p ) = N i ( p ) Σ i = 1 F N i ( p )
α i I = τ i N i B i
α i I = U i Σ i V i T
α i I = Σ j = 1 r σ i , j u i , j v i , j
f ̂ i ( p ) = N ̂ i ( p ) Σ i = 1 F N ̂ i ( p )
f i ( p ) = c i f ̂ i ( p ) f ̂ i ( p ) ( c i 1 ) + 1
B 1 ( λ ) = exp [ ( λ λ μ 1 ) 2 2 λ σ 1 ]
I λ p t = B 1 ( λ ) N 1 ( p ) e ( t τ 1 ) + B 2 ( λ ) N 2 ( p ) e ( t τ 2 )
A λ p = Σ t I ( t ) sin ( ω t ) Σ t I ( t )
B λ p = Σ t I ( t ) cos ( ω t ) Σ t I ( t )
A α 1 τ 1 τ 2 = α 1 ω 0 τ 1 1 + ( ω 0 τ 1 ) 2 + ( 1 α 1 ) ω 0 τ 2 1 + ( ω 0 τ 2 ) 2
B α 1 τ 1 τ 2 = α 1 1 + ( ω 0 τ 1 ) 2 + ( 1 α 1 ) 1 + ( ω 0 τ 2 ) 2
FoM = Σ i stdev ( n i ) mean ( n i ) N ph

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