Abstract

We report a spectrally resolved fluorescence lifetime imaging system based on time gated single photon detection with a fixed gate width of 200 ps and 7 spectral channels. Time gated systems can operate at high count rates but usually have large gate widths and sample only part of the fluorescence decay curve. In the system presented in this work, the fluorescence signal is sampled using a high speed transceiver. An error analysis is carried out to characterize the performance of both lifetime and spectral detection. The effect of gate width and spectral channel width on the accuracy of estimated lifetimes and spectral widths is described. The performance of the whole instrument is evaluated at count rates of up to 12 MHz. Accurate fluorescence lifetimes (error < 2%) are recorded at count rates as high as 5 MHz. This is limited by the PMT performance, not by the electronics. Analysis of the large spectral lifetime image sets is challenging and time-consuming. Here, we demonstrate the use of lifetime and spectral phasors for analyzing images of fibroblast cells with 2 different labeled components. The phasor approach provides a fast and intuitive way of analyzing the results of spectrally resolved fluorescence lifetime imaging experiments.

© 2013 OSA

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2012

2011

M. R. Hight, D. D. Nolting, E. T. McKinley, A. D. Lander, S. K. Wyatt, M. Gonyea, P. Zhao, and H. C. Manning, “Multispectral fluorescence imaging to assess pH in biological specimens,” J. Biomed. Opt.16(1), 016007–016007 (2011).
[CrossRef] [PubMed]

F. Fereidouni, A. Esposito, G. A. Blab, and H. C. Gerritsen, “A modified phasor approach for analyzing time-gated fluorescence lifetime images,” J. Microsc.244(3), 248–258 (2011).
[CrossRef] [PubMed]

D. Strat, F. Dolp, B. von Einem, C. Steinmetz, C. A. F. von Arnim, and A. Rueck, “Spectrally resolved fluorescence lifetime imaging microscopy: Forster resonant energy transfer global analysis with a one- and two-exponential donor model,” J. Biomed. Opt.16(2), 026002 (2011).
[CrossRef] [PubMed]

Y. C. Chen and R. M. Clegg, “Spectral resolution in conjunction with polar plots improves the accuracy and reliability of FLIM measurements and estimates of FRET efficiency,” J. Microsc.244(1), 21–37 (2011).
[CrossRef] [PubMed]

R. W. K. Leung, S. C. A. Yeh, and Q. Fang, “Effects of incomplete decay in fluorescence lifetime estimation,” Biomed. Opt. Express2(9), 2517–2531 (2011).
[CrossRef] [PubMed]

2010

2009

S. Schlachter, S. Schwedler, A. Esposito, G. S. Kaminski Schierle, G. D. Moggridge, and C. F. Kaminski, “A method to unmix multiple fluorophores in microscopy images with minimal a priori information,” Opt. Express17(25), 22747–22760 (2009).
[CrossRef] [PubMed]

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).

B. Q. Spring and R. M. Clegg, “Image analysis for denoising full-field frequency-domain fluorescence lifetime images,” J. Microsc.235(2), 221–237 (2009).
[CrossRef] [PubMed]

2008

M. A. Digman, V. R. Caiolfa, M. Zamai, and E. Gratton, “The phasor approach to fluorescence lifetime imaging analysis,” Biophys. J.94(2), L14–L16 (2008).
[CrossRef] [PubMed]

2007

M. Jose, D. K. Nair, C. Reissner, R. Hartig, and W. Zuschratter, “Photophysics of Clomeleon by FLIM: discriminating excited state reactions along neuronal development,” Biophys. J.92(6), 2237–2254 (2007).
[CrossRef] [PubMed]

P. D. Beule, D. M. Owen, H. B. Manning, C. B. Talbot, J. Requejo-Isidro, C. Dunsby, J. Mcginty, R. K. P. Benninger, D. S. Elson, I. Munro, M. John Lever, P. Anand, M. A. A. Neil, and P. M. W. French, “Rapid hyperspectral fluorescence lifetime imaging,” Microsc. Res. Tech.70(5), 481–484 (2007).
[CrossRef] [PubMed]

A. Rück, Ch. Hülshoff, I. Kinzler, W. Becker, and R. Steiner, “SLIM: a new method for molecular imaging,” Microsc. Res. Tech.70(5), 485–492 (2007).
[CrossRef] [PubMed]

Y. Chen, J. P. Mauldin, R. N. Day, and A. Periasamy, “Characterization of spectral FRET imaging microscopy for monitoring nuclear protein interactions,” J. Microsc.228(2), 139–152 (2007).
[CrossRef] [PubMed]

2006

2004

J. A. Jo, Q. Fang, T. Papaioannou, and L. Marcu, “Fast model-free deconvolution of fluorescence decay for analysis of biological systems,” J. Biomed. Opt.9(4), 743–752 (2004).
[CrossRef] [PubMed]

D. K. Bird, K. W. Eliceiri, C. H. Fan, and J. G. White, “Simultaneous two-photon spectral and lifetime fluorescence microscopy,” Appl. Opt.43(27), 5173–5182 (2004).
[CrossRef] [PubMed]

A. V. Agronskaia, L. Tertoolen, and H. C. Gerritsen, “Fast fluorescence lifetime imaging of calcium in living cells,” J. Biomed. Opt.9(6), 1230–1237 (2004).
[CrossRef] [PubMed]

2003

V. E. Centonze, M. Sun, A. Masuda, H. Gerritsen, and B. Herman, “Fluorescence resonance energy transfer imaging microscopy,” Methods Enzymol.360, 542–560 (2003).
[CrossRef] [PubMed]

2002

Q. S. Hanley, D. J. Arndt-Jovin, and T. M. Jovin, “Spectrally resolved fluorescence lifetime imaging microscopy,” Appl. Spectrosc.56(2), 155–166 (2002).
[CrossRef]

M. Tramier, I. Gautier, T. Piolot, S. Ravalet, K. Kemnitz, J. Coppey, C. Durieux, V. Mignotte, and M. Coppey-Moisan, “Picosecond-hetero-FRET microscopy to probe protein-protein interactions in live cells,” Biophys. J.83(6), 3570–3577 (2002).
[CrossRef] [PubMed]

H. C. Gerritsen, M. A. Asselbergs, A. V. Agronskaia, and W. G. Van Sark, “Fluorescence lifetime imaging in scanning microscopes: acquisition speed, photon economy and lifetime resolution,” J. Microsc.206(3), 218–224 (2002).
[CrossRef] [PubMed]

1995

R. Sanders, A. Draaijer, H. C. Gerritsen, P. M. Houpt, and Y. K. Levine, “Quantitative pH imaging in cells using confocal fluorescence lifetime imaging microscopy,” Anal. Biochem.227(2), 302–308 (1995).
[CrossRef] [PubMed]

1992

J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, and M. L. Johnson, “Fluorescence lifetime imaging of free and protein-bound NADH,” Proc. Natl. Acad. Sci. U.S.A.89(4), 1271–1275 (1992).
[CrossRef] [PubMed]

M. Köllner and J. Wolfrum, “How many photons are necessary for fluorescence-lifetime measurements?” Chem. Phys. Lett.200(1-2), 199–204 (1992).
[CrossRef]

1986

D. Bebelaar, “Time response of various types of photomultipliers and its wavelength dependence in time‐correlated single‐photon counting with an ultimate resolution of 47 ps FWHM,” Rev. Sci. Instrum.57(6), 1116–1125 (1986).
[CrossRef]

1979

D. O'Connor, W. Ware, and J. Andre, “Deconvolution of fluorescence decay curves. A critical comparison of techniques,” J. Phys. Chem.83(10), 1333–1343 (1979).
[CrossRef]

1975

A. Gafni, R. L. Modlin, and L. Brand, “Analysis of fluorescence decay curves by means of the Laplace transformation,” Biophys. J.15(3), 263–280 (1975).
[CrossRef] [PubMed]

Agronskaia, A. V.

A. V. Agronskaia, L. Tertoolen, and H. C. Gerritsen, “Fast fluorescence lifetime imaging of calcium in living cells,” J. Biomed. Opt.9(6), 1230–1237 (2004).
[CrossRef] [PubMed]

H. C. Gerritsen, M. A. Asselbergs, A. V. Agronskaia, and W. G. Van Sark, “Fluorescence lifetime imaging in scanning microscopes: acquisition speed, photon economy and lifetime resolution,” J. Microsc.206(3), 218–224 (2002).
[CrossRef] [PubMed]

Anand, P.

P. D. Beule, D. M. Owen, H. B. Manning, C. B. Talbot, J. Requejo-Isidro, C. Dunsby, J. Mcginty, R. K. P. Benninger, D. S. Elson, I. Munro, M. John Lever, P. Anand, M. A. A. Neil, and P. M. W. French, “Rapid hyperspectral fluorescence lifetime imaging,” Microsc. Res. Tech.70(5), 481–484 (2007).
[CrossRef] [PubMed]

Andre, J.

D. O'Connor, W. Ware, and J. Andre, “Deconvolution of fluorescence decay curves. A critical comparison of techniques,” J. Phys. Chem.83(10), 1333–1343 (1979).
[CrossRef]

Applegate, B. E.

Arlt, J.

Arndt-Jovin, D. J.

Asselbergs, M. A.

H. C. Gerritsen, M. A. Asselbergs, A. V. Agronskaia, and W. G. Van Sark, “Fluorescence lifetime imaging in scanning microscopes: acquisition speed, photon economy and lifetime resolution,” J. Microsc.206(3), 218–224 (2002).
[CrossRef] [PubMed]

Bader, A. N.

Bebelaar, D.

D. Bebelaar, “Time response of various types of photomultipliers and its wavelength dependence in time‐correlated single‐photon counting with an ultimate resolution of 47 ps FWHM,” Rev. Sci. Instrum.57(6), 1116–1125 (1986).
[CrossRef]

Becker, W.

A. Rück, Ch. Hülshoff, I. Kinzler, W. Becker, and R. Steiner, “SLIM: a new method for molecular imaging,” Microsc. Res. Tech.70(5), 485–492 (2007).
[CrossRef] [PubMed]

W. Becker, A. Bergmann, C. Biskup, T. Zimmer, N. Klöcker, and K. Benndorf, ”Multi-wavelength TCSPC lifetime imaging” in Proc. SPIEAnonymous 79–84, (2002).

Behne, M.

A. Celli, S. Sanchez, M. Behne, T. Hazlett, E. Gratton, and T. Mauro, “The epidermal Ca2 gradient: Measurement using the phasor representation of fluorescent lifetime imaging,” Biophys. J.98(5), 911–921 (2010).
[CrossRef] [PubMed]

Benndorf, K.

W. Becker, A. Bergmann, C. Biskup, T. Zimmer, N. Klöcker, and K. Benndorf, ”Multi-wavelength TCSPC lifetime imaging” in Proc. SPIEAnonymous 79–84, (2002).

Benninger, R. K. P.

P. D. Beule, D. M. Owen, H. B. Manning, C. B. Talbot, J. Requejo-Isidro, C. Dunsby, J. Mcginty, R. K. P. Benninger, D. S. Elson, I. Munro, M. John Lever, P. Anand, M. A. A. Neil, and P. M. W. French, “Rapid hyperspectral fluorescence lifetime imaging,” Microsc. Res. Tech.70(5), 481–484 (2007).
[CrossRef] [PubMed]

Bergmann, A.

W. Becker, A. Bergmann, C. Biskup, T. Zimmer, N. Klöcker, and K. Benndorf, ”Multi-wavelength TCSPC lifetime imaging” in Proc. SPIEAnonymous 79–84, (2002).

Beule, P. D.

P. D. Beule, D. M. Owen, H. B. Manning, C. B. Talbot, J. Requejo-Isidro, C. Dunsby, J. Mcginty, R. K. P. Benninger, D. S. Elson, I. Munro, M. John Lever, P. Anand, M. A. A. Neil, and P. M. W. French, “Rapid hyperspectral fluorescence lifetime imaging,” Microsc. Res. Tech.70(5), 481–484 (2007).
[CrossRef] [PubMed]

Bird, D. K.

Biskup, C.

W. Becker, A. Bergmann, C. Biskup, T. Zimmer, N. Klöcker, and K. Benndorf, ”Multi-wavelength TCSPC lifetime imaging” in Proc. SPIEAnonymous 79–84, (2002).

Blab, G. A.

F. Fereidouni, A. Esposito, G. A. Blab, and H. C. Gerritsen, “A modified phasor approach for analyzing time-gated fluorescence lifetime images,” J. Microsc.244(3), 248–258 (2011).
[CrossRef] [PubMed]

Brand, L.

A. Gafni, R. L. Modlin, and L. Brand, “Analysis of fluorescence decay curves by means of the Laplace transformation,” Biophys. J.15(3), 263–280 (1975).
[CrossRef] [PubMed]

Buts, A.

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(2), L14–L16 (2008).
[CrossRef] [PubMed]

Celli, A.

A. Celli, S. Sanchez, M. Behne, T. Hazlett, E. Gratton, and T. Mauro, “The epidermal Ca2 gradient: Measurement using the phasor representation of fluorescent lifetime imaging,” Biophys. J.98(5), 911–921 (2010).
[CrossRef] [PubMed]

Centonze, V. E.

V. E. Centonze, M. Sun, A. Masuda, H. Gerritsen, and B. Herman, “Fluorescence resonance energy transfer imaging microscopy,” Methods Enzymol.360, 542–560 (2003).
[CrossRef] [PubMed]

Charbon, E.

Chen, Y.

Y. Chen, J. P. Mauldin, R. N. Day, and A. Periasamy, “Characterization of spectral FRET imaging microscopy for monitoring nuclear protein interactions,” J. Microsc.228(2), 139–152 (2007).
[CrossRef] [PubMed]

Chen, Y. C.

Y. C. Chen and R. M. Clegg, “Spectral resolution in conjunction with polar plots improves the accuracy and reliability of FLIM measurements and estimates of FRET efficiency,” J. Microsc.244(1), 21–37 (2011).
[CrossRef] [PubMed]

Clegg, R. M.

Y. C. Chen and R. M. Clegg, “Spectral resolution in conjunction with polar plots improves the accuracy and reliability of FLIM measurements and estimates of FRET efficiency,” J. Microsc.244(1), 21–37 (2011).
[CrossRef] [PubMed]

B. Q. Spring and R. M. Clegg, “Image analysis for denoising full-field frequency-domain fluorescence lifetime images,” J. Microsc.235(2), 221–237 (2009).
[CrossRef] [PubMed]

Coppey, J.

M. Tramier, I. Gautier, T. Piolot, S. Ravalet, K. Kemnitz, J. Coppey, C. Durieux, V. Mignotte, and M. Coppey-Moisan, “Picosecond-hetero-FRET microscopy to probe protein-protein interactions in live cells,” Biophys. J.83(6), 3570–3577 (2002).
[CrossRef] [PubMed]

Coppey-Moisan, M.

M. Tramier, I. Gautier, T. Piolot, S. Ravalet, K. Kemnitz, J. Coppey, C. Durieux, V. Mignotte, and M. Coppey-Moisan, “Picosecond-hetero-FRET microscopy to probe protein-protein interactions in live cells,” Biophys. J.83(6), 3570–3577 (2002).
[CrossRef] [PubMed]

Day, R. N.

Y. Chen, J. P. Mauldin, R. N. Day, and A. Periasamy, “Characterization of spectral FRET imaging microscopy for monitoring nuclear protein interactions,” J. Microsc.228(2), 139–152 (2007).
[CrossRef] [PubMed]

de Bruijn, H. S.

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(2), L14–L16 (2008).
[CrossRef] [PubMed]

Dolp, F.

D. Strat, F. Dolp, B. von Einem, C. Steinmetz, C. A. F. von Arnim, and A. Rueck, “Spectrally resolved fluorescence lifetime imaging microscopy: Forster resonant energy transfer global analysis with a one- and two-exponential donor model,” J. Biomed. Opt.16(2), 026002 (2011).
[CrossRef] [PubMed]

Draaijer, A.

R. Sanders, A. Draaijer, H. C. Gerritsen, P. M. Houpt, and Y. K. Levine, “Quantitative pH imaging in cells using confocal fluorescence lifetime imaging microscopy,” Anal. Biochem.227(2), 302–308 (1995).
[CrossRef] [PubMed]

Dunsby, C.

P. D. Beule, D. M. Owen, H. B. Manning, C. B. Talbot, J. Requejo-Isidro, C. Dunsby, J. Mcginty, R. K. P. Benninger, D. S. Elson, I. Munro, M. John Lever, P. Anand, M. A. A. Neil, and P. M. W. French, “Rapid hyperspectral fluorescence lifetime imaging,” Microsc. Res. Tech.70(5), 481–484 (2007).
[CrossRef] [PubMed]

Durieux, C.

M. Tramier, I. Gautier, T. Piolot, S. Ravalet, K. Kemnitz, J. Coppey, C. Durieux, V. Mignotte, and M. Coppey-Moisan, “Picosecond-hetero-FRET microscopy to probe protein-protein interactions in live cells,” Biophys. J.83(6), 3570–3577 (2002).
[CrossRef] [PubMed]

Eliceiri, K. W.

Elson, D. S.

P. D. Beule, D. M. Owen, H. B. Manning, C. B. Talbot, J. Requejo-Isidro, C. Dunsby, J. Mcginty, R. K. P. Benninger, D. S. Elson, I. Munro, M. John Lever, P. Anand, M. A. A. Neil, and P. M. W. French, “Rapid hyperspectral fluorescence lifetime imaging,” Microsc. Res. Tech.70(5), 481–484 (2007).
[CrossRef] [PubMed]

Esposito, A.

Fan, C. H.

Fang, Q.

R. W. K. Leung, S. C. A. Yeh, and Q. Fang, “Effects of incomplete decay in fluorescence lifetime estimation,” Biomed. Opt. Express2(9), 2517–2531 (2011).
[CrossRef] [PubMed]

J. A. Jo, Q. Fang, T. Papaioannou, and L. Marcu, “Fast model-free deconvolution of fluorescence decay for analysis of biological systems,” J. Biomed. Opt.9(4), 743–752 (2004).
[CrossRef] [PubMed]

Fereidouni, F.

F. Fereidouni, A. N. Bader, and H. C. Gerritsen, “Spectral phasor analysis allows rapid and reliable unmixing of fluorescence microscopy spectral images,” Opt. Express20(12), 12729–12741 (2012).
[CrossRef] [PubMed]

F. Fereidouni, A. Esposito, G. A. Blab, and H. C. Gerritsen, “A modified phasor approach for analyzing time-gated fluorescence lifetime images,” J. Microsc.244(3), 248–258 (2011).
[CrossRef] [PubMed]

French, P. M. W.

P. D. Beule, D. M. Owen, H. B. Manning, C. B. Talbot, J. Requejo-Isidro, C. Dunsby, J. Mcginty, R. K. P. Benninger, D. S. Elson, I. Munro, M. John Lever, P. Anand, M. A. A. Neil, and P. M. W. French, “Rapid hyperspectral fluorescence lifetime imaging,” Microsc. Res. Tech.70(5), 481–484 (2007).
[CrossRef] [PubMed]

Gafni, A.

A. Gafni, R. L. Modlin, and L. Brand, “Analysis of fluorescence decay curves by means of the Laplace transformation,” Biophys. J.15(3), 263–280 (1975).
[CrossRef] [PubMed]

Gautier, I.

M. Tramier, I. Gautier, T. Piolot, S. Ravalet, K. Kemnitz, J. Coppey, C. Durieux, V. Mignotte, and M. Coppey-Moisan, “Picosecond-hetero-FRET microscopy to probe protein-protein interactions in live cells,” Biophys. J.83(6), 3570–3577 (2002).
[CrossRef] [PubMed]

Gerritsen, H.

V. E. Centonze, M. Sun, A. Masuda, H. Gerritsen, and B. Herman, “Fluorescence resonance energy transfer imaging microscopy,” Methods Enzymol.360, 542–560 (2003).
[CrossRef] [PubMed]

Gerritsen, H. C.

F. Fereidouni, A. N. Bader, and H. C. Gerritsen, “Spectral phasor analysis allows rapid and reliable unmixing of fluorescence microscopy spectral images,” Opt. Express20(12), 12729–12741 (2012).
[CrossRef] [PubMed]

F. Fereidouni, A. Esposito, G. A. Blab, and H. C. Gerritsen, “A modified phasor approach for analyzing time-gated fluorescence lifetime images,” J. Microsc.244(3), 248–258 (2011).
[CrossRef] [PubMed]

J. A. Palero, H. S. de Bruijn, A. van der Ploeg-van den Heuvel, H. J. Sterenborg, and H. C. Gerritsen, “In vivo nonlinear spectral imaging in mouse skin,” Opt. Express14(10), 4395–4402 (2006).
[CrossRef] [PubMed]

A. V. Agronskaia, L. Tertoolen, and H. C. Gerritsen, “Fast fluorescence lifetime imaging of calcium in living cells,” J. Biomed. Opt.9(6), 1230–1237 (2004).
[CrossRef] [PubMed]

H. C. Gerritsen, M. A. Asselbergs, A. V. Agronskaia, and W. G. Van Sark, “Fluorescence lifetime imaging in scanning microscopes: acquisition speed, photon economy and lifetime resolution,” J. Microsc.206(3), 218–224 (2002).
[CrossRef] [PubMed]

R. Sanders, A. Draaijer, H. C. Gerritsen, P. M. Houpt, and Y. K. Levine, “Quantitative pH imaging in cells using confocal fluorescence lifetime imaging microscopy,” Anal. Biochem.227(2), 302–308 (1995).
[CrossRef] [PubMed]

Gonyea, M.

M. R. Hight, D. D. Nolting, E. T. McKinley, A. D. Lander, S. K. Wyatt, M. Gonyea, P. Zhao, and H. C. Manning, “Multispectral fluorescence imaging to assess pH in biological specimens,” J. Biomed. Opt.16(1), 016007–016007 (2011).
[CrossRef] [PubMed]

Gratton, E.

A. Celli, S. Sanchez, M. Behne, T. Hazlett, E. Gratton, and T. Mauro, “The epidermal Ca2 gradient: Measurement using the phasor representation of fluorescent lifetime imaging,” Biophys. J.98(5), 911–921 (2010).
[CrossRef] [PubMed]

M. A. Digman, V. R. Caiolfa, M. Zamai, and E. Gratton, “The phasor approach to fluorescence lifetime imaging analysis,” Biophys. J.94(2), L14–L16 (2008).
[CrossRef] [PubMed]

Hanley, Q. S.

Hartig, R.

M. Jose, D. K. Nair, C. Reissner, R. Hartig, and W. Zuschratter, “Photophysics of Clomeleon by FLIM: discriminating excited state reactions along neuronal development,” Biophys. J.92(6), 2237–2254 (2007).
[CrossRef] [PubMed]

D. K. Nair, M. Jose, T. Kuner, W. Zuschratter, and R. Hartig, “FRET-FLIM at nanometer spectral resolution from living cells,” Opt. Express14(25), 12217–12229 (2006).
[CrossRef] [PubMed]

Hazlett, T.

A. Celli, S. Sanchez, M. Behne, T. Hazlett, E. Gratton, and T. Mauro, “The epidermal Ca2 gradient: Measurement using the phasor representation of fluorescent lifetime imaging,” Biophys. J.98(5), 911–921 (2010).
[CrossRef] [PubMed]

Henderson, R.

Herman, B.

V. E. Centonze, M. Sun, A. Masuda, H. Gerritsen, and B. Herman, “Fluorescence resonance energy transfer imaging microscopy,” Methods Enzymol.360, 542–560 (2003).
[CrossRef] [PubMed]

Hight, M. R.

M. R. Hight, D. D. Nolting, E. T. McKinley, A. D. Lander, S. K. Wyatt, M. Gonyea, P. Zhao, and H. C. Manning, “Multispectral fluorescence imaging to assess pH in biological specimens,” J. Biomed. Opt.16(1), 016007–016007 (2011).
[CrossRef] [PubMed]

Houpt, P. M.

R. Sanders, A. Draaijer, H. C. Gerritsen, P. M. Houpt, and Y. K. Levine, “Quantitative pH imaging in cells using confocal fluorescence lifetime imaging microscopy,” Anal. Biochem.227(2), 302–308 (1995).
[CrossRef] [PubMed]

Hülshoff, Ch.

A. Rück, Ch. Hülshoff, I. Kinzler, W. Becker, and R. Steiner, “SLIM: a new method for molecular imaging,” Microsc. Res. Tech.70(5), 485–492 (2007).
[CrossRef] [PubMed]

Jo, J. A.

S. Shrestha, B. E. Applegate, J. Park, X. Xiao, P. Pande, and J. A. Jo, “High-speed multispectral fluorescence lifetime imaging implementation for in vivo applications,” Opt. Lett.35(15), 2558–2560 (2010).
[CrossRef] [PubMed]

J. A. Jo, Q. Fang, T. Papaioannou, and L. Marcu, “Fast model-free deconvolution of fluorescence decay for analysis of biological systems,” J. Biomed. Opt.9(4), 743–752 (2004).
[CrossRef] [PubMed]

John Lever, M.

P. D. Beule, D. M. Owen, H. B. Manning, C. B. Talbot, J. Requejo-Isidro, C. Dunsby, J. Mcginty, R. K. P. Benninger, D. S. Elson, I. Munro, M. John Lever, P. Anand, M. A. A. Neil, and P. M. W. French, “Rapid hyperspectral fluorescence lifetime imaging,” Microsc. Res. Tech.70(5), 481–484 (2007).
[CrossRef] [PubMed]

Johnson, M. L.

J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, and M. L. Johnson, “Fluorescence lifetime imaging of free and protein-bound NADH,” Proc. Natl. Acad. Sci. U.S.A.89(4), 1271–1275 (1992).
[CrossRef] [PubMed]

Jose, M.

M. Jose, D. K. Nair, C. Reissner, R. Hartig, and W. Zuschratter, “Photophysics of Clomeleon by FLIM: discriminating excited state reactions along neuronal development,” Biophys. J.92(6), 2237–2254 (2007).
[CrossRef] [PubMed]

D. K. Nair, M. Jose, T. Kuner, W. Zuschratter, and R. Hartig, “FRET-FLIM at nanometer spectral resolution from living cells,” Opt. Express14(25), 12217–12229 (2006).
[CrossRef] [PubMed]

Jovin, T. M.

Kaminski, C. F.

Kaminski Schierle, G. S.

Kemnitz, K.

M. Tramier, I. Gautier, T. Piolot, S. Ravalet, K. Kemnitz, J. Coppey, C. Durieux, V. Mignotte, and M. Coppey-Moisan, “Picosecond-hetero-FRET microscopy to probe protein-protein interactions in live cells,” Biophys. J.83(6), 3570–3577 (2002).
[CrossRef] [PubMed]

Kinzler, I.

A. Rück, Ch. Hülshoff, I. Kinzler, W. Becker, and R. Steiner, “SLIM: a new method for molecular imaging,” Microsc. Res. Tech.70(5), 485–492 (2007).
[CrossRef] [PubMed]

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).

Klöcker, N.

W. Becker, A. Bergmann, C. Biskup, T. Zimmer, N. Klöcker, and K. Benndorf, ”Multi-wavelength TCSPC lifetime imaging” in Proc. SPIEAnonymous 79–84, (2002).

Köllner, M.

M. Köllner and J. Wolfrum, “How many photons are necessary for fluorescence-lifetime measurements?” Chem. Phys. Lett.200(1-2), 199–204 (1992).
[CrossRef]

Kuner, T.

Lakowicz, J. R.

J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, and M. L. Johnson, “Fluorescence lifetime imaging of free and protein-bound NADH,” Proc. Natl. Acad. Sci. U.S.A.89(4), 1271–1275 (1992).
[CrossRef] [PubMed]

Lander, A. D.

M. R. Hight, D. D. Nolting, E. T. McKinley, A. D. Lander, S. K. Wyatt, M. Gonyea, P. Zhao, and H. C. Manning, “Multispectral fluorescence imaging to assess pH in biological specimens,” J. Biomed. Opt.16(1), 016007–016007 (2011).
[CrossRef] [PubMed]

Leung, R. W. K.

Levine, Y. K.

R. Sanders, A. Draaijer, H. C. Gerritsen, P. M. Houpt, and Y. K. Levine, “Quantitative pH imaging in cells using confocal fluorescence lifetime imaging microscopy,” Anal. Biochem.227(2), 302–308 (1995).
[CrossRef] [PubMed]

Li, D. U.

Manning, H. B.

P. D. Beule, D. M. Owen, H. B. Manning, C. B. Talbot, J. Requejo-Isidro, C. Dunsby, J. Mcginty, R. K. P. Benninger, D. S. Elson, I. Munro, M. John Lever, P. Anand, M. A. A. Neil, and P. M. W. French, “Rapid hyperspectral fluorescence lifetime imaging,” Microsc. Res. Tech.70(5), 481–484 (2007).
[CrossRef] [PubMed]

Manning, H. C.

M. R. Hight, D. D. Nolting, E. T. McKinley, A. D. Lander, S. K. Wyatt, M. Gonyea, P. Zhao, and H. C. Manning, “Multispectral fluorescence imaging to assess pH in biological specimens,” J. Biomed. Opt.16(1), 016007–016007 (2011).
[CrossRef] [PubMed]

Marcu, L.

J. A. Jo, Q. Fang, T. Papaioannou, and L. Marcu, “Fast model-free deconvolution of fluorescence decay for analysis of biological systems,” J. Biomed. Opt.9(4), 743–752 (2004).
[CrossRef] [PubMed]

Masuda, A.

V. E. Centonze, M. Sun, A. Masuda, H. Gerritsen, and B. Herman, “Fluorescence resonance energy transfer imaging microscopy,” Methods Enzymol.360, 542–560 (2003).
[CrossRef] [PubMed]

Mauldin, J. P.

Y. Chen, J. P. Mauldin, R. N. Day, and A. Periasamy, “Characterization of spectral FRET imaging microscopy for monitoring nuclear protein interactions,” J. Microsc.228(2), 139–152 (2007).
[CrossRef] [PubMed]

Mauro, T.

A. Celli, S. Sanchez, M. Behne, T. Hazlett, E. Gratton, and T. Mauro, “The epidermal Ca2 gradient: Measurement using the phasor representation of fluorescent lifetime imaging,” Biophys. J.98(5), 911–921 (2010).
[CrossRef] [PubMed]

Mcginty, J.

P. D. Beule, D. M. Owen, H. B. Manning, C. B. Talbot, J. Requejo-Isidro, C. Dunsby, J. Mcginty, R. K. P. Benninger, D. S. Elson, I. Munro, M. John Lever, P. Anand, M. A. A. Neil, and P. M. W. French, “Rapid hyperspectral fluorescence lifetime imaging,” Microsc. Res. Tech.70(5), 481–484 (2007).
[CrossRef] [PubMed]

McKinley, E. T.

M. R. Hight, D. D. Nolting, E. T. McKinley, A. D. Lander, S. K. Wyatt, M. Gonyea, P. Zhao, and H. C. Manning, “Multispectral fluorescence imaging to assess pH in biological specimens,” J. Biomed. Opt.16(1), 016007–016007 (2011).
[CrossRef] [PubMed]

Mignotte, V.

M. Tramier, I. Gautier, T. Piolot, S. Ravalet, K. Kemnitz, J. Coppey, C. Durieux, V. Mignotte, and M. Coppey-Moisan, “Picosecond-hetero-FRET microscopy to probe protein-protein interactions in live cells,” Biophys. J.83(6), 3570–3577 (2002).
[CrossRef] [PubMed]

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).

Modlin, R. L.

A. Gafni, R. L. Modlin, and L. Brand, “Analysis of fluorescence decay curves by means of the Laplace transformation,” Biophys. J.15(3), 263–280 (1975).
[CrossRef] [PubMed]

Moggridge, G. D.

Munro, I.

P. D. Beule, D. M. Owen, H. B. Manning, C. B. Talbot, J. Requejo-Isidro, C. Dunsby, J. Mcginty, R. K. P. Benninger, D. S. Elson, I. Munro, M. John Lever, P. Anand, M. A. A. Neil, and P. M. W. French, “Rapid hyperspectral fluorescence lifetime imaging,” Microsc. Res. Tech.70(5), 481–484 (2007).
[CrossRef] [PubMed]

Nair, D. K.

M. Jose, D. K. Nair, C. Reissner, R. Hartig, and W. Zuschratter, “Photophysics of Clomeleon by FLIM: discriminating excited state reactions along neuronal development,” Biophys. J.92(6), 2237–2254 (2007).
[CrossRef] [PubMed]

D. K. Nair, M. Jose, T. Kuner, W. Zuschratter, and R. Hartig, “FRET-FLIM at nanometer spectral resolution from living cells,” Opt. Express14(25), 12217–12229 (2006).
[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).

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).

Neil, M. A. A.

P. D. Beule, D. M. Owen, H. B. Manning, C. B. Talbot, J. Requejo-Isidro, C. Dunsby, J. Mcginty, R. K. P. Benninger, D. S. Elson, I. Munro, M. John Lever, P. Anand, M. A. A. Neil, and P. M. W. French, “Rapid hyperspectral fluorescence lifetime imaging,” Microsc. Res. Tech.70(5), 481–484 (2007).
[CrossRef] [PubMed]

Nolting, D. D.

M. R. Hight, D. D. Nolting, E. T. McKinley, A. D. Lander, S. K. Wyatt, M. Gonyea, P. Zhao, and H. C. Manning, “Multispectral fluorescence imaging to assess pH in biological specimens,” J. Biomed. Opt.16(1), 016007–016007 (2011).
[CrossRef] [PubMed]

Nowaczyk, K.

J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, and M. L. Johnson, “Fluorescence lifetime imaging of free and protein-bound NADH,” Proc. Natl. Acad. Sci. U.S.A.89(4), 1271–1275 (1992).
[CrossRef] [PubMed]

O'Connor, D.

D. O'Connor, W. Ware, and J. Andre, “Deconvolution of fluorescence decay curves. A critical comparison of techniques,” J. Phys. Chem.83(10), 1333–1343 (1979).
[CrossRef]

Owen, D. M.

P. D. Beule, D. M. Owen, H. B. Manning, C. B. Talbot, J. Requejo-Isidro, C. Dunsby, J. Mcginty, R. K. P. Benninger, D. S. Elson, I. Munro, M. John Lever, P. Anand, M. A. A. Neil, and P. M. W. French, “Rapid hyperspectral fluorescence lifetime imaging,” Microsc. Res. Tech.70(5), 481–484 (2007).
[CrossRef] [PubMed]

Palero, J. A.

Pande, P.

Papaioannou, T.

J. A. Jo, Q. Fang, T. Papaioannou, and L. Marcu, “Fast model-free deconvolution of fluorescence decay for analysis of biological systems,” J. Biomed. Opt.9(4), 743–752 (2004).
[CrossRef] [PubMed]

Park, J.

Periasamy, A.

Y. Chen, J. P. Mauldin, R. N. Day, and A. Periasamy, “Characterization of spectral FRET imaging microscopy for monitoring nuclear protein interactions,” J. Microsc.228(2), 139–152 (2007).
[CrossRef] [PubMed]

Piolot, T.

M. Tramier, I. Gautier, T. Piolot, S. Ravalet, K. Kemnitz, J. Coppey, C. Durieux, V. Mignotte, and M. Coppey-Moisan, “Picosecond-hetero-FRET microscopy to probe protein-protein interactions in live cells,” Biophys. J.83(6), 3570–3577 (2002).
[CrossRef] [PubMed]

Ravalet, S.

M. Tramier, I. Gautier, T. Piolot, S. Ravalet, K. Kemnitz, J. Coppey, C. Durieux, V. Mignotte, and M. Coppey-Moisan, “Picosecond-hetero-FRET microscopy to probe protein-protein interactions in live cells,” Biophys. J.83(6), 3570–3577 (2002).
[CrossRef] [PubMed]

Reissner, C.

M. Jose, D. K. Nair, C. Reissner, R. Hartig, and W. Zuschratter, “Photophysics of Clomeleon by FLIM: discriminating excited state reactions along neuronal development,” Biophys. J.92(6), 2237–2254 (2007).
[CrossRef] [PubMed]

Requejo-Isidro, J.

P. D. Beule, D. M. Owen, H. B. Manning, C. B. Talbot, J. Requejo-Isidro, C. Dunsby, J. Mcginty, R. K. P. Benninger, D. S. Elson, I. Munro, M. John Lever, P. Anand, M. A. A. Neil, and P. M. W. French, “Rapid hyperspectral fluorescence lifetime imaging,” Microsc. Res. Tech.70(5), 481–484 (2007).
[CrossRef] [PubMed]

Richardson, J.

Rück, A.

A. Rück, Ch. Hülshoff, I. Kinzler, W. Becker, and R. Steiner, “SLIM: a new method for molecular imaging,” Microsc. Res. Tech.70(5), 485–492 (2007).
[CrossRef] [PubMed]

Rueck, A.

D. Strat, F. Dolp, B. von Einem, C. Steinmetz, C. A. F. von Arnim, and A. Rueck, “Spectrally resolved fluorescence lifetime imaging microscopy: Forster resonant energy transfer global analysis with a one- and two-exponential donor model,” J. Biomed. Opt.16(2), 026002 (2011).
[CrossRef] [PubMed]

Sanchez, S.

A. Celli, S. Sanchez, M. Behne, T. Hazlett, E. Gratton, and T. Mauro, “The epidermal Ca2 gradient: Measurement using the phasor representation of fluorescent lifetime imaging,” Biophys. J.98(5), 911–921 (2010).
[CrossRef] [PubMed]

Sanders, R.

R. Sanders, A. Draaijer, H. C. Gerritsen, P. M. Houpt, and Y. K. Levine, “Quantitative pH imaging in cells using confocal fluorescence lifetime imaging microscopy,” Anal. Biochem.227(2), 302–308 (1995).
[CrossRef] [PubMed]

Schlachter, S.

Schwedler, S.

Shrestha, S.

Spring, B. Q.

B. Q. Spring and R. M. Clegg, “Image analysis for denoising full-field frequency-domain fluorescence lifetime images,” J. Microsc.235(2), 221–237 (2009).
[CrossRef] [PubMed]

Steiner, R.

A. Rück, Ch. Hülshoff, I. Kinzler, W. Becker, and R. Steiner, “SLIM: a new method for molecular imaging,” Microsc. Res. Tech.70(5), 485–492 (2007).
[CrossRef] [PubMed]

Steinmetz, C.

D. Strat, F. Dolp, B. von Einem, C. Steinmetz, C. A. F. von Arnim, and A. Rueck, “Spectrally resolved fluorescence lifetime imaging microscopy: Forster resonant energy transfer global analysis with a one- and two-exponential donor model,” J. Biomed. Opt.16(2), 026002 (2011).
[CrossRef] [PubMed]

Sterenborg, H. J.

Stoppa, D.

Strat, D.

D. Strat, F. Dolp, B. von Einem, C. Steinmetz, C. A. F. von Arnim, and A. Rueck, “Spectrally resolved fluorescence lifetime imaging microscopy: Forster resonant energy transfer global analysis with a one- and two-exponential donor model,” J. Biomed. Opt.16(2), 026002 (2011).
[CrossRef] [PubMed]

Sun, M.

V. E. Centonze, M. Sun, A. Masuda, H. Gerritsen, and B. Herman, “Fluorescence resonance energy transfer imaging microscopy,” Methods Enzymol.360, 542–560 (2003).
[CrossRef] [PubMed]

Szmacinski, H.

J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, and M. L. Johnson, “Fluorescence lifetime imaging of free and protein-bound NADH,” Proc. Natl. Acad. Sci. U.S.A.89(4), 1271–1275 (1992).
[CrossRef] [PubMed]

Talbot, C. B.

P. D. Beule, D. M. Owen, H. B. Manning, C. B. Talbot, J. Requejo-Isidro, C. Dunsby, J. Mcginty, R. K. P. Benninger, D. S. Elson, I. Munro, M. John Lever, P. Anand, M. A. A. Neil, and P. M. W. French, “Rapid hyperspectral fluorescence lifetime imaging,” Microsc. Res. Tech.70(5), 481–484 (2007).
[CrossRef] [PubMed]

Tertoolen, L.

A. V. Agronskaia, L. Tertoolen, and H. C. Gerritsen, “Fast fluorescence lifetime imaging of calcium in living cells,” J. Biomed. Opt.9(6), 1230–1237 (2004).
[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).

Tramier, M.

M. Tramier, I. Gautier, T. Piolot, S. Ravalet, K. Kemnitz, J. Coppey, C. Durieux, V. Mignotte, and M. Coppey-Moisan, “Picosecond-hetero-FRET microscopy to probe protein-protein interactions in live cells,” Biophys. J.83(6), 3570–3577 (2002).
[CrossRef] [PubMed]

van der Ploeg-van den Heuvel, A.

Van Sark, W. G.

H. C. Gerritsen, M. A. Asselbergs, A. V. Agronskaia, and W. G. Van Sark, “Fluorescence lifetime imaging in scanning microscopes: acquisition speed, photon economy and lifetime resolution,” J. Microsc.206(3), 218–224 (2002).
[CrossRef] [PubMed]

von Arnim, C. A. F.

D. Strat, F. Dolp, B. von Einem, C. Steinmetz, C. A. F. von Arnim, and A. Rueck, “Spectrally resolved fluorescence lifetime imaging microscopy: Forster resonant energy transfer global analysis with a one- and two-exponential donor model,” J. Biomed. Opt.16(2), 026002 (2011).
[CrossRef] [PubMed]

von Einem, B.

D. Strat, F. Dolp, B. von Einem, C. Steinmetz, C. A. F. von Arnim, and A. Rueck, “Spectrally resolved fluorescence lifetime imaging microscopy: Forster resonant energy transfer global analysis with a one- and two-exponential donor model,” J. Biomed. Opt.16(2), 026002 (2011).
[CrossRef] [PubMed]

Walker, R.

Ware, W.

D. O'Connor, W. Ware, and J. Andre, “Deconvolution of fluorescence decay curves. A critical comparison of techniques,” J. Phys. Chem.83(10), 1333–1343 (1979).
[CrossRef]

White, J. G.

Wolfrum, J.

M. Köllner and J. Wolfrum, “How many photons are necessary for fluorescence-lifetime measurements?” Chem. Phys. Lett.200(1-2), 199–204 (1992).
[CrossRef]

Wyatt, S. K.

M. R. Hight, D. D. Nolting, E. T. McKinley, A. D. Lander, S. K. Wyatt, M. Gonyea, P. Zhao, and H. C. Manning, “Multispectral fluorescence imaging to assess pH in biological specimens,” J. Biomed. Opt.16(1), 016007–016007 (2011).
[CrossRef] [PubMed]

Xiao, X.

Yeh, S. C. A.

Zamai, M.

M. A. Digman, V. R. Caiolfa, M. Zamai, and E. Gratton, “The phasor approach to fluorescence lifetime imaging analysis,” Biophys. J.94(2), L14–L16 (2008).
[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).

Zhao, P.

M. R. Hight, D. D. Nolting, E. T. McKinley, A. D. Lander, S. K. Wyatt, M. Gonyea, P. Zhao, and H. C. Manning, “Multispectral fluorescence imaging to assess pH in biological specimens,” J. Biomed. Opt.16(1), 016007–016007 (2011).
[CrossRef] [PubMed]

Zimmer, T.

W. Becker, A. Bergmann, C. Biskup, T. Zimmer, N. Klöcker, and K. Benndorf, ”Multi-wavelength TCSPC lifetime imaging” in Proc. SPIEAnonymous 79–84, (2002).

Zuschratter, W.

M. Jose, D. K. Nair, C. Reissner, R. Hartig, and W. Zuschratter, “Photophysics of Clomeleon by FLIM: discriminating excited state reactions along neuronal development,” Biophys. J.92(6), 2237–2254 (2007).
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Figures (8)

Fig. 1
Fig. 1

The Schematic diagram of the Lambda-Tau electronics.

Fig. 2
Fig. 2

a) Plot of the modified semi-circle for different gate numbers. b) The phasor of a bi-exponential decay curve with lifetimes τ 1 /T=0.2 , τ 2 /T=0.4 and fractional intensity α=0.5 (open circle).

Fig. 3
Fig. 3

Spectral phasor plots for Gaussian spectra recorded with K spectral channels and with widths 0, K/8 and K/4. k0 indicates the peak position (wavelength) of the spectrum.

Fig. 4
Fig. 4

a) Figure of merit for total time windows of 12.5ns, 20ns and 50 ns and a gate width of 200 ps and b) for a fixed total time window of 50ns and K = 1000, 500 and 250 corresponding to gate widths of 50ps, 100ps and 200 ps respectively.

Fig. 5
Fig. 5

The figure of merit for spectral widths for different numbers of spectral channels.

Fig. 6
Fig. 6

a) Measured and fitted fluorescence decay curves. b) Fitted lifetimes as a function of detection count rate using a single detection channel. c) As in b) using the signal from 4 combined channels. The dashed line indicates an accuracy of 98%.

Fig. 7
Fig. 7

The phasor plots and fluorescence lifetime images calculated using Eq. (3).

Fig. 8
Fig. 8

Spectral and temporal phasor based segmentation.

Equations (11)

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R( τ )= 1 cos( π K )sin( π K )coth( T 2Kτ )j
R(τ)= 1 1j 2π T τ .
τ= T 2KArccoth( G S cot( π K ) ) .
τ 1,2 = T/2K Arccoth( ± 12 u 2 4uvcos( π K )2 u 2 cos( 2π K )±1 2usin( π K ) ) .
F τ = Δτ τ / ΔN N ,
F τ = Δτ τ N ,
var N (τ)= 1 N τ 2 K 2 r 2 [ 1exp(r) ] ( exp( r/K )[ 1exp( r ) ] [ exp( r/K )1 ] 2 K 2 exp( r )1 ) 1 ,
F τ = var N (τ) τ N = var 1 (τ) .
var 1 (τ)= K 2 r 2 [ 1exp(r) ] ( exp( r/K )[ 1exp( r ) ] [ exp( r/K )1 ] 2 K 2 exp( r )1 ) 1 .
N var 1 (τ) (desired accuracy) 2 .
var N (σ)= 1 N ( i=1 k erf( 2 L 4kσ (k2i+2) )erf( 2 L 4kσ (k2i) ) erf( 2 L 4σ ) ) 1 .

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