Abstract

Spectrally resolved fluorescence lifetime imaging microscopy (λFLIM) has powerful potential for biochemical and medical imaging applications. However, long acquisition times, low spectral resolution and complexity of λFLIM often narrow its use to specialized laboratories. Therefore, we demonstrate here a simple spectral FLIM based on a solid-state detector array providing in-pixel histrogramming and delivering faster acquisition, larger dynamic range, and higher spectral elements than state-of-the-art λFLIM. We successfully apply this novel microscopy system to biochemical and medical imaging demonstrating that solid-state detectors are a key strategic technology to enable complex assays in biomedical laboratories and the clinic.

© 2015 Optical Society of America

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References

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2015 (3)

2014 (3)

J. M. Pavia, M. Wolf, and E. Charbon, “Measurement and modeling of microlenses fabricated on single-photon avalanche diode arrays for fill factor recovery,” Opt. Express 22(4), 4202–4213 (2014).
[Crossref] [PubMed]

T. S. Blacker, Z. F. Mann, J. E. Gale, M. Ziegler, A. J. Bain, G. Szabadkai, and M. R. Duchen, “Separating NADH and NADPH fluorescence in live cells and tissues using FLIM,” Nat. Commun. 5, 3936 (2014).
[Crossref] [PubMed]

F. Fereidouni, A. N. Bader, A. Colonna, and H. C. Gerritsen, “Phasor analysis of multiphoton spectral images distinguishes autofluorescence components of in vivo human skin,” J. Biophotonics 7(8), 589–596 (2014).
[PubMed]

2013 (4)

A. Esposito, M. Popleteeva, and A. R. Venkitaraman, “Maximizing the biochemical resolving power of fluorescence microscopy,” PLoS One 8(10), e77392 (2013).
[Crossref] [PubMed]

F. Fereidouni, K. Reitsma, and H. C. Gerritsen, “High speed multispectral fluorescence lifetime imaging,” Opt. Express 21(10), 11769–11782 (2013).
[Crossref] [PubMed]

F. Fereidouni, G. A. Blab, and H. C. Gerritsen, “Blind unmixing of spectrally resolved lifetime images,” J. Biomed. Opt. 18(8), 086006 (2013).
[Crossref] [PubMed]

D. Bronzi, F. Villa, S. Bellisai, S. Tisa, A. Tosi, G. Ripamonti, F. Zappa, S. Weyers, D. Durini, W. Brockherde, and U. Paschen, “Large Area CMOS SPADs with very low dark counting rate,” Quantum Sensing and Nanophotonic Devices X, 8631 (2013).

2012 (6)

M. Zhao, R. Huang, and L. Peng, “Quantitative multi-color FRET measurements by Fourier lifetime excitation-emission matrix spectroscopy,” Opt. Express 20(24), 26806–26827 (2012).
[Crossref] [PubMed]

D. M. Shcherbakova, M. A. Hink, L. Joosen, T. W. Gadella, and V. V. Verkhusha, “An orange fluorescent protein with a large Stokes shift for single-excitation multicolor FCCS and FRET imaging,” J. Am. Chem. Soc. 134(18), 7913–7923 (2012).
[Crossref] [PubMed]

Q. Zhao, B. Schelen, R. Schouten, R. van den Oever, R. Leenen, H. van Kuijk, I. Peters, F. Polderdijk, J. Bosiers, M. Raspe, K. Jalink, J. Geert Sander de Jong, B. van Geest, K. Stoop, and I. T. Young, “Modulated electron-multiplied fluorescence lifetime imaging microscope: all-solid-state camera for fluorescence lifetime imaging,” J. Biomed. Opt. 17(12), 126020 (2012).
[Crossref] [PubMed]

S. S. Kiwanuka, T. K. Laurila, J. H. Frank, A. Esposito, K. Blomberg von der Geest, L. Pancheri, D. Stoppa, and C. F. Kaminski, “Development of broadband cavity ring-down spectroscopy for biomedical diagnostics of liquid analytes,” Anal. Chem. 84(13), 5489–5493 (2012).
[Crossref] [PubMed]

F. Fereidouni, A. N. Bader, and H. C. Gerritsen, “Spectral phasor analysis allows rapid and reliable unmixing of fluorescence microscopy spectral images,” Opt. Express 20(12), 12729–12741 (2012).
[Crossref] [PubMed]

R. Patalay, C. Talbot, Y. Alexandrov, M. O. Lenz, S. Kumar, S. Warren, I. Munro, M. A. Neil, K. König, P. M. French, A. Chu, G. W. Stamp, and C. Dunsby, “Multiphoton multispectral fluorescence lifetime tomography for the evaluation of basal cell carcinomas,” PLoS One 7(9), e43460 (2012).
[Crossref] [PubMed]

2011 (4)

N. Komatsu, K. Aoki, M. Yamada, H. Yukinaga, Y. Fujita, Y. Kamioka, and M. Matsuda, “Development of an optimized backbone of FRET biosensors for kinases and GTPases,” Mol. Biol. Cell 22(23), 4647–4656 (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]

A. Esposito, A. N. Bader, S. C. Schlachter, D. J. van den Heuvel, G. S. Schierle, A. R. Venkitaraman, C. F. Kaminski, and H. C. Gerritsen, “Design and application of a confocal microscope for spectrally resolved anisotropy imaging,” Opt. Express 19(3), 2546–2555 (2011).
[Crossref] [PubMed]

D. D. U. Li, J. Arlt, D. Tyndall, R. Walker, J. Richardson, D. Stoppa, E. Charbon, and R. K. Henderson, “Video-rate fluorescence lifetime imaging camera with CMOS single-photon avalanche diode arrays and high-speed imaging algorithm,” J. Biomed. Opt. 16(9), 096012 (2011).
[Crossref] [PubMed]

2010 (2)

M. Gersbach, R. Trimananda, Y. Maruyama, M. Fishburn, D. Stoppa, J. Richardson, R. Walker, R. K. Henderson, and E. Charbon, “High frame-rate TCSPC-FLIM using a novel SPAD-based image sensor,” Proc. SPIE 7780, 77801H (2010).

F. Skoulidis, L. D. Cassidy, V. Pisupati, J. G. Jonasson, H. Bjarnason, J. E. Eyfjord, F. A. Karreth, M. Lim, L. M. Barber, S. A. Clatworthy, S. E. Davies, K. P. Olive, D. A. Tuveson, and A. R. Venkitaraman, “Germline Brca2 heterozygosity promotes Kras(G12D) -driven carcinogenesis in a murine model of familial pancreatic cancer,” Cancer Cell 18(5), 499–509 (2010).
[Crossref] [PubMed]

2009 (2)

2008 (3)

H. W. Ai, K. L. Hazelwood, M. W. Davidson, and R. E. Campbell, “Fluorescent protein FRET pairs for ratiometric imaging of dual biosensors,” Nat. Methods 5(5), 401–403 (2008).
[Crossref] [PubMed]

D. M. Grant, W. Zhang, E. J. McGhee, T. D. Bunney, C. B. Talbot, S. Kumar, I. Munro, C. Dunsby, M. A. Neil, M. Katan, and P. M. French, “Multiplexed FRET to image multiple signaling events in live cells,” Biophys. J. 95(10), L69–L71 (2008).
[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]

2007 (2)

S. R. Kantelhardt, J. Leppert, J. Krajewski, N. Petkus, E. Reusche, V. M. Tronnier, G. Hüttmann, and A. Giese, “Imaging of brain and brain tumor specimens by time-resolved multiphoton excitation microscopy ex vivo,” Neuro-oncol. 9(2), 103–112 (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]

2006 (3)

2005 (1)

Q. S. Hanley and A. H. Clayton, “AB-plot assisted determination of fluorophore mixtures in a fluorescence lifetime microscope using spectra or quenchers,” J. Microsc. 218(1), 62–67 (2005).
[Crossref] [PubMed]

2004 (3)

A. H. Clayton, Q. S. Hanley, and P. J. Verveer, “Graphical representation and multicomponent analysis of single-frequency fluorescence lifetime imaging microscopy data,” J. Microsc. 213(1), 1–5 (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]

W. Becker, A. Bergmann, G. Biscotti, K. König, I. Riemann, L. Kelbauskas, and C. Biskup, “High-speed FLIM data acquisition by time-correlated single photon counting,” Proc. SPIE 5223, 1–14 (2004).
[Crossref]

2003 (1)

E. A. Jares-Erijman and T. M. Jovin, “FRET imaging,” Nat. Biotechnol. 21(11), 1387–1395 (2003).
[Crossref] [PubMed]

2002 (4)

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]

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]

W. Becker, A. Bergmann, C. Biskup, T. Zimmer, N. Klocker, and K. Benndorf, “Multi-wavelength TCSPC lifetime imaging,” Multiphoton Microsc. Biomed. Sci. II 4620, 79–84 (2002).
[Crossref]

A. C. Mitchell, J. E. Wall, J. G. Murray, and C. G. Morgan, “Measurement of nanosecond time-resolved fluorescence with a directly gated interline CCD camera,” J. Microsc. 206(3), 233–238 (2002).
[Crossref] [PubMed]

2001 (1)

1996 (2)

D. McLoskey, D. J. S. Birch, A. Sanderson, K. Suhling, E. Welch, and P. J. Hicks, “Multiplexed single-photon counting. 1. A time-correlated fluorescence lifetime camera,” Rev. Sci. Instrum. 67(6), 2228–2237 (1996).
[Crossref]

K. Suhling, D. McLoskey, and D. J. S. Birch, “Multiplexed single-photon counting.2. The statistical theory of time-correlated measurements,” Rev. Sci. Instrum. 67(6), 2238–2246 (1996).
[Crossref]

1992 (1)

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

1971 (1)

W. R. Ware, S. K. Lee, G. J. Brant, and P. P. Chow, “Nanosecond time-resolved rmission rpectroscopy - spectral shifts due to solvent-excited solute relaxation,” J. Chem. Phys. 54(11), 4729–4737 (1971).
[Crossref]

Agronskaia, A. V.

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]

Ai, H. W.

H. W. Ai, K. L. Hazelwood, M. W. Davidson, and R. E. Campbell, “Fluorescent protein FRET pairs for ratiometric imaging of dual biosensors,” Nat. Methods 5(5), 401–403 (2008).
[Crossref] [PubMed]

Alexandrov, Y.

R. Patalay, C. Talbot, Y. Alexandrov, M. O. Lenz, S. Kumar, S. Warren, I. Munro, M. A. Neil, K. König, P. M. French, A. Chu, G. W. Stamp, and C. Dunsby, “Multiphoton multispectral fluorescence lifetime tomography for the evaluation of basal cell carcinomas,” PLoS One 7(9), e43460 (2012).
[Crossref] [PubMed]

Ameer-Beg, S.

Ameer-Beg, S. M.

Aoki, K.

N. Komatsu, K. Aoki, M. Yamada, H. Yukinaga, Y. Fujita, Y. Kamioka, and M. Matsuda, “Development of an optimized backbone of FRET biosensors for kinases and GTPases,” Mol. Biol. Cell 22(23), 4647–4656 (2011).
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Arlt, J.

D. D. U. Li, J. Arlt, D. Tyndall, R. Walker, J. Richardson, D. Stoppa, E. Charbon, and R. K. Henderson, “Video-rate fluorescence lifetime imaging camera with CMOS single-photon avalanche diode arrays and high-speed imaging algorithm,” J. Biomed. Opt. 16(9), 096012 (2011).
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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).
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Avezov, E.

W. Chen, E. Avezov, S. C. Schlachter, F. Gielen, R. F. Laine, H. P. Harding, F. Hollfelder, D. Ron, and C. F. Kaminski, “A method to quantify FRET stoichiometry with phasor plot analysis and acceptor lifetime ingrowth,” Biophys. J. 108(5), 999–1002 (2015).
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Bader, A. N.

Bain, A. J.

T. S. Blacker, Z. F. Mann, J. E. Gale, M. Ziegler, A. J. Bain, G. Szabadkai, and M. R. Duchen, “Separating NADH and NADPH fluorescence in live cells and tissues using FLIM,” Nat. Commun. 5, 3936 (2014).
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F. Skoulidis, L. D. Cassidy, V. Pisupati, J. G. Jonasson, H. Bjarnason, J. E. Eyfjord, F. A. Karreth, M. Lim, L. M. Barber, S. A. Clatworthy, S. E. Davies, K. P. Olive, D. A. Tuveson, and A. R. Venkitaraman, “Germline Brca2 heterozygosity promotes Kras(G12D) -driven carcinogenesis in a murine model of familial pancreatic cancer,” Cancer Cell 18(5), 499–509 (2010).
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Barber, P.

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).
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W. Becker, A. Bergmann, G. Biscotti, K. König, I. Riemann, L. Kelbauskas, and C. Biskup, “High-speed FLIM data acquisition by time-correlated single photon counting,” Proc. SPIE 5223, 1–14 (2004).
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W. Becker, A. Bergmann, C. Biskup, T. Zimmer, N. Klocker, and K. Benndorf, “Multi-wavelength TCSPC lifetime imaging,” Multiphoton Microsc. Biomed. Sci. II 4620, 79–84 (2002).
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Bellisai, S.

D. Bronzi, F. Villa, S. Bellisai, S. Tisa, A. Tosi, G. Ripamonti, F. Zappa, S. Weyers, D. Durini, W. Brockherde, and U. Paschen, “Large Area CMOS SPADs with very low dark counting rate,” Quantum Sensing and Nanophotonic Devices X, 8631 (2013).

Benndorf, K.

W. Becker, A. Bergmann, C. Biskup, T. Zimmer, N. Klocker, and K. Benndorf, “Multi-wavelength TCSPC lifetime imaging,” Multiphoton Microsc. Biomed. Sci. II 4620, 79–84 (2002).
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Bergmann, A.

W. Becker, A. Bergmann, G. Biscotti, K. König, I. Riemann, L. Kelbauskas, and C. Biskup, “High-speed FLIM data acquisition by time-correlated single photon counting,” Proc. SPIE 5223, 1–14 (2004).
[Crossref]

W. Becker, A. Bergmann, C. Biskup, T. Zimmer, N. Klocker, and K. Benndorf, “Multi-wavelength TCSPC lifetime imaging,” Multiphoton Microsc. Biomed. Sci. II 4620, 79–84 (2002).
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Birch, D. J. S.

D. McLoskey, D. J. S. Birch, A. Sanderson, K. Suhling, E. Welch, and P. J. Hicks, “Multiplexed single-photon counting. 1. A time-correlated fluorescence lifetime camera,” Rev. Sci. Instrum. 67(6), 2228–2237 (1996).
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K. Suhling, D. McLoskey, and D. J. S. Birch, “Multiplexed single-photon counting.2. The statistical theory of time-correlated measurements,” Rev. Sci. Instrum. 67(6), 2238–2246 (1996).
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Bird, D. K.

Biscotti, G.

W. Becker, A. Bergmann, G. Biscotti, K. König, I. Riemann, L. Kelbauskas, and C. Biskup, “High-speed FLIM data acquisition by time-correlated single photon counting,” Proc. SPIE 5223, 1–14 (2004).
[Crossref]

Biskup, C.

W. Becker, A. Bergmann, G. Biscotti, K. König, I. Riemann, L. Kelbauskas, and C. Biskup, “High-speed FLIM data acquisition by time-correlated single photon counting,” Proc. SPIE 5223, 1–14 (2004).
[Crossref]

W. Becker, A. Bergmann, C. Biskup, T. Zimmer, N. Klocker, and K. Benndorf, “Multi-wavelength TCSPC lifetime imaging,” Multiphoton Microsc. Biomed. Sci. II 4620, 79–84 (2002).
[Crossref]

Bjarnason, H.

F. Skoulidis, L. D. Cassidy, V. Pisupati, J. G. Jonasson, H. Bjarnason, J. E. Eyfjord, F. A. Karreth, M. Lim, L. M. Barber, S. A. Clatworthy, S. E. Davies, K. P. Olive, D. A. Tuveson, and A. R. Venkitaraman, “Germline Brca2 heterozygosity promotes Kras(G12D) -driven carcinogenesis in a murine model of familial pancreatic cancer,” Cancer Cell 18(5), 499–509 (2010).
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Blab, G. A.

F. Fereidouni, G. A. Blab, and H. C. Gerritsen, “Blind unmixing of spectrally resolved lifetime images,” J. Biomed. Opt. 18(8), 086006 (2013).
[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]

Blacker, T. S.

T. S. Blacker, Z. F. Mann, J. E. Gale, M. Ziegler, A. J. Bain, G. Szabadkai, and M. R. Duchen, “Separating NADH and NADPH fluorescence in live cells and tissues using FLIM,” Nat. Commun. 5, 3936 (2014).
[Crossref] [PubMed]

Blomberg von der Geest, K.

S. S. Kiwanuka, T. K. Laurila, J. H. Frank, A. Esposito, K. Blomberg von der Geest, L. Pancheri, D. Stoppa, and C. F. Kaminski, “Development of broadband cavity ring-down spectroscopy for biomedical diagnostics of liquid analytes,” Anal. Chem. 84(13), 5489–5493 (2012).
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Borghetti, F.

F. Borghetti, D. Mosconi, L. Pancheri, and D. Stoppa, “CMOS single-photon avalanche diode sensor for fluorescence lifetime imaging,” in IEEE International Image Sensors Workshop, 2007), 7–10.

Bosiers, J.

Q. Zhao, B. Schelen, R. Schouten, R. van den Oever, R. Leenen, H. van Kuijk, I. Peters, F. Polderdijk, J. Bosiers, M. Raspe, K. Jalink, J. Geert Sander de Jong, B. van Geest, K. Stoop, and I. T. Young, “Modulated electron-multiplied fluorescence lifetime imaging microscope: all-solid-state camera for fluorescence lifetime imaging,” J. Biomed. Opt. 17(12), 126020 (2012).
[Crossref] [PubMed]

Brant, G. J.

W. R. Ware, S. K. Lee, G. J. Brant, and P. P. Chow, “Nanosecond time-resolved rmission rpectroscopy - spectral shifts due to solvent-excited solute relaxation,” J. Chem. Phys. 54(11), 4729–4737 (1971).
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Brockherde, W.

D. Bronzi, F. Villa, S. Bellisai, S. Tisa, A. Tosi, G. Ripamonti, F. Zappa, S. Weyers, D. Durini, W. Brockherde, and U. Paschen, “Large Area CMOS SPADs with very low dark counting rate,” Quantum Sensing and Nanophotonic Devices X, 8631 (2013).

Bronzi, D.

D. Bronzi, F. Villa, S. Bellisai, S. Tisa, A. Tosi, G. Ripamonti, F. Zappa, S. Weyers, D. Durini, W. Brockherde, and U. Paschen, “Large Area CMOS SPADs with very low dark counting rate,” Quantum Sensing and Nanophotonic Devices X, 8631 (2013).

Bunney, T. D.

D. M. Grant, W. Zhang, E. J. McGhee, T. D. Bunney, C. B. Talbot, S. Kumar, I. Munro, C. Dunsby, M. A. Neil, M. Katan, and P. M. French, “Multiplexed FRET to image multiple signaling events in live cells,” Biophys. J. 95(10), L69–L71 (2008).
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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).
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Campbell, R. E.

H. W. Ai, K. L. Hazelwood, M. W. Davidson, and R. E. Campbell, “Fluorescent protein FRET pairs for ratiometric imaging of dual biosensors,” Nat. Methods 5(5), 401–403 (2008).
[Crossref] [PubMed]

Cassidy, L. D.

F. Skoulidis, L. D. Cassidy, V. Pisupati, J. G. Jonasson, H. Bjarnason, J. E. Eyfjord, F. A. Karreth, M. Lim, L. M. Barber, S. A. Clatworthy, S. E. Davies, K. P. Olive, D. A. Tuveson, and A. R. Venkitaraman, “Germline Brca2 heterozygosity promotes Kras(G12D) -driven carcinogenesis in a murine model of familial pancreatic cancer,” Cancer Cell 18(5), 499–509 (2010).
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Charbon, E.

J. M. Pavia, M. Wolf, and E. Charbon, “Measurement and modeling of microlenses fabricated on single-photon avalanche diode arrays for fill factor recovery,” Opt. Express 22(4), 4202–4213 (2014).
[Crossref] [PubMed]

D. D. U. Li, J. Arlt, D. Tyndall, R. Walker, J. Richardson, D. Stoppa, E. Charbon, and R. K. Henderson, “Video-rate fluorescence lifetime imaging camera with CMOS single-photon avalanche diode arrays and high-speed imaging algorithm,” J. Biomed. Opt. 16(9), 096012 (2011).
[Crossref] [PubMed]

M. Gersbach, R. Trimananda, Y. Maruyama, M. Fishburn, D. Stoppa, J. Richardson, R. Walker, R. K. Henderson, and E. Charbon, “High frame-rate TCSPC-FLIM using a novel SPAD-based image sensor,” Proc. SPIE 7780, 77801H (2010).

Chen, W.

W. Chen, E. Avezov, S. C. Schlachter, F. Gielen, R. F. Laine, H. P. Harding, F. Hollfelder, D. Ron, and C. F. Kaminski, “A method to quantify FRET stoichiometry with phasor plot analysis and acceptor lifetime ingrowth,” Biophys. J. 108(5), 999–1002 (2015).
[Crossref] [PubMed]

Chow, P. P.

W. R. Ware, S. K. Lee, G. J. Brant, and P. P. Chow, “Nanosecond time-resolved rmission rpectroscopy - spectral shifts due to solvent-excited solute relaxation,” J. Chem. Phys. 54(11), 4729–4737 (1971).
[Crossref]

Chu, A.

R. Patalay, C. Talbot, Y. Alexandrov, M. O. Lenz, S. Kumar, S. Warren, I. Munro, M. A. Neil, K. König, P. M. French, A. Chu, G. W. Stamp, and C. Dunsby, “Multiphoton multispectral fluorescence lifetime tomography for the evaluation of basal cell carcinomas,” PLoS One 7(9), e43460 (2012).
[Crossref] [PubMed]

Clatworthy, S. A.

F. Skoulidis, L. D. Cassidy, V. Pisupati, J. G. Jonasson, H. Bjarnason, J. E. Eyfjord, F. A. Karreth, M. Lim, L. M. Barber, S. A. Clatworthy, S. E. Davies, K. P. Olive, D. A. Tuveson, and A. R. Venkitaraman, “Germline Brca2 heterozygosity promotes Kras(G12D) -driven carcinogenesis in a murine model of familial pancreatic cancer,” Cancer Cell 18(5), 499–509 (2010).
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Clayton, A. H.

Q. S. Hanley and A. H. Clayton, “AB-plot assisted determination of fluorophore mixtures in a fluorescence lifetime microscope using spectra or quenchers,” J. Microsc. 218(1), 62–67 (2005).
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A. H. Clayton, Q. S. Hanley, and P. J. Verveer, “Graphical representation and multicomponent analysis of single-frequency fluorescence lifetime imaging microscopy data,” J. Microsc. 213(1), 1–5 (2004).
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Coelho, S.

Colonna, A.

F. Fereidouni, A. N. Bader, A. Colonna, and H. C. Gerritsen, “Phasor analysis of multiphoton spectral images distinguishes autofluorescence components of in vivo human skin,” J. Biophotonics 7(8), 589–596 (2014).
[PubMed]

Davidson, M. W.

H. W. Ai, K. L. Hazelwood, M. W. Davidson, and R. E. Campbell, “Fluorescent protein FRET pairs for ratiometric imaging of dual biosensors,” Nat. Methods 5(5), 401–403 (2008).
[Crossref] [PubMed]

Davies, S. E.

F. Skoulidis, L. D. Cassidy, V. Pisupati, J. G. Jonasson, H. Bjarnason, J. E. Eyfjord, F. A. Karreth, M. Lim, L. M. Barber, S. A. Clatworthy, S. E. Davies, K. P. Olive, D. A. Tuveson, and A. R. Venkitaraman, “Germline Brca2 heterozygosity promotes Kras(G12D) -driven carcinogenesis in a murine model of familial pancreatic cancer,” Cancer Cell 18(5), 499–509 (2010).
[Crossref] [PubMed]

de Bruijn, H. S.

de Grauw, C. J.

Devauges, V.

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]

Duchen, M. R.

T. S. Blacker, Z. F. Mann, J. E. Gale, M. Ziegler, A. J. Bain, G. Szabadkai, and M. R. Duchen, “Separating NADH and NADPH fluorescence in live cells and tissues using FLIM,” Nat. Commun. 5, 3936 (2014).
[Crossref] [PubMed]

Dunsby, C.

R. Patalay, C. Talbot, Y. Alexandrov, M. O. Lenz, S. Kumar, S. Warren, I. Munro, M. A. Neil, K. König, P. M. French, A. Chu, G. W. Stamp, and C. Dunsby, “Multiphoton multispectral fluorescence lifetime tomography for the evaluation of basal cell carcinomas,” PLoS One 7(9), e43460 (2012).
[Crossref] [PubMed]

D. M. Grant, W. Zhang, E. J. McGhee, T. D. Bunney, C. B. Talbot, S. Kumar, I. Munro, C. Dunsby, M. A. Neil, M. Katan, and P. M. French, “Multiplexed FRET to image multiple signaling events in live cells,” Biophys. J. 95(10), L69–L71 (2008).
[Crossref] [PubMed]

Durini, D.

D. Bronzi, F. Villa, S. Bellisai, S. Tisa, A. Tosi, G. Ripamonti, F. Zappa, S. Weyers, D. Durini, W. Brockherde, and U. Paschen, “Large Area CMOS SPADs with very low dark counting rate,” Quantum Sensing and Nanophotonic Devices X, 8631 (2013).

Dutton, N.

Eliceiri, K. W.

Esposito, A.

A. Esposito, M. Popleteeva, and A. R. Venkitaraman, “Maximizing the biochemical resolving power of fluorescence microscopy,” PLoS One 8(10), e77392 (2013).
[Crossref] [PubMed]

S. S. Kiwanuka, T. K. Laurila, J. H. Frank, A. Esposito, K. Blomberg von der Geest, L. Pancheri, D. Stoppa, and C. F. Kaminski, “Development of broadband cavity ring-down spectroscopy for biomedical diagnostics of liquid analytes,” Anal. Chem. 84(13), 5489–5493 (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]

A. Esposito, A. N. Bader, S. C. Schlachter, D. J. van den Heuvel, G. S. Schierle, A. R. Venkitaraman, C. F. Kaminski, and H. C. Gerritsen, “Design and application of a confocal microscope for spectrally resolved anisotropy imaging,” Opt. Express 19(3), 2546–2555 (2011).
[Crossref] [PubMed]

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. Express 17(25), 22747–22760 (2009).
[Crossref] [PubMed]

A. Esposito, H. C. Gerritsen, T. Oggier, F. Lustenberger, and F. S. Wouters, “Innovating lifetime microscopy: a compact and simple tool for life sciences, screening, and diagnostics,” J. Biomed. Opt. 11(3), 034016 (2006).
[Crossref] [PubMed]

Eyfjord, J. E.

F. Skoulidis, L. D. Cassidy, V. Pisupati, J. G. Jonasson, H. Bjarnason, J. E. Eyfjord, F. A. Karreth, M. Lim, L. M. Barber, S. A. Clatworthy, S. E. Davies, K. P. Olive, D. A. Tuveson, and A. R. Venkitaraman, “Germline Brca2 heterozygosity promotes Kras(G12D) -driven carcinogenesis in a murine model of familial pancreatic cancer,” Cancer Cell 18(5), 499–509 (2010).
[Crossref] [PubMed]

Fan, C. H.

Fereidouni, F.

F. Fereidouni, A. N. Bader, A. Colonna, and H. C. Gerritsen, “Phasor analysis of multiphoton spectral images distinguishes autofluorescence components of in vivo human skin,” J. Biophotonics 7(8), 589–596 (2014).
[PubMed]

F. Fereidouni, K. Reitsma, and H. C. Gerritsen, “High speed multispectral fluorescence lifetime imaging,” Opt. Express 21(10), 11769–11782 (2013).
[Crossref] [PubMed]

F. Fereidouni, G. A. Blab, and H. C. Gerritsen, “Blind unmixing of spectrally resolved lifetime images,” J. Biomed. Opt. 18(8), 086006 (2013).
[Crossref] [PubMed]

F. Fereidouni, A. N. Bader, and H. C. Gerritsen, “Spectral phasor analysis allows rapid and reliable unmixing of fluorescence microscopy spectral images,” Opt. Express 20(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]

Fishburn, M.

M. Gersbach, R. Trimananda, Y. Maruyama, M. Fishburn, D. Stoppa, J. Richardson, R. Walker, R. K. Henderson, and E. Charbon, “High frame-rate TCSPC-FLIM using a novel SPAD-based image sensor,” Proc. SPIE 7780, 77801H (2010).

Forde, T. S.

Frank, J. H.

S. S. Kiwanuka, T. K. Laurila, J. H. Frank, A. Esposito, K. Blomberg von der Geest, L. Pancheri, D. Stoppa, and C. F. Kaminski, “Development of broadband cavity ring-down spectroscopy for biomedical diagnostics of liquid analytes,” Anal. Chem. 84(13), 5489–5493 (2012).
[Crossref] [PubMed]

French, P. M.

R. Patalay, C. Talbot, Y. Alexandrov, M. O. Lenz, S. Kumar, S. Warren, I. Munro, M. A. Neil, K. König, P. M. French, A. Chu, G. W. Stamp, and C. Dunsby, “Multiphoton multispectral fluorescence lifetime tomography for the evaluation of basal cell carcinomas,” PLoS One 7(9), e43460 (2012).
[Crossref] [PubMed]

D. M. Grant, W. Zhang, E. J. McGhee, T. D. Bunney, C. B. Talbot, S. Kumar, I. Munro, C. Dunsby, M. A. Neil, M. Katan, and P. M. French, “Multiplexed FRET to image multiple signaling events in live cells,” Biophys. J. 95(10), L69–L71 (2008).
[Crossref] [PubMed]

Fujita, Y.

N. Komatsu, K. Aoki, M. Yamada, H. Yukinaga, Y. Fujita, Y. Kamioka, and M. Matsuda, “Development of an optimized backbone of FRET biosensors for kinases and GTPases,” Mol. Biol. Cell 22(23), 4647–4656 (2011).
[Crossref] [PubMed]

Gadella, T. W.

D. M. Shcherbakova, M. A. Hink, L. Joosen, T. W. Gadella, and V. V. Verkhusha, “An orange fluorescent protein with a large Stokes shift for single-excitation multicolor FCCS and FRET imaging,” J. Am. Chem. Soc. 134(18), 7913–7923 (2012).
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Gale, J. E.

T. S. Blacker, Z. F. Mann, J. E. Gale, M. Ziegler, A. J. Bain, G. Szabadkai, and M. R. Duchen, “Separating NADH and NADPH fluorescence in live cells and tissues using FLIM,” Nat. Commun. 5, 3936 (2014).
[Crossref] [PubMed]

Geert Sander de Jong, J.

Q. Zhao, B. Schelen, R. Schouten, R. van den Oever, R. Leenen, H. van Kuijk, I. Peters, F. Polderdijk, J. Bosiers, M. Raspe, K. Jalink, J. Geert Sander de Jong, B. van Geest, K. Stoop, and I. T. Young, “Modulated electron-multiplied fluorescence lifetime imaging microscope: all-solid-state camera for fluorescence lifetime imaging,” J. Biomed. Opt. 17(12), 126020 (2012).
[Crossref] [PubMed]

Gerritsen, H. C.

F. Fereidouni, A. N. Bader, A. Colonna, and H. C. Gerritsen, “Phasor analysis of multiphoton spectral images distinguishes autofluorescence components of in vivo human skin,” J. Biophotonics 7(8), 589–596 (2014).
[PubMed]

F. Fereidouni, K. Reitsma, and H. C. Gerritsen, “High speed multispectral fluorescence lifetime imaging,” Opt. Express 21(10), 11769–11782 (2013).
[Crossref] [PubMed]

F. Fereidouni, G. A. Blab, and H. C. Gerritsen, “Blind unmixing of spectrally resolved lifetime images,” J. Biomed. Opt. 18(8), 086006 (2013).
[Crossref] [PubMed]

F. Fereidouni, A. N. Bader, and H. C. Gerritsen, “Spectral phasor analysis allows rapid and reliable unmixing of fluorescence microscopy spectral images,” Opt. Express 20(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]

A. Esposito, A. N. Bader, S. C. Schlachter, D. J. van den Heuvel, G. S. Schierle, A. R. Venkitaraman, C. F. Kaminski, and H. C. Gerritsen, “Design and application of a confocal microscope for spectrally resolved anisotropy imaging,” Opt. Express 19(3), 2546–2555 (2011).
[Crossref] [PubMed]

J. A. Palero, H. S. de Bruijn, A. van der Ploeg-van den Heuvel, H. J. C. M. Sterenborg, and H. C. Gerritsen, “In vivo nonlinear spectral imaging in mouse skin,” Opt. Express 14(10), 4395–4402 (2006).
[Crossref] [PubMed]

A. Esposito, H. C. Gerritsen, T. Oggier, F. Lustenberger, and F. S. Wouters, “Innovating lifetime microscopy: a compact and simple tool for life sciences, screening, and diagnostics,” J. Biomed. Opt. 11(3), 034016 (2006).
[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]

C. J. de Grauw and H. C. Gerritsen, “Multiple time-gate module for fluorescence lifetime imaging,” Appl. Spectrosc. 55(6), 670–678 (2001).
[Crossref]

Gersbach, M.

M. Gersbach, R. Trimananda, Y. Maruyama, M. Fishburn, D. Stoppa, J. Richardson, R. Walker, R. K. Henderson, and E. Charbon, “High frame-rate TCSPC-FLIM using a novel SPAD-based image sensor,” Proc. SPIE 7780, 77801H (2010).

Gielen, F.

W. Chen, E. Avezov, S. C. Schlachter, F. Gielen, R. F. Laine, H. P. Harding, F. Hollfelder, D. Ron, and C. F. Kaminski, “A method to quantify FRET stoichiometry with phasor plot analysis and acceptor lifetime ingrowth,” Biophys. J. 108(5), 999–1002 (2015).
[Crossref] [PubMed]

Giese, A.

S. R. Kantelhardt, J. Leppert, J. Krajewski, N. Petkus, E. Reusche, V. M. Tronnier, G. Hüttmann, and A. Giese, “Imaging of brain and brain tumor specimens by time-resolved multiphoton excitation microscopy ex vivo,” Neuro-oncol. 9(2), 103–112 (2007).
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Grant, D. M.

D. M. Grant, W. Zhang, E. J. McGhee, T. D. Bunney, C. B. Talbot, S. Kumar, I. Munro, C. Dunsby, M. A. Neil, M. Katan, and P. M. French, “Multiplexed FRET to image multiple signaling events in live cells,” Biophys. J. 95(10), L69–L71 (2008).
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S. P. Poland, N. Krstajić, J. Monypenny, S. Coelho, D. Tyndall, R. J. Walker, V. Devauges, J. Richardson, N. Dutton, P. Barber, D. D. Li, K. Suhling, T. Ng, R. K. Henderson, and S. M. Ameer-Beg, “A high speed multifocal multiphoton fluorescence lifetime imaging microscope for live-cell FRET imaging,” Biomed. Opt. Express 6(2), 277–296 (2015).
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D. D. U. Li, J. Arlt, D. Tyndall, R. Walker, J. Richardson, D. Stoppa, E. Charbon, and R. K. Henderson, “Video-rate fluorescence lifetime imaging camera with CMOS single-photon avalanche diode arrays and high-speed imaging algorithm,” J. Biomed. Opt. 16(9), 096012 (2011).
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M. Gersbach, R. Trimananda, Y. Maruyama, M. Fishburn, D. Stoppa, J. Richardson, R. Walker, R. K. Henderson, and E. Charbon, “High frame-rate TCSPC-FLIM using a novel SPAD-based image sensor,” Proc. SPIE 7780, 77801H (2010).

Riemann, I.

W. Becker, A. Bergmann, G. Biscotti, K. König, I. Riemann, L. Kelbauskas, and C. Biskup, “High-speed FLIM data acquisition by time-correlated single photon counting,” Proc. SPIE 5223, 1–14 (2004).
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D. Bronzi, F. Villa, S. Bellisai, S. Tisa, A. Tosi, G. Ripamonti, F. Zappa, S. Weyers, D. Durini, W. Brockherde, and U. Paschen, “Large Area CMOS SPADs with very low dark counting rate,” Quantum Sensing and Nanophotonic Devices X, 8631 (2013).

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W. Chen, E. Avezov, S. C. Schlachter, F. Gielen, R. F. Laine, H. P. Harding, F. Hollfelder, D. Ron, and C. F. Kaminski, “A method to quantify FRET stoichiometry with phasor plot analysis and acceptor lifetime ingrowth,” Biophys. J. 108(5), 999–1002 (2015).
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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).
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D. McLoskey, D. J. S. Birch, A. Sanderson, K. Suhling, E. Welch, and P. J. Hicks, “Multiplexed single-photon counting. 1. A time-correlated fluorescence lifetime camera,” Rev. Sci. Instrum. 67(6), 2228–2237 (1996).
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Schlachter, S.

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W. Chen, E. Avezov, S. C. Schlachter, F. Gielen, R. F. Laine, H. P. Harding, F. Hollfelder, D. Ron, and C. F. Kaminski, “A method to quantify FRET stoichiometry with phasor plot analysis and acceptor lifetime ingrowth,” Biophys. J. 108(5), 999–1002 (2015).
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A. Esposito, A. N. Bader, S. C. Schlachter, D. J. van den Heuvel, G. S. Schierle, A. R. Venkitaraman, C. F. Kaminski, and H. C. Gerritsen, “Design and application of a confocal microscope for spectrally resolved anisotropy imaging,” Opt. Express 19(3), 2546–2555 (2011).
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D. M. Shcherbakova, M. A. Hink, L. Joosen, T. W. Gadella, and V. V. Verkhusha, “An orange fluorescent protein with a large Stokes shift for single-excitation multicolor FCCS and FRET imaging,” J. Am. Chem. Soc. 134(18), 7913–7923 (2012).
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D. D. U. Li, J. Arlt, D. Tyndall, R. Walker, J. Richardson, D. Stoppa, E. Charbon, and R. K. Henderson, “Video-rate fluorescence lifetime imaging camera with CMOS single-photon avalanche diode arrays and high-speed imaging algorithm,” J. Biomed. Opt. 16(9), 096012 (2011).
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White, J. G.

Wolf, M.

Wolfrum, J.

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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).
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D. Bronzi, F. Villa, S. Bellisai, S. Tisa, A. Tosi, G. Ripamonti, F. Zappa, S. Weyers, D. Durini, W. Brockherde, and U. Paschen, “Large Area CMOS SPADs with very low dark counting rate,” Quantum Sensing and Nanophotonic Devices X, 8631 (2013).

Zhang, W.

D. M. Grant, W. Zhang, E. J. McGhee, T. D. Bunney, C. B. Talbot, S. Kumar, I. Munro, C. Dunsby, M. A. Neil, M. Katan, and P. M. French, “Multiplexed FRET to image multiple signaling events in live cells,” Biophys. J. 95(10), L69–L71 (2008).
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Zhao, Q.

Q. Zhao, B. Schelen, R. Schouten, R. van den Oever, R. Leenen, H. van Kuijk, I. Peters, F. Polderdijk, J. Bosiers, M. Raspe, K. Jalink, J. Geert Sander de Jong, B. van Geest, K. Stoop, and I. T. Young, “Modulated electron-multiplied fluorescence lifetime imaging microscope: all-solid-state camera for fluorescence lifetime imaging,” J. Biomed. Opt. 17(12), 126020 (2012).
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T. S. Blacker, Z. F. Mann, J. E. Gale, M. Ziegler, A. J. Bain, G. Szabadkai, and M. R. Duchen, “Separating NADH and NADPH fluorescence in live cells and tissues using FLIM,” Nat. Commun. 5, 3936 (2014).
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Anal. Chem. (1)

S. S. Kiwanuka, T. K. Laurila, J. H. Frank, A. Esposito, K. Blomberg von der Geest, L. Pancheri, D. Stoppa, and C. F. Kaminski, “Development of broadband cavity ring-down spectroscopy for biomedical diagnostics of liquid analytes,” Anal. Chem. 84(13), 5489–5493 (2012).
[Crossref] [PubMed]

Appl. Opt. (1)

Appl. Spectrosc. (3)

Biomed. Opt. Express (1)

Biophys. J. (3)

D. M. Grant, W. Zhang, E. J. McGhee, T. D. Bunney, C. B. Talbot, S. Kumar, I. Munro, C. Dunsby, M. A. Neil, M. Katan, and P. M. French, “Multiplexed FRET to image multiple signaling events in live cells,” Biophys. J. 95(10), L69–L71 (2008).
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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).
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W. Chen, E. Avezov, S. C. Schlachter, F. Gielen, R. F. Laine, H. P. Harding, F. Hollfelder, D. Ron, and C. F. Kaminski, “A method to quantify FRET stoichiometry with phasor plot analysis and acceptor lifetime ingrowth,” Biophys. J. 108(5), 999–1002 (2015).
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Cancer Cell (1)

F. Skoulidis, L. D. Cassidy, V. Pisupati, J. G. Jonasson, H. Bjarnason, J. E. Eyfjord, F. A. Karreth, M. Lim, L. M. Barber, S. A. Clatworthy, S. E. Davies, K. P. Olive, D. A. Tuveson, and A. R. Venkitaraman, “Germline Brca2 heterozygosity promotes Kras(G12D) -driven carcinogenesis in a murine model of familial pancreatic cancer,” Cancer Cell 18(5), 499–509 (2010).
[Crossref] [PubMed]

Chem. Phys. Lett. (1)

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

J. Am. Chem. Soc. (1)

D. M. Shcherbakova, M. A. Hink, L. Joosen, T. W. Gadella, and V. V. Verkhusha, “An orange fluorescent protein with a large Stokes shift for single-excitation multicolor FCCS and FRET imaging,” J. Am. Chem. Soc. 134(18), 7913–7923 (2012).
[Crossref] [PubMed]

J. Biomed. Opt. (4)

A. Esposito, H. C. Gerritsen, T. Oggier, F. Lustenberger, and F. S. Wouters, “Innovating lifetime microscopy: a compact and simple tool for life sciences, screening, and diagnostics,” J. Biomed. Opt. 11(3), 034016 (2006).
[Crossref] [PubMed]

Q. Zhao, B. Schelen, R. Schouten, R. van den Oever, R. Leenen, H. van Kuijk, I. Peters, F. Polderdijk, J. Bosiers, M. Raspe, K. Jalink, J. Geert Sander de Jong, B. van Geest, K. Stoop, and I. T. Young, “Modulated electron-multiplied fluorescence lifetime imaging microscope: all-solid-state camera for fluorescence lifetime imaging,” J. Biomed. Opt. 17(12), 126020 (2012).
[Crossref] [PubMed]

D. D. U. Li, J. Arlt, D. Tyndall, R. Walker, J. Richardson, D. Stoppa, E. Charbon, and R. K. Henderson, “Video-rate fluorescence lifetime imaging camera with CMOS single-photon avalanche diode arrays and high-speed imaging algorithm,” J. Biomed. Opt. 16(9), 096012 (2011).
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F. Fereidouni, G. A. Blab, and H. C. Gerritsen, “Blind unmixing of spectrally resolved lifetime images,” J. Biomed. Opt. 18(8), 086006 (2013).
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J. Biophotonics (1)

F. Fereidouni, A. N. Bader, A. Colonna, and H. C. Gerritsen, “Phasor analysis of multiphoton spectral images distinguishes autofluorescence components of in vivo human skin,” J. Biophotonics 7(8), 589–596 (2014).
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W. R. Ware, S. K. Lee, G. J. Brant, and P. P. Chow, “Nanosecond time-resolved rmission rpectroscopy - spectral shifts due to solvent-excited solute relaxation,” J. Chem. Phys. 54(11), 4729–4737 (1971).
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J. Microsc. (5)

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).
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A. H. Clayton, Q. S. Hanley, and P. J. Verveer, “Graphical representation and multicomponent analysis of single-frequency fluorescence lifetime imaging microscopy data,” J. Microsc. 213(1), 1–5 (2004).
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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).
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A. C. Mitchell, J. E. Wall, J. G. Murray, and C. G. Morgan, “Measurement of nanosecond time-resolved fluorescence with a directly gated interline CCD camera,” J. Microsc. 206(3), 233–238 (2002).
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Q. S. Hanley and A. H. Clayton, “AB-plot assisted determination of fluorophore mixtures in a fluorescence lifetime microscope using spectra or quenchers,” J. Microsc. 218(1), 62–67 (2005).
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Microsc. Res. Tech. (1)

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).
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Mol. Biol. Cell (1)

N. Komatsu, K. Aoki, M. Yamada, H. Yukinaga, Y. Fujita, Y. Kamioka, and M. Matsuda, “Development of an optimized backbone of FRET biosensors for kinases and GTPases,” Mol. Biol. Cell 22(23), 4647–4656 (2011).
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Multiphoton Microsc. Biomed. Sci. II (1)

W. Becker, A. Bergmann, C. Biskup, T. Zimmer, N. Klocker, and K. Benndorf, “Multi-wavelength TCSPC lifetime imaging,” Multiphoton Microsc. Biomed. Sci. II 4620, 79–84 (2002).
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Nat. Biotechnol. (1)

E. A. Jares-Erijman and T. M. Jovin, “FRET imaging,” Nat. Biotechnol. 21(11), 1387–1395 (2003).
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Nat. Commun. (1)

T. S. Blacker, Z. F. Mann, J. E. Gale, M. Ziegler, A. J. Bain, G. Szabadkai, and M. R. Duchen, “Separating NADH and NADPH fluorescence in live cells and tissues using FLIM,” Nat. Commun. 5, 3936 (2014).
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Nat. Methods (1)

H. W. Ai, K. L. Hazelwood, M. W. Davidson, and R. E. Campbell, “Fluorescent protein FRET pairs for ratiometric imaging of dual biosensors,” Nat. Methods 5(5), 401–403 (2008).
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Neuro-oncol. (1)

S. R. Kantelhardt, J. Leppert, J. Krajewski, N. Petkus, E. Reusche, V. M. Tronnier, G. Hüttmann, and A. Giese, “Imaging of brain and brain tumor specimens by time-resolved multiphoton excitation microscopy ex vivo,” Neuro-oncol. 9(2), 103–112 (2007).
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Opt. Express (8)

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. Express 17(25), 22747–22760 (2009).
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F. Fereidouni, K. Reitsma, and H. C. Gerritsen, “High speed multispectral fluorescence lifetime imaging,” Opt. Express 21(10), 11769–11782 (2013).
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N. Krstajić, J. Levitt, S. Poland, S. Ameer-Beg, and R. Henderson, “256 × 2 SPAD line sensor for time resolved fluorescence spectroscopy,” Opt. Express 23(5), 5653–5669 (2015).
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F. Fereidouni, A. N. Bader, and H. C. Gerritsen, “Spectral phasor analysis allows rapid and reliable unmixing of fluorescence microscopy spectral images,” Opt. Express 20(12), 12729–12741 (2012).
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Proc. SPIE (2)

W. Becker, A. Bergmann, G. Biscotti, K. König, I. Riemann, L. Kelbauskas, and C. Biskup, “High-speed FLIM data acquisition by time-correlated single photon counting,” Proc. SPIE 5223, 1–14 (2004).
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M. Gersbach, R. Trimananda, Y. Maruyama, M. Fishburn, D. Stoppa, J. Richardson, R. Walker, R. K. Henderson, and E. Charbon, “High frame-rate TCSPC-FLIM using a novel SPAD-based image sensor,” Proc. SPIE 7780, 77801H (2010).

Quantum Sensing and Nanophotonic Devices (1)

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L. Pancheri and D. Stoppa, “A SPAD-based pixel linear array for high-speed time-gated fluorescence lifetime imaging,” 2009 P ESSCIRC, 429–432 (2009).

W. Becker, The bh TCSPC Handbook, 6th ed. (Becker and Hicki, 2014).

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F. Borghetti, D. Mosconi, L. Pancheri, and D. Stoppa, “CMOS single-photon avalanche diode sensor for fluorescence lifetime imaging,” in IEEE International Image Sensors Workshop, 2007), 7–10.

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Figures (5)

Fig. 1
Fig. 1

System diagram: (a) The design of the λFLIM system, comprising a Ti:Sapphire laser, coupling optics, a laser scanning confocal microscope and a simple direct vision spectrograph based on the linear array of SPADs. (b) Diagram representing the read-out protocol of the SPAD array. A field-programmable gate array (FPGA) handles the complex sequence of triggers to provide a data stream to the computer via a standard USB connection. (c) Example of the laser comb (514-561-564-633nm) used for a spectral calibration. (d) Fluorescence lifetime estimation of the green plastic slide used as a reference for time calibration; the vertical and horizontal lines mark the delay and the minimum lifetime estimates, respectively. (e) Comparison of photon detection probability (blue curves) between a multi-alkali PMT photocathode and a SPAD built in CMOS technology and relative sensitivity ratio (red curves) obtained by dividing the CMOS to the PMT sensitivity (values with maximum normalized to one).

Fig. 2
Fig. 2

Fast acquisition speed and high dynamic range. Photon counts (a, d and g), colour (b, e and h) and fluorescence lifetime images (c, f and i) of the reference sample Convallaria majalis measured by a PMT-based system in 360 seconds (a-c) and the SPAD-based system in ~8 seconds (d-f) or in ~1 minute (g-i) acquisition time. Laser power was adjusted to maximize count rates for each instrument and condition (1-1.5mW to obtain higher count rates for the SPAD-based measurements and ~300µW to avoid pulse pile-up in TCSPC). (j) Histograms of the estimated fluorescence lifetimes (histogram area normalized to one) and (k) emission spectra (each curve normalized to their maximum) for each of the images shown. Time-resolved emission spectra measured with SPADs or TCSPC are shown in panel (l) and (m), respectively. Scale bar: 20µm.

Fig. 3
Fig. 3

Simple and efficient blind unmixing. Decay curves represented with phasor plots by discrete sine and cosine transforms (DST and DCT) in time (τ, panel (a)), spectral dimension (λ, panel (b)) and two-dimensional transforms (τλ, panel (c)) enables the identification of populations of fluorophores without any a priori information. (d) Relative intensities of the two fluorophores used to stain the sample shown in Figs. 2(g)-2(i) obtained by global analysis of phasor-transformed data and blind unmixing. Dots and letters in panels (a-c) indicate the unmixed phasors of noise (N, not shown in panel (d)), and two fluorophores represented in magenta (M) and green (G). The insert in panel (d) shows a 3x magnification of the image to display the level of separation achieved with this technique (brightness and contrast adjusted for best display).

Fig. 4
Fig. 4

Label-free tissue imaging. Photon counts (a, h), true-colour (b, i) and fluorescence lifetime (c, j) images of unstained liver tissue excised from a tumorogenic murine model. Images in the top row were measured with excitation at 840nm. The other images were acquired with excitation at 920nm, with the first time-gate used to detect the second harmonic signal generated by collagen (blue color in (i)). Blind unmixing performed with the use of phasors (e-g, l-n) exhibits maximum image contrast within a single image; panels (d) and (k) show the fractional intensities of the unmixed components. A region in panel (k) is shown with a 3X magnification to illustrate the level of separation between different regions that this technique can achieve (brightness and contrast adjusted for best display). The letters in the phasor plots mark the pure unmixed phasors of noise (N), and fluorophores which fractional intensities are displayed in blue (B), green (G), red (corresponding to a dominant second harmonic signal, R) and magenta (M) in panels (d) and (k). Scale bar: 20µm.

Fig. 5
Fig. 5

Biochemical imaging. HeLa cells expressing the fluorescent protein mAmetrine (a-c) and the fusion construct of mAmetrine and tdTomato (d-f) exhibiting ~30% FRET efficiency. Photon counts (a, d), true colour images (b, e) and fluorescence lifetime images (c, f) are shown. The histogram of the estimated fluorescence lifetimes is shown in panel (g). Scale bar: 20µm.

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v x D A k x 2 + ( u x D v x A D + A k x w x D ) A k x u x A D A k y w x A D = 0
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