Abstract

We demonstrate a high-speed confocal fluorescence lifetime imaging microscopy (FLIM) whose accuracy and photon economy are as good as that of a time-correlated single photon counting (TCSPC). It is based on a new lifetime determination scheme, the analog mean delay (AMD) method. Due to the technical advantages of multiple fluorescence photon detection capability, accurate lifetime determination scheme and high photon detection efficiency, the AMD method can be the most effective method for high-speed confocal FLIM. The feasibility of real-time confocal FLIM with the AMD method has been demonstrated by observing the dynamic reaction of calcium channels in a RBL-2H3 cell with respect to 4αPDD stimulus. We have achieved the photon detection rate of 125 times faster than a conventional TCSPC based system in this experiment.

© 2011 OSA

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2009 (1)

2008 (3)

T. H. Chia, A. Williamson, D. D. Spencer, and M. J. Levene, “Multiphoton fluorescence lifetime imaging of intrinsic fluorescence in human and rat brain tissue reveals spatially distinct NADH binding,” Opt. Express 16(6), 4237–4249 (2008), http://www.opticsinfobase.org/abstract.cfm?URI=oe-16-6-4237 .
[CrossRef] [PubMed]

H.-J. van Manen, P. Verkuijlen, P. Wittendorp, V. Subramaniam, T. K. van den Berg, D. Roos, and C. Otto, “Refractive index sensing of green fluorescent proteins in living cells using fluorescence lifetime imaging microscopy,” Biophys. J. 94(8), L67–L69 (2008).
[CrossRef] [PubMed]

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] [PubMed]

2007 (2)

R. K. Benninger, O. Hofmann, B. Onfelt, I. Munro, C. Dunsby, D. M. Davis, M. A. Neil, P. M. French, and A. J. de Mello, “Fluorescence-lifetime imaging of DNA-dye interactions within continuous-flow microfluidic systems,” Angew. Chem. Int. Ed. Engl. 46(13), 2228–2231 (2007).
[CrossRef] [PubMed]

A. Esposito, H. C. Gerritsen, and F. S. Wouters, “Optimizing frequency-domain fluorescence lifetime sensing for high-throughput applications: photon economy and acquisition speed,” J. Opt. Soc. Am. A 24(10), 3261–3273 (2007).
[CrossRef]

2005 (2)

K. Yoshiki, H. Azuma, K. Yoshioka, M. Hashimoto, and T. Araki, “Finding of Optimal Calcium Ion Probes for Fluorescence Lifetime Measurement,” Opt. Rev. 12(5), 415–419 (2005).
[CrossRef]

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

2004 (2)

W. Becker, A. Bergmann, M. A. Hink, K. König, K. Benndorf, and C. Biskup, “Fluorescence lifetime imaging by time-correlated single-photon counting,” Microsc. Res. Tech. 63(1), 58–66 (2004).
[CrossRef]

J. Neefjes and N. P. Dantuma, “Fluorescent probes for proteolysis: tools for drug discovery,” Nat. Rev. Drug Discov. 3(1), 58–69 (2004).
[CrossRef] [PubMed]

2003 (1)

2002 (1)

H. Watanabe, J. B. Davis, D. Smart, J. C. Jerman, G. D. Smith, P. Hayes, J. Vriens, W. Cairns, U. Wissenbach, J. Prenen, V. Flockerzi, G. Droogmans, C. D. Benham, and B. Nilius, “Activation of TRPV4 channels (hVRL-2/mTRP12) by phorbol derivatives,” J. Biol. Chem. 277(16), 13569–13577 (2002).
[CrossRef] [PubMed]

2001 (1)

W. Becker, A. Bergmann, H. Wabnitz, D. Grosenick, and A. Liebert, “High count rate multichannel TCSPC for optical tomography,” Proc. SPIE 4431, 249–254 (2001).
[CrossRef]

1999 (1)

P. I. H. Bastiaens and A. Squire, “Fluorescence lifetime imaging microscopy: spatial resolution of biochemical processes in the cell,” Trends Cell Biol. 9(2), 48–52 (1999).
[CrossRef] [PubMed]

1949 (1)

C. E. Shannon, “Communication in the presence of noise,” Proc. IRE 37(1), 10–21 (1949).
[CrossRef]

Araki, T.

K. Yoshiki, H. Azuma, K. Yoshioka, M. Hashimoto, and T. Araki, “Finding of Optimal Calcium Ion Probes for Fluorescence Lifetime Measurement,” Opt. Rev. 12(5), 415–419 (2005).
[CrossRef]

Azuma, H.

K. Yoshiki, H. Azuma, K. Yoshioka, M. Hashimoto, and T. Araki, “Finding of Optimal Calcium Ion Probes for Fluorescence Lifetime Measurement,” Opt. Rev. 12(5), 415–419 (2005).
[CrossRef]

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] [PubMed]

Bastiaens, P. I. H.

P. I. H. Bastiaens and A. Squire, “Fluorescence lifetime imaging microscopy: spatial resolution of biochemical processes in the cell,” Trends Cell Biol. 9(2), 48–52 (1999).
[CrossRef] [PubMed]

Becker, W.

W. Becker, A. Bergmann, M. A. Hink, K. König, K. Benndorf, and C. Biskup, “Fluorescence lifetime imaging by time-correlated single-photon counting,” Microsc. Res. Tech. 63(1), 58–66 (2004).
[CrossRef]

W. Becker, A. Bergmann, H. Wabnitz, D. Grosenick, and A. Liebert, “High count rate multichannel TCSPC for optical tomography,” Proc. SPIE 4431, 249–254 (2001).
[CrossRef]

Benham, C. D.

H. Watanabe, J. B. Davis, D. Smart, J. C. Jerman, G. D. Smith, P. Hayes, J. Vriens, W. Cairns, U. Wissenbach, J. Prenen, V. Flockerzi, G. Droogmans, C. D. Benham, and B. Nilius, “Activation of TRPV4 channels (hVRL-2/mTRP12) by phorbol derivatives,” J. Biol. Chem. 277(16), 13569–13577 (2002).
[CrossRef] [PubMed]

Benndorf, K.

W. Becker, A. Bergmann, M. A. Hink, K. König, K. Benndorf, and C. Biskup, “Fluorescence lifetime imaging by time-correlated single-photon counting,” Microsc. Res. Tech. 63(1), 58–66 (2004).
[CrossRef]

Benninger, R. K.

R. K. Benninger, O. Hofmann, B. Onfelt, I. Munro, C. Dunsby, D. M. Davis, M. A. Neil, P. M. French, and A. J. de Mello, “Fluorescence-lifetime imaging of DNA-dye interactions within continuous-flow microfluidic systems,” Angew. Chem. Int. Ed. Engl. 46(13), 2228–2231 (2007).
[CrossRef] [PubMed]

Bergmann, A.

W. Becker, A. Bergmann, M. A. Hink, K. König, K. Benndorf, and C. Biskup, “Fluorescence lifetime imaging by time-correlated single-photon counting,” Microsc. Res. Tech. 63(1), 58–66 (2004).
[CrossRef]

W. Becker, A. Bergmann, H. Wabnitz, D. Grosenick, and A. Liebert, “High count rate multichannel TCSPC for optical tomography,” Proc. SPIE 4431, 249–254 (2001).
[CrossRef]

Biskup, C.

W. Becker, A. Bergmann, M. A. Hink, K. König, K. Benndorf, and C. Biskup, “Fluorescence lifetime imaging by time-correlated single-photon counting,” Microsc. Res. Tech. 63(1), 58–66 (2004).
[CrossRef]

Cairns, W.

H. Watanabe, J. B. Davis, D. Smart, J. C. Jerman, G. D. Smith, P. Hayes, J. Vriens, W. Cairns, U. Wissenbach, J. Prenen, V. Flockerzi, G. Droogmans, C. D. Benham, and B. Nilius, “Activation of TRPV4 channels (hVRL-2/mTRP12) by phorbol derivatives,” J. Biol. Chem. 277(16), 13569–13577 (2002).
[CrossRef] [PubMed]

Carlsson, K.

Chia, T. H.

Dantuma, N. P.

J. Neefjes and N. P. Dantuma, “Fluorescent probes for proteolysis: tools for drug discovery,” Nat. Rev. Drug Discov. 3(1), 58–69 (2004).
[CrossRef] [PubMed]

Davis, D. M.

R. K. Benninger, O. Hofmann, B. Onfelt, I. Munro, C. Dunsby, D. M. Davis, M. A. Neil, P. M. French, and A. J. de Mello, “Fluorescence-lifetime imaging of DNA-dye interactions within continuous-flow microfluidic systems,” Angew. Chem. Int. Ed. Engl. 46(13), 2228–2231 (2007).
[CrossRef] [PubMed]

Davis, J. B.

H. Watanabe, J. B. Davis, D. Smart, J. C. Jerman, G. D. Smith, P. Hayes, J. Vriens, W. Cairns, U. Wissenbach, J. Prenen, V. Flockerzi, G. Droogmans, C. D. Benham, and B. Nilius, “Activation of TRPV4 channels (hVRL-2/mTRP12) by phorbol derivatives,” J. Biol. Chem. 277(16), 13569–13577 (2002).
[CrossRef] [PubMed]

de Mello, A. J.

R. K. Benninger, O. Hofmann, B. Onfelt, I. Munro, C. Dunsby, D. M. Davis, M. A. Neil, P. M. French, and A. J. de Mello, “Fluorescence-lifetime imaging of DNA-dye interactions within continuous-flow microfluidic systems,” Angew. Chem. Int. Ed. Engl. 46(13), 2228–2231 (2007).
[CrossRef] [PubMed]

Droogmans, G.

H. Watanabe, J. B. Davis, D. Smart, J. C. Jerman, G. D. Smith, P. Hayes, J. Vriens, W. Cairns, U. Wissenbach, J. Prenen, V. Flockerzi, G. Droogmans, C. D. Benham, and B. Nilius, “Activation of TRPV4 channels (hVRL-2/mTRP12) by phorbol derivatives,” J. Biol. Chem. 277(16), 13569–13577 (2002).
[CrossRef] [PubMed]

Dunsby, C.

R. K. Benninger, O. Hofmann, B. Onfelt, I. Munro, C. Dunsby, D. M. Davis, M. A. Neil, P. M. French, and A. J. de Mello, “Fluorescence-lifetime imaging of DNA-dye interactions within continuous-flow microfluidic systems,” Angew. Chem. Int. Ed. Engl. 46(13), 2228–2231 (2007).
[CrossRef] [PubMed]

Esposito, 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] [PubMed]

A. Esposito, H. C. Gerritsen, and F. S. Wouters, “Optimizing frequency-domain fluorescence lifetime sensing for high-throughput applications: photon economy and acquisition speed,” J. Opt. Soc. Am. A 24(10), 3261–3273 (2007).
[CrossRef]

Flockerzi, V.

H. Watanabe, J. B. Davis, D. Smart, J. C. Jerman, G. D. Smith, P. Hayes, J. Vriens, W. Cairns, U. Wissenbach, J. Prenen, V. Flockerzi, G. Droogmans, C. D. Benham, and B. Nilius, “Activation of TRPV4 channels (hVRL-2/mTRP12) by phorbol derivatives,” J. Biol. Chem. 277(16), 13569–13577 (2002).
[CrossRef] [PubMed]

French, P. M.

R. K. Benninger, O. Hofmann, B. Onfelt, I. Munro, C. Dunsby, D. M. Davis, M. A. Neil, P. M. French, and A. J. de Mello, “Fluorescence-lifetime imaging of DNA-dye interactions within continuous-flow microfluidic systems,” Angew. Chem. Int. Ed. Engl. 46(13), 2228–2231 (2007).
[CrossRef] [PubMed]

Gerritsen, H. C.

Grosenick, D.

W. Becker, A. Bergmann, H. Wabnitz, D. Grosenick, and A. Liebert, “High count rate multichannel TCSPC for optical tomography,” Proc. SPIE 4431, 249–254 (2001).
[CrossRef]

Hashimoto, M.

K. Yoshiki, H. Azuma, K. Yoshioka, M. Hashimoto, and T. Araki, “Finding of Optimal Calcium Ion Probes for Fluorescence Lifetime Measurement,” Opt. Rev. 12(5), 415–419 (2005).
[CrossRef]

Hayes, P.

H. Watanabe, J. B. Davis, D. Smart, J. C. Jerman, G. D. Smith, P. Hayes, J. Vriens, W. Cairns, U. Wissenbach, J. Prenen, V. Flockerzi, G. Droogmans, C. D. Benham, and B. Nilius, “Activation of TRPV4 channels (hVRL-2/mTRP12) by phorbol derivatives,” J. Biol. Chem. 277(16), 13569–13577 (2002).
[CrossRef] [PubMed]

Hink, M. A.

W. Becker, A. Bergmann, M. A. Hink, K. König, K. Benndorf, and C. Biskup, “Fluorescence lifetime imaging by time-correlated single-photon counting,” Microsc. Res. Tech. 63(1), 58–66 (2004).
[CrossRef]

Hofmann, O.

R. K. Benninger, O. Hofmann, B. Onfelt, I. Munro, C. Dunsby, D. M. Davis, M. A. Neil, P. M. French, and A. J. de Mello, “Fluorescence-lifetime imaging of DNA-dye interactions within continuous-flow microfluidic systems,” Angew. Chem. Int. Ed. Engl. 46(13), 2228–2231 (2007).
[CrossRef] [PubMed]

Jerman, J. C.

H. Watanabe, J. B. Davis, D. Smart, J. C. Jerman, G. D. Smith, P. Hayes, J. Vriens, W. Cairns, U. Wissenbach, J. Prenen, V. Flockerzi, G. Droogmans, C. D. Benham, and B. Nilius, “Activation of TRPV4 channels (hVRL-2/mTRP12) by phorbol derivatives,” J. Biol. Chem. 277(16), 13569–13577 (2002).
[CrossRef] [PubMed]

Kaminski, C. F.

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] [PubMed]

Kim, D. Y.

König, K.

W. Becker, A. Bergmann, M. A. Hink, K. König, K. Benndorf, and C. Biskup, “Fluorescence lifetime imaging by time-correlated single-photon counting,” Microsc. Res. Tech. 63(1), 58–66 (2004).
[CrossRef]

Levene, M. J.

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] [PubMed]

Liebert, A.

W. Becker, A. Bergmann, H. Wabnitz, D. Grosenick, and A. Liebert, “High count rate multichannel TCSPC for optical tomography,” Proc. SPIE 4431, 249–254 (2001).
[CrossRef]

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] [PubMed]

Moon, S.

Munro, I.

R. K. Benninger, O. Hofmann, B. Onfelt, I. Munro, C. Dunsby, D. M. Davis, M. A. Neil, P. M. French, and A. J. de Mello, “Fluorescence-lifetime imaging of DNA-dye interactions within continuous-flow microfluidic systems,” Angew. Chem. Int. Ed. Engl. 46(13), 2228–2231 (2007).
[CrossRef] [PubMed]

Neefjes, J.

J. Neefjes and N. P. Dantuma, “Fluorescent probes for proteolysis: tools for drug discovery,” Nat. Rev. Drug Discov. 3(1), 58–69 (2004).
[CrossRef] [PubMed]

Neil, M. A.

R. K. Benninger, O. Hofmann, B. Onfelt, I. Munro, C. Dunsby, D. M. Davis, M. A. Neil, P. M. French, and A. J. de Mello, “Fluorescence-lifetime imaging of DNA-dye interactions within continuous-flow microfluidic systems,” Angew. Chem. Int. Ed. Engl. 46(13), 2228–2231 (2007).
[CrossRef] [PubMed]

Nilius, B.

H. Watanabe, J. B. Davis, D. Smart, J. C. Jerman, G. D. Smith, P. Hayes, J. Vriens, W. Cairns, U. Wissenbach, J. Prenen, V. Flockerzi, G. Droogmans, C. D. Benham, and B. Nilius, “Activation of TRPV4 channels (hVRL-2/mTRP12) by phorbol derivatives,” J. Biol. Chem. 277(16), 13569–13577 (2002).
[CrossRef] [PubMed]

Onfelt, B.

R. K. Benninger, O. Hofmann, B. Onfelt, I. Munro, C. Dunsby, D. M. Davis, M. A. Neil, P. M. French, and A. J. de Mello, “Fluorescence-lifetime imaging of DNA-dye interactions within continuous-flow microfluidic systems,” Angew. Chem. Int. Ed. Engl. 46(13), 2228–2231 (2007).
[CrossRef] [PubMed]

Otto, C.

H.-J. van Manen, P. Verkuijlen, P. Wittendorp, V. Subramaniam, T. K. van den Berg, D. Roos, and C. Otto, “Refractive index sensing of green fluorescent proteins in living cells using fluorescence lifetime imaging microscopy,” Biophys. J. 94(8), L67–L69 (2008).
[CrossRef] [PubMed]

Periasamy, A.

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

Philip, J.

Prenen, J.

H. Watanabe, J. B. Davis, D. Smart, J. C. Jerman, G. D. Smith, P. Hayes, J. Vriens, W. Cairns, U. Wissenbach, J. Prenen, V. Flockerzi, G. Droogmans, C. D. Benham, and B. Nilius, “Activation of TRPV4 channels (hVRL-2/mTRP12) by phorbol derivatives,” J. Biol. Chem. 277(16), 13569–13577 (2002).
[CrossRef] [PubMed]

Roos, D.

H.-J. van Manen, P. Verkuijlen, P. Wittendorp, V. Subramaniam, T. K. van den Berg, D. Roos, and C. Otto, “Refractive index sensing of green fluorescent proteins in living cells using fluorescence lifetime imaging microscopy,” Biophys. J. 94(8), L67–L69 (2008).
[CrossRef] [PubMed]

Schlachter, S.

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] [PubMed]

Shannon, C. E.

C. E. Shannon, “Communication in the presence of noise,” Proc. IRE 37(1), 10–21 (1949).
[CrossRef]

Smart, D.

H. Watanabe, J. B. Davis, D. Smart, J. C. Jerman, G. D. Smith, P. Hayes, J. Vriens, W. Cairns, U. Wissenbach, J. Prenen, V. Flockerzi, G. Droogmans, C. D. Benham, and B. Nilius, “Activation of TRPV4 channels (hVRL-2/mTRP12) by phorbol derivatives,” J. Biol. Chem. 277(16), 13569–13577 (2002).
[CrossRef] [PubMed]

Smith, G. D.

H. Watanabe, J. B. Davis, D. Smart, J. C. Jerman, G. D. Smith, P. Hayes, J. Vriens, W. Cairns, U. Wissenbach, J. Prenen, V. Flockerzi, G. Droogmans, C. D. Benham, and B. Nilius, “Activation of TRPV4 channels (hVRL-2/mTRP12) by phorbol derivatives,” J. Biol. Chem. 277(16), 13569–13577 (2002).
[CrossRef] [PubMed]

Spencer, D. D.

Squire, A.

P. I. H. Bastiaens and A. Squire, “Fluorescence lifetime imaging microscopy: spatial resolution of biochemical processes in the cell,” Trends Cell Biol. 9(2), 48–52 (1999).
[CrossRef] [PubMed]

Subramaniam, V.

H.-J. van Manen, P. Verkuijlen, P. Wittendorp, V. Subramaniam, T. K. van den Berg, D. Roos, and C. Otto, “Refractive index sensing of green fluorescent proteins in living cells using fluorescence lifetime imaging microscopy,” Biophys. J. 94(8), L67–L69 (2008).
[CrossRef] [PubMed]

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] [PubMed]

van den Berg, T. K.

H.-J. van Manen, P. Verkuijlen, P. Wittendorp, V. Subramaniam, T. K. van den Berg, D. Roos, and C. Otto, “Refractive index sensing of green fluorescent proteins in living cells using fluorescence lifetime imaging microscopy,” Biophys. J. 94(8), L67–L69 (2008).
[CrossRef] [PubMed]

van Manen, H.-J.

H.-J. van Manen, P. Verkuijlen, P. Wittendorp, V. Subramaniam, T. K. van den Berg, D. Roos, and C. Otto, “Refractive index sensing of green fluorescent proteins in living cells using fluorescence lifetime imaging microscopy,” Biophys. J. 94(8), L67–L69 (2008).
[CrossRef] [PubMed]

Verkuijlen, P.

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H. Watanabe, J. B. Davis, D. Smart, J. C. Jerman, G. D. Smith, P. Hayes, J. Vriens, W. Cairns, U. Wissenbach, J. Prenen, V. Flockerzi, G. Droogmans, C. D. Benham, and B. Nilius, “Activation of TRPV4 channels (hVRL-2/mTRP12) by phorbol derivatives,” J. Biol. Chem. 277(16), 13569–13577 (2002).
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H. Watanabe, J. B. Davis, D. Smart, J. C. Jerman, G. D. Smith, P. Hayes, J. Vriens, W. Cairns, U. Wissenbach, J. Prenen, V. Flockerzi, G. Droogmans, C. D. Benham, and B. Nilius, “Activation of TRPV4 channels (hVRL-2/mTRP12) by phorbol derivatives,” J. Biol. Chem. 277(16), 13569–13577 (2002).
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H.-J. van Manen, P. Verkuijlen, P. Wittendorp, V. Subramaniam, T. K. van den Berg, D. Roos, and C. Otto, “Refractive index sensing of green fluorescent proteins in living cells using fluorescence lifetime imaging microscopy,” Biophys. J. 94(8), L67–L69 (2008).
[CrossRef] [PubMed]

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Wouters, F. S.

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K. Yoshiki, H. Azuma, K. Yoshioka, M. Hashimoto, and T. Araki, “Finding of Optimal Calcium Ion Probes for Fluorescence Lifetime Measurement,” Opt. Rev. 12(5), 415–419 (2005).
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K. Yoshiki, H. Azuma, K. Yoshioka, M. Hashimoto, and T. Araki, “Finding of Optimal Calcium Ion Probes for Fluorescence Lifetime Measurement,” Opt. Rev. 12(5), 415–419 (2005).
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Biophys. J. (1)

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[CrossRef] [PubMed]

J. Biol. Chem. (1)

H. Watanabe, J. B. Davis, D. Smart, J. C. Jerman, G. D. Smith, P. Hayes, J. Vriens, W. Cairns, U. Wissenbach, J. Prenen, V. Flockerzi, G. Droogmans, C. D. Benham, and B. Nilius, “Activation of TRPV4 channels (hVRL-2/mTRP12) by phorbol derivatives,” J. Biol. Chem. 277(16), 13569–13577 (2002).
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Microsc. Res. Tech. (1)

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Opt. Express (2)

Opt. Rev. (1)

K. Yoshiki, H. Azuma, K. Yoshioka, M. Hashimoto, and T. Araki, “Finding of Optimal Calcium Ion Probes for Fluorescence Lifetime Measurement,” Opt. Rev. 12(5), 415–419 (2005).
[CrossRef]

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W. Becker, A. Bergmann, H. Wabnitz, D. Grosenick, and A. Liebert, “High count rate multichannel TCSPC for optical tomography,” Proc. SPIE 4431, 249–254 (2001).
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Figures (5)

Fig. 1
Fig. 1

Schematic diagram of high-speed confocal FLIM by the AMD method.

Fig. 2
Fig. 2

(a) Simulated IRF and fluorescence signal sampled by 10 GHz, (b) Frequency components of the signals shown in (a), (c) Simulated IRF and fluorescence signal sampled by 100 MHz, (d) Lifetimes extracted by AMD method with the signals sampled by 10 GHz and 100 MHz.

Fig. 3
Fig. 3

(a) Fluorescence lifetime of Alexa fluor 488 and figure of merit in regard to the number of photons. (b) Standard deviation in lifetime as a function of the photon number used for lifetime measurement in our AMD-FLIM system. Total SD, Shot SD, and Sys SD represent Δτtot , Δτshot , and Δτsys , respectively.

Fig. 4
Fig. 4

(a) Calibration between intensity of analog pulse signal and the number of photons (b) Fluorescence intensity (top) and lifetime (bottom) images of Alexa fluor 488.

Fig. 5
Fig. 5

Real-time dynamic FLIM images. (a) Average fluorescence intensity and lifetime variations inside a cell due calcium channel opening. (b) fluorescence intensity (top) and lifetime (bottom) images from 0.52 s to 2.34 s with frame rate of 7.7 frames/sec.

Equations (3)

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F = N σ τ / τ
τ = < T e > < T e 0 > ( t i e ( t ) d t i e ( t ) d t ) ( t i i r f ( t ) d t i i r f ( t ) d t )
Δ τ t o t = Δ τ s h o t 2 + Δ τ s y s 2 .

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