Abstract

We present the instrumental development of a versatile total internal reflection fluorescence lifetime imaging microscopy setup illuminated by a supercontinuum laser source. It enables performing wide-field fluorescence lifetime imaging with subwavelength axial resolution for a large range of fluorophores. The short overall acquisition time and the axial resolution are well suited for dynamic neurobiological applications.

© 2009 Optical Society of America

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    [CrossRef] [PubMed]
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  26. K. Suhling, J. Siegel, P. Lanigan, S. Lévêque-Fort, S. Webb, D. Phillips, D. Davis, and P. French, “Time-resolved fluorescence anisotropy imaging applied to live cells,” Opt. Lett. 29, 584-586 (2004).
    [CrossRef] [PubMed]
  27. X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir, and S. Weiss, “Quantum dots for live cells, in vivo imaging, and diagnostics,” Science 307, 538-544 (2005).
    [CrossRef] [PubMed]

2008 (2)

C. von Arnim, B. von Einem, P. Weber, M. Wagner, D. Schwanzar, R. Spoelgen, W. Strauss, and H. Schneckenburger, “Impact of cholesterol level upon APP and BACE proximity and APP cleavage,” Biochem. Biophys. Res. Comm. 370, 207-212 (2008).
[CrossRef] [PubMed]

E. Auksorius, B. Boruah, C. Dunsby, P. Lanigan, G. Kennedy, M. Neil, and P. French, “Stimulated emission depletion microscopy with a supercontinuum source and fluorescence lifetime imaging,” Opt. Lett. 33, 113-115 (2008).
[CrossRef] [PubMed]

2007 (3)

2006 (3)

C. Leterrier, J. Lainé, M. Darmon, H. Boudin, J. Rossier, and Z. Lenkei, “Constitutive activation drives compartment-selective endocytosis and axonal targeting of type 1 cannabinoid receptors,” J. Neurosci. 26, 3141-3153 (2006).
[CrossRef] [PubMed]

K. S. Vetrievel and G. Thinakaran, “Amyloidogenic processing of beta-amyloid precursor protein in intracellular compartments,” Neurology 66, S69-S73 (2006).
[CrossRef]

H. Kano and H. Hamaguchi, “In-vivo multi-nonlinear optical imaging of a living cell using a supercontinuum light source generated from a photonic crystal fiber,” Opt. Express 14, 2798-2804 (2006).
[CrossRef] [PubMed]

2005 (4)

X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir, and S. Weiss, “Quantum dots for live cells, in vivo imaging, and diagnostics,” Science 307, 538-544 (2005).
[CrossRef] [PubMed]

S. Leveque-Fort, D. Papadopoulos, S. Forget, F. Balembois, and P. Georges, “Fluorescence lifetime imaging with a low repetition rate passively mode-locked diode-pumped Nd:YVO4 oscillator,” Opt. Lett. 30, 168-170 (2005).
[CrossRef] [PubMed]

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

I. Munro, J. McGinty, N. Galletly, J. Requejo-Isidro, P. M. P. Lanigan, D. S. Elson, C. Dunsby, M. A. A. Neil, M. J. Lever, G. W. H. Stamp, and P. M. W. French, “Toward the clinical application of time domain fluorescence lifetime imaging,” J Biomed. Opt. 10, 051403 (2005).
[CrossRef] [PubMed]

2004 (5)

H. Schneckenburger, M. Wagner, M. Kretzschmar, W. Strauss, and R. Sailer, “Laser-assisted fluorescence microscopy for measuring cell membrane dynamics,” Photochem. Photobiol. Sci. 3, 817-822 (2004).
[CrossRef] [PubMed]

M. Gee, L. Lensun, T. Smith, and C. Scholes, “Time-resolved evanescent wave-induced fluorescence anisotropy for the determination of molecular conformational changes of proteins at an interface,” Eur. Biophys. J. 33, 130-139 (2004).
[CrossRef]

Y. Chen and A. Periasamy, “Characterization of two-photon excitation fluorescence lifetime imaging microscopy for protein localization,” Microsc. Res. Tech. 63, 72-80 (2004).
[CrossRef]

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

K. Suhling, J. Siegel, P. Lanigan, S. Lévêque-Fort, S. Webb, D. Phillips, D. Davis, and P. French, “Time-resolved fluorescence anisotropy imaging applied to live cells,” Opt. Lett. 29, 584-586 (2004).
[CrossRef] [PubMed]

2001 (5)

J. Siegel, D. S. Elson, S. E. D. Webb, D. Parsons-Karavassilis, S. Lévêque-Fort, M. J. Cole, M. J. Lever, P. M. W. French, M. A. A. Neil, R. Juskaitis, L. O. Sucharov, and T. Wilson, “Whole-field five-dimensional fluorescence microscopy combining lifetime and spectral resolution with optical sectioning,” Opt. Lett. 26, 1338-1340 (2001).
[CrossRef]

D. Axelrod, “Selective imaging of surface fluorescence with very high aperture microscope objectives,” J Biomed. Opt. 6, 6-13 (2001).
[CrossRef] [PubMed]

J. Zhang, Y. Ma, S. S. Taylor, and R. Y. Tsien, “Genetically encoded reporters of protein kinase A activity reveal impact of substrate tethering,” Proc. Nat. Acad. Sci. 98, 14997-15002 (2001).
[CrossRef] [PubMed]

S. Scheuermann, B. Hambsch, L. Hesse, J. Stumm, C. Schmidt, D. Beher, T. A. Bayer, K. Beyreuther, and G. Multhaup, “Homodimerization of amyloid precursor protein and its implication in the amyloidogenic pathway of Alzheimer's disease,” J. Biol. Chem. 276, 33923-33929 (2001).
[CrossRef] [PubMed]

D. Axelrod, “Total internal reflection fluorescence microscopy in cell biology,” Traffic 2, 764-774 (2001).
[CrossRef] [PubMed]

1999 (1)

K. K. Sharman, A. Periasamy, H. Ashworth, J. N. Demas, and N. H. Snow, “Error analysis of the rapid lifetime determination method for double-exponential decays and new windowing schemes,” Anal. Chem. 71, 947-952 (1999).
[CrossRef] [PubMed]

1998 (1)

J. Burmeister, L. Olivier, W. Reichert, and G. Truskey, “Application of total internal reflection fluorescence microscopy to study cell adhesion to biomaterials,” Biomaterials 19, 307-325 (1998).
[CrossRef] [PubMed]

1997 (1)

N. Thompson and B. Lagerholm, “Total internal reflection fluorescence: applications in cellular biophysics,” Curr. Opin. Biotechnol. 8, 58-64 (1997).
[CrossRef] [PubMed]

1981 (1)

D. Axelrod, “Cell-substrate contacts illuminated by total internal reflection fluorescence,” J. Cell Biol. 89, 141-145 (1981).
[CrossRef] [PubMed]

Ashworth, H.

K. K. Sharman, A. Periasamy, H. Ashworth, J. N. Demas, and N. H. Snow, “Error analysis of the rapid lifetime determination method for double-exponential decays and new windowing schemes,” Anal. Chem. 71, 947-952 (1999).
[CrossRef] [PubMed]

Auksorius, E.

Axelrod, D.

D. Axelrod, “Selective imaging of surface fluorescence with very high aperture microscope objectives,” J Biomed. Opt. 6, 6-13 (2001).
[CrossRef] [PubMed]

D. Axelrod, “Total internal reflection fluorescence microscopy in cell biology,” Traffic 2, 764-774 (2001).
[CrossRef] [PubMed]

D. Axelrod, “Cell-substrate contacts illuminated by total internal reflection fluorescence,” J. Cell Biol. 89, 141-145 (1981).
[CrossRef] [PubMed]

Balembois, F.

Bayer, T. A.

S. Scheuermann, B. Hambsch, L. Hesse, J. Stumm, C. Schmidt, D. Beher, T. A. Bayer, K. Beyreuther, and G. Multhaup, “Homodimerization of amyloid precursor protein and its implication in the amyloidogenic pathway of Alzheimer's disease,” J. Biol. Chem. 276, 33923-33929 (2001).
[CrossRef] [PubMed]

Becker, W.

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

Beher, D.

S. Scheuermann, B. Hambsch, L. Hesse, J. Stumm, C. Schmidt, D. Beher, T. A. Bayer, K. Beyreuther, and G. Multhaup, “Homodimerization of amyloid precursor protein and its implication in the amyloidogenic pathway of Alzheimer's disease,” J. Biol. Chem. 276, 33923-33929 (2001).
[CrossRef] [PubMed]

Benndorf, K.

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

Bentolila, L. A.

X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir, and S. Weiss, “Quantum dots for live cells, in vivo imaging, and diagnostics,” Science 307, 538-544 (2005).
[CrossRef] [PubMed]

Bergmann, A.

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

Beyreuther, K.

S. Scheuermann, B. Hambsch, L. Hesse, J. Stumm, C. Schmidt, D. Beher, T. A. Bayer, K. Beyreuther, and G. Multhaup, “Homodimerization of amyloid precursor protein and its implication in the amyloidogenic pathway of Alzheimer's disease,” J. Biol. Chem. 276, 33923-33929 (2001).
[CrossRef] [PubMed]

Biskup, C.

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

Boruah, B.

Boudin, H.

C. Leterrier, J. Lainé, M. Darmon, H. Boudin, J. Rossier, and Z. Lenkei, “Constitutive activation drives compartment-selective endocytosis and axonal targeting of type 1 cannabinoid receptors,” J. Neurosci. 26, 3141-3153 (2006).
[CrossRef] [PubMed]

Bulina, M.

E. Merzlyak, J. Goedhart, D. Shcherbo, M. Bulina, A. Shcheglov, A. Fradkov, A. Gaintzea, K. Lukyanov, S. Lukyanov, T. Gadella, and D. Chudakov, “Bright monomeric red fluorescent protein with an extended fluorescence lifetime,” Nat. Methods 4, 555-557 (2007).
[CrossRef] [PubMed]

Burmeister, J.

J. Burmeister, L. Olivier, W. Reichert, and G. Truskey, “Application of total internal reflection fluorescence microscopy to study cell adhesion to biomaterials,” Biomaterials 19, 307-325 (1998).
[CrossRef] [PubMed]

Chen, Y.

Y. Chen and A. Periasamy, “Characterization of two-photon excitation fluorescence lifetime imaging microscopy for protein localization,” Microsc. Res. Tech. 63, 72-80 (2004).
[CrossRef]

Chudakov, D.

E. Merzlyak, J. Goedhart, D. Shcherbo, M. Bulina, A. Shcheglov, A. Fradkov, A. Gaintzea, K. Lukyanov, S. Lukyanov, T. Gadella, and D. Chudakov, “Bright monomeric red fluorescent protein with an extended fluorescence lifetime,” Nat. Methods 4, 555-557 (2007).
[CrossRef] [PubMed]

Cole, M. J.

Darmon, M.

C. Leterrier, J. Lainé, M. Darmon, H. Boudin, J. Rossier, and Z. Lenkei, “Constitutive activation drives compartment-selective endocytosis and axonal targeting of type 1 cannabinoid receptors,” J. Neurosci. 26, 3141-3153 (2006).
[CrossRef] [PubMed]

Davis, D.

Demas, J. N.

K. K. Sharman, A. Periasamy, H. Ashworth, J. N. Demas, and N. H. Snow, “Error analysis of the rapid lifetime determination method for double-exponential decays and new windowing schemes,” Anal. Chem. 71, 947-952 (1999).
[CrossRef] [PubMed]

Doose, S.

X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir, and S. Weiss, “Quantum dots for live cells, in vivo imaging, and diagnostics,” Science 307, 538-544 (2005).
[CrossRef] [PubMed]

Dunsby, C.

E. Auksorius, B. Boruah, C. Dunsby, P. Lanigan, G. Kennedy, M. Neil, and P. French, “Stimulated emission depletion microscopy with a supercontinuum source and fluorescence lifetime imaging,” Opt. Lett. 33, 113-115 (2008).
[CrossRef] [PubMed]

I. Munro, J. McGinty, N. Galletly, J. Requejo-Isidro, P. M. P. Lanigan, D. S. Elson, C. Dunsby, M. A. A. Neil, M. J. Lever, G. W. H. Stamp, and P. M. W. French, “Toward the clinical application of time domain fluorescence lifetime imaging,” J Biomed. Opt. 10, 051403 (2005).
[CrossRef] [PubMed]

Elson, D. S.

I. Munro, J. McGinty, N. Galletly, J. Requejo-Isidro, P. M. P. Lanigan, D. S. Elson, C. Dunsby, M. A. A. Neil, M. J. Lever, G. W. H. Stamp, and P. M. W. French, “Toward the clinical application of time domain fluorescence lifetime imaging,” J Biomed. Opt. 10, 051403 (2005).
[CrossRef] [PubMed]

J. Siegel, D. S. Elson, S. E. D. Webb, D. Parsons-Karavassilis, S. Lévêque-Fort, M. J. Cole, M. J. Lever, P. M. W. French, M. A. A. Neil, R. Juskaitis, L. O. Sucharov, and T. Wilson, “Whole-field five-dimensional fluorescence microscopy combining lifetime and spectral resolution with optical sectioning,” Opt. Lett. 26, 1338-1340 (2001).
[CrossRef]

Fiutowski, J.

Forget, S.

Fradkov, A.

E. Merzlyak, J. Goedhart, D. Shcherbo, M. Bulina, A. Shcheglov, A. Fradkov, A. Gaintzea, K. Lukyanov, S. Lukyanov, T. Gadella, and D. Chudakov, “Bright monomeric red fluorescent protein with an extended fluorescence lifetime,” Nat. Methods 4, 555-557 (2007).
[CrossRef] [PubMed]

French, P.

French, P. M. W.

I. Munro, J. McGinty, N. Galletly, J. Requejo-Isidro, P. M. P. Lanigan, D. S. Elson, C. Dunsby, M. A. A. Neil, M. J. Lever, G. W. H. Stamp, and P. M. W. French, “Toward the clinical application of time domain fluorescence lifetime imaging,” J Biomed. Opt. 10, 051403 (2005).
[CrossRef] [PubMed]

J. Siegel, D. S. Elson, S. E. D. Webb, D. Parsons-Karavassilis, S. Lévêque-Fort, M. J. Cole, M. J. Lever, P. M. W. French, M. A. A. Neil, R. Juskaitis, L. O. Sucharov, and T. Wilson, “Whole-field five-dimensional fluorescence microscopy combining lifetime and spectral resolution with optical sectioning,” Opt. Lett. 26, 1338-1340 (2001).
[CrossRef]

Gadella, T.

E. Merzlyak, J. Goedhart, D. Shcherbo, M. Bulina, A. Shcheglov, A. Fradkov, A. Gaintzea, K. Lukyanov, S. Lukyanov, T. Gadella, and D. Chudakov, “Bright monomeric red fluorescent protein with an extended fluorescence lifetime,” Nat. Methods 4, 555-557 (2007).
[CrossRef] [PubMed]

Gaintzea, A.

E. Merzlyak, J. Goedhart, D. Shcherbo, M. Bulina, A. Shcheglov, A. Fradkov, A. Gaintzea, K. Lukyanov, S. Lukyanov, T. Gadella, and D. Chudakov, “Bright monomeric red fluorescent protein with an extended fluorescence lifetime,” Nat. Methods 4, 555-557 (2007).
[CrossRef] [PubMed]

Galletly, N.

I. Munro, J. McGinty, N. Galletly, J. Requejo-Isidro, P. M. P. Lanigan, D. S. Elson, C. Dunsby, M. A. A. Neil, M. J. Lever, G. W. H. Stamp, and P. M. W. French, “Toward the clinical application of time domain fluorescence lifetime imaging,” J Biomed. Opt. 10, 051403 (2005).
[CrossRef] [PubMed]

Gambhir, S. S.

X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir, and S. Weiss, “Quantum dots for live cells, in vivo imaging, and diagnostics,” Science 307, 538-544 (2005).
[CrossRef] [PubMed]

Gee, M.

M. Gee, L. Lensun, T. Smith, and C. Scholes, “Time-resolved evanescent wave-induced fluorescence anisotropy for the determination of molecular conformational changes of proteins at an interface,” Eur. Biophys. J. 33, 130-139 (2004).
[CrossRef]

Georges, P.

Goedhart, J.

E. Merzlyak, J. Goedhart, D. Shcherbo, M. Bulina, A. Shcheglov, A. Fradkov, A. Gaintzea, K. Lukyanov, S. Lukyanov, T. Gadella, and D. Chudakov, “Bright monomeric red fluorescent protein with an extended fluorescence lifetime,” Nat. Methods 4, 555-557 (2007).
[CrossRef] [PubMed]

Hamaguchi, H.

Hambsch, B.

S. Scheuermann, B. Hambsch, L. Hesse, J. Stumm, C. Schmidt, D. Beher, T. A. Bayer, K. Beyreuther, and G. Multhaup, “Homodimerization of amyloid precursor protein and its implication in the amyloidogenic pathway of Alzheimer's disease,” J. Biol. Chem. 276, 33923-33929 (2001).
[CrossRef] [PubMed]

Herman, P.

P. Herman, H. J. Lin, and J. R. Lakowicz, “Lifetime based imaging,” in Biomedical Photonics Handbook (CRC Press, 2003).
[CrossRef]

Hesse, L.

S. Scheuermann, B. Hambsch, L. Hesse, J. Stumm, C. Schmidt, D. Beher, T. A. Bayer, K. Beyreuther, and G. Multhaup, “Homodimerization of amyloid precursor protein and its implication in the amyloidogenic pathway of Alzheimer's disease,” J. Biol. Chem. 276, 33923-33929 (2001).
[CrossRef] [PubMed]

Hink, M.

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

Jozefowski, L.

Juskaitis, R.

Kannari, F.

Kano, H.

Kawalec, T.

Kennedy, G.

König, K.

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

Kono, T.

Kretzschmar, M.

H. Schneckenburger, M. Wagner, M. Kretzschmar, W. Strauss, and R. Sailer, “Laser-assisted fluorescence microscopy for measuring cell membrane dynamics,” Photochem. Photobiol. Sci. 3, 817-822 (2004).
[CrossRef] [PubMed]

Lagerholm, B.

N. Thompson and B. Lagerholm, “Total internal reflection fluorescence: applications in cellular biophysics,” Curr. Opin. Biotechnol. 8, 58-64 (1997).
[CrossRef] [PubMed]

Lainé, J.

C. Leterrier, J. Lainé, M. Darmon, H. Boudin, J. Rossier, and Z. Lenkei, “Constitutive activation drives compartment-selective endocytosis and axonal targeting of type 1 cannabinoid receptors,” J. Neurosci. 26, 3141-3153 (2006).
[CrossRef] [PubMed]

Lakowicz, J. R.

P. Herman, H. J. Lin, and J. R. Lakowicz, “Lifetime based imaging,” in Biomedical Photonics Handbook (CRC Press, 2003).
[CrossRef]

Lanigan, P.

Lanigan, P. M. P.

I. Munro, J. McGinty, N. Galletly, J. Requejo-Isidro, P. M. P. Lanigan, D. S. Elson, C. Dunsby, M. A. A. Neil, M. J. Lever, G. W. H. Stamp, and P. M. W. French, “Toward the clinical application of time domain fluorescence lifetime imaging,” J Biomed. Opt. 10, 051403 (2005).
[CrossRef] [PubMed]

Lenkei, Z.

C. Leterrier, J. Lainé, M. Darmon, H. Boudin, J. Rossier, and Z. Lenkei, “Constitutive activation drives compartment-selective endocytosis and axonal targeting of type 1 cannabinoid receptors,” J. Neurosci. 26, 3141-3153 (2006).
[CrossRef] [PubMed]

Lensun, L.

M. Gee, L. Lensun, T. Smith, and C. Scholes, “Time-resolved evanescent wave-induced fluorescence anisotropy for the determination of molecular conformational changes of proteins at an interface,” Eur. Biophys. J. 33, 130-139 (2004).
[CrossRef]

Leterrier, C.

C. Leterrier, J. Lainé, M. Darmon, H. Boudin, J. Rossier, and Z. Lenkei, “Constitutive activation drives compartment-selective endocytosis and axonal targeting of type 1 cannabinoid receptors,” J. Neurosci. 26, 3141-3153 (2006).
[CrossRef] [PubMed]

Leveque-Fort, S.

Lévêque-Fort, S.

Lever, M. J.

I. Munro, J. McGinty, N. Galletly, J. Requejo-Isidro, P. M. P. Lanigan, D. S. Elson, C. Dunsby, M. A. A. Neil, M. J. Lever, G. W. H. Stamp, and P. M. W. French, “Toward the clinical application of time domain fluorescence lifetime imaging,” J Biomed. Opt. 10, 051403 (2005).
[CrossRef] [PubMed]

J. Siegel, D. S. Elson, S. E. D. Webb, D. Parsons-Karavassilis, S. Lévêque-Fort, M. J. Cole, M. J. Lever, P. M. W. French, M. A. A. Neil, R. Juskaitis, L. O. Sucharov, and T. Wilson, “Whole-field five-dimensional fluorescence microscopy combining lifetime and spectral resolution with optical sectioning,” Opt. Lett. 26, 1338-1340 (2001).
[CrossRef]

Li, J. J.

X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir, and S. Weiss, “Quantum dots for live cells, in vivo imaging, and diagnostics,” Science 307, 538-544 (2005).
[CrossRef] [PubMed]

Lin, H. J.

P. Herman, H. J. Lin, and J. R. Lakowicz, “Lifetime based imaging,” in Biomedical Photonics Handbook (CRC Press, 2003).
[CrossRef]

Lukyanov, K.

E. Merzlyak, J. Goedhart, D. Shcherbo, M. Bulina, A. Shcheglov, A. Fradkov, A. Gaintzea, K. Lukyanov, S. Lukyanov, T. Gadella, and D. Chudakov, “Bright monomeric red fluorescent protein with an extended fluorescence lifetime,” Nat. Methods 4, 555-557 (2007).
[CrossRef] [PubMed]

Lukyanov, S.

E. Merzlyak, J. Goedhart, D. Shcherbo, M. Bulina, A. Shcheglov, A. Fradkov, A. Gaintzea, K. Lukyanov, S. Lukyanov, T. Gadella, and D. Chudakov, “Bright monomeric red fluorescent protein with an extended fluorescence lifetime,” Nat. Methods 4, 555-557 (2007).
[CrossRef] [PubMed]

Ma, Y.

J. Zhang, Y. Ma, S. S. Taylor, and R. Y. Tsien, “Genetically encoded reporters of protein kinase A activity reveal impact of substrate tethering,” Proc. Nat. Acad. Sci. 98, 14997-15002 (2001).
[CrossRef] [PubMed]

McGinty, J.

I. Munro, J. McGinty, N. Galletly, J. Requejo-Isidro, P. M. P. Lanigan, D. S. Elson, C. Dunsby, M. A. A. Neil, M. J. Lever, G. W. H. Stamp, and P. M. W. French, “Toward the clinical application of time domain fluorescence lifetime imaging,” J Biomed. Opt. 10, 051403 (2005).
[CrossRef] [PubMed]

Merzlyak, E.

E. Merzlyak, J. Goedhart, D. Shcherbo, M. Bulina, A. Shcheglov, A. Fradkov, A. Gaintzea, K. Lukyanov, S. Lukyanov, T. Gadella, and D. Chudakov, “Bright monomeric red fluorescent protein with an extended fluorescence lifetime,” Nat. Methods 4, 555-557 (2007).
[CrossRef] [PubMed]

Michalet, X.

X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir, and S. Weiss, “Quantum dots for live cells, in vivo imaging, and diagnostics,” Science 307, 538-544 (2005).
[CrossRef] [PubMed]

Midorikawa, K.

Miyawaki, A.

Mizuno, H.

Multhaup, G.

S. Scheuermann, B. Hambsch, L. Hesse, J. Stumm, C. Schmidt, D. Beher, T. A. Bayer, K. Beyreuther, and G. Multhaup, “Homodimerization of amyloid precursor protein and its implication in the amyloidogenic pathway of Alzheimer's disease,” J. Biol. Chem. 276, 33923-33929 (2001).
[CrossRef] [PubMed]

Munro, I.

I. Munro, J. McGinty, N. Galletly, J. Requejo-Isidro, P. M. P. Lanigan, D. S. Elson, C. Dunsby, M. A. A. Neil, M. J. Lever, G. W. H. Stamp, and P. M. W. French, “Toward the clinical application of time domain fluorescence lifetime imaging,” J Biomed. Opt. 10, 051403 (2005).
[CrossRef] [PubMed]

Neil, M.

Neil, M. A. A.

I. Munro, J. McGinty, N. Galletly, J. Requejo-Isidro, P. M. P. Lanigan, D. S. Elson, C. Dunsby, M. A. A. Neil, M. J. Lever, G. W. H. Stamp, and P. M. W. French, “Toward the clinical application of time domain fluorescence lifetime imaging,” J Biomed. Opt. 10, 051403 (2005).
[CrossRef] [PubMed]

J. Siegel, D. S. Elson, S. E. D. Webb, D. Parsons-Karavassilis, S. Lévêque-Fort, M. J. Cole, M. J. Lever, P. M. W. French, M. A. A. Neil, R. Juskaitis, L. O. Sucharov, and T. Wilson, “Whole-field five-dimensional fluorescence microscopy combining lifetime and spectral resolution with optical sectioning,” Opt. Lett. 26, 1338-1340 (2001).
[CrossRef]

Olivier, L.

J. Burmeister, L. Olivier, W. Reichert, and G. Truskey, “Application of total internal reflection fluorescence microscopy to study cell adhesion to biomaterials,” Biomaterials 19, 307-325 (1998).
[CrossRef] [PubMed]

Papadopoulos, D.

Parsons-Karavassilis, D.

Periasamy, A.

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

Y. Chen and A. Periasamy, “Characterization of two-photon excitation fluorescence lifetime imaging microscopy for protein localization,” Microsc. Res. Tech. 63, 72-80 (2004).
[CrossRef]

K. K. Sharman, A. Periasamy, H. Ashworth, J. N. Demas, and N. H. Snow, “Error analysis of the rapid lifetime determination method for double-exponential decays and new windowing schemes,” Anal. Chem. 71, 947-952 (1999).
[CrossRef] [PubMed]

Phillips, D.

Pinaud, F. F.

X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir, and S. Weiss, “Quantum dots for live cells, in vivo imaging, and diagnostics,” Science 307, 538-544 (2005).
[CrossRef] [PubMed]

Reichert, W.

J. Burmeister, L. Olivier, W. Reichert, and G. Truskey, “Application of total internal reflection fluorescence microscopy to study cell adhesion to biomaterials,” Biomaterials 19, 307-325 (1998).
[CrossRef] [PubMed]

Requejo-Isidro, J.

I. Munro, J. McGinty, N. Galletly, J. Requejo-Isidro, P. M. P. Lanigan, D. S. Elson, C. Dunsby, M. A. A. Neil, M. J. Lever, G. W. H. Stamp, and P. M. W. French, “Toward the clinical application of time domain fluorescence lifetime imaging,” J Biomed. Opt. 10, 051403 (2005).
[CrossRef] [PubMed]

Rossier, J.

C. Leterrier, J. Lainé, M. Darmon, H. Boudin, J. Rossier, and Z. Lenkei, “Constitutive activation drives compartment-selective endocytosis and axonal targeting of type 1 cannabinoid receptors,” J. Neurosci. 26, 3141-3153 (2006).
[CrossRef] [PubMed]

Rubahn, H. G.

Sailer, R.

H. Schneckenburger, M. Wagner, M. Kretzschmar, W. Strauss, and R. Sailer, “Laser-assisted fluorescence microscopy for measuring cell membrane dynamics,” Photochem. Photobiol. Sci. 3, 817-822 (2004).
[CrossRef] [PubMed]

Scheuermann, S.

S. Scheuermann, B. Hambsch, L. Hesse, J. Stumm, C. Schmidt, D. Beher, T. A. Bayer, K. Beyreuther, and G. Multhaup, “Homodimerization of amyloid precursor protein and its implication in the amyloidogenic pathway of Alzheimer's disease,” J. Biol. Chem. 276, 33923-33929 (2001).
[CrossRef] [PubMed]

Schmidt, C.

S. Scheuermann, B. Hambsch, L. Hesse, J. Stumm, C. Schmidt, D. Beher, T. A. Bayer, K. Beyreuther, and G. Multhaup, “Homodimerization of amyloid precursor protein and its implication in the amyloidogenic pathway of Alzheimer's disease,” J. Biol. Chem. 276, 33923-33929 (2001).
[CrossRef] [PubMed]

Schneckenburger, H.

C. von Arnim, B. von Einem, P. Weber, M. Wagner, D. Schwanzar, R. Spoelgen, W. Strauss, and H. Schneckenburger, “Impact of cholesterol level upon APP and BACE proximity and APP cleavage,” Biochem. Biophys. Res. Comm. 370, 207-212 (2008).
[CrossRef] [PubMed]

H. Schneckenburger, M. Wagner, M. Kretzschmar, W. Strauss, and R. Sailer, “Laser-assisted fluorescence microscopy for measuring cell membrane dynamics,” Photochem. Photobiol. Sci. 3, 817-822 (2004).
[CrossRef] [PubMed]

Scholes, C.

M. Gee, L. Lensun, T. Smith, and C. Scholes, “Time-resolved evanescent wave-induced fluorescence anisotropy for the determination of molecular conformational changes of proteins at an interface,” Eur. Biophys. J. 33, 130-139 (2004).
[CrossRef]

Schwanzar, D.

C. von Arnim, B. von Einem, P. Weber, M. Wagner, D. Schwanzar, R. Spoelgen, W. Strauss, and H. Schneckenburger, “Impact of cholesterol level upon APP and BACE proximity and APP cleavage,” Biochem. Biophys. Res. Comm. 370, 207-212 (2008).
[CrossRef] [PubMed]

Sharman, K. K.

K. K. Sharman, A. Periasamy, H. Ashworth, J. N. Demas, and N. H. Snow, “Error analysis of the rapid lifetime determination method for double-exponential decays and new windowing schemes,” Anal. Chem. 71, 947-952 (1999).
[CrossRef] [PubMed]

Shcheglov, A.

E. Merzlyak, J. Goedhart, D. Shcherbo, M. Bulina, A. Shcheglov, A. Fradkov, A. Gaintzea, K. Lukyanov, S. Lukyanov, T. Gadella, and D. Chudakov, “Bright monomeric red fluorescent protein with an extended fluorescence lifetime,” Nat. Methods 4, 555-557 (2007).
[CrossRef] [PubMed]

Shcherbo, D.

E. Merzlyak, J. Goedhart, D. Shcherbo, M. Bulina, A. Shcheglov, A. Fradkov, A. Gaintzea, K. Lukyanov, S. Lukyanov, T. Gadella, and D. Chudakov, “Bright monomeric red fluorescent protein with an extended fluorescence lifetime,” Nat. Methods 4, 555-557 (2007).
[CrossRef] [PubMed]

Siegel, J.

Smith, T.

M. Gee, L. Lensun, T. Smith, and C. Scholes, “Time-resolved evanescent wave-induced fluorescence anisotropy for the determination of molecular conformational changes of proteins at an interface,” Eur. Biophys. J. 33, 130-139 (2004).
[CrossRef]

Snow, N. H.

K. K. Sharman, A. Periasamy, H. Ashworth, J. N. Demas, and N. H. Snow, “Error analysis of the rapid lifetime determination method for double-exponential decays and new windowing schemes,” Anal. Chem. 71, 947-952 (1999).
[CrossRef] [PubMed]

Spoelgen, R.

C. von Arnim, B. von Einem, P. Weber, M. Wagner, D. Schwanzar, R. Spoelgen, W. Strauss, and H. Schneckenburger, “Impact of cholesterol level upon APP and BACE proximity and APP cleavage,” Biochem. Biophys. Res. Comm. 370, 207-212 (2008).
[CrossRef] [PubMed]

Stamp, G. W. H.

I. Munro, J. McGinty, N. Galletly, J. Requejo-Isidro, P. M. P. Lanigan, D. S. Elson, C. Dunsby, M. A. A. Neil, M. J. Lever, G. W. H. Stamp, and P. M. W. French, “Toward the clinical application of time domain fluorescence lifetime imaging,” J Biomed. Opt. 10, 051403 (2005).
[CrossRef] [PubMed]

Strauss, W.

C. von Arnim, B. von Einem, P. Weber, M. Wagner, D. Schwanzar, R. Spoelgen, W. Strauss, and H. Schneckenburger, “Impact of cholesterol level upon APP and BACE proximity and APP cleavage,” Biochem. Biophys. Res. Comm. 370, 207-212 (2008).
[CrossRef] [PubMed]

H. Schneckenburger, M. Wagner, M. Kretzschmar, W. Strauss, and R. Sailer, “Laser-assisted fluorescence microscopy for measuring cell membrane dynamics,” Photochem. Photobiol. Sci. 3, 817-822 (2004).
[CrossRef] [PubMed]

Stumm, J.

S. Scheuermann, B. Hambsch, L. Hesse, J. Stumm, C. Schmidt, D. Beher, T. A. Bayer, K. Beyreuther, and G. Multhaup, “Homodimerization of amyloid precursor protein and its implication in the amyloidogenic pathway of Alzheimer's disease,” J. Biol. Chem. 276, 33923-33929 (2001).
[CrossRef] [PubMed]

Sucharov, L. O.

Suda, A.

Suhling, K.

Sundaresan, G.

X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir, and S. Weiss, “Quantum dots for live cells, in vivo imaging, and diagnostics,” Science 307, 538-544 (2005).
[CrossRef] [PubMed]

Tada, J.

Taylor, S. S.

J. Zhang, Y. Ma, S. S. Taylor, and R. Y. Tsien, “Genetically encoded reporters of protein kinase A activity reveal impact of substrate tethering,” Proc. Nat. Acad. Sci. 98, 14997-15002 (2001).
[CrossRef] [PubMed]

Thinakaran, G.

K. S. Vetrievel and G. Thinakaran, “Amyloidogenic processing of beta-amyloid precursor protein in intracellular compartments,” Neurology 66, S69-S73 (2006).
[CrossRef]

Thompson, N.

N. Thompson and B. Lagerholm, “Total internal reflection fluorescence: applications in cellular biophysics,” Curr. Opin. Biotechnol. 8, 58-64 (1997).
[CrossRef] [PubMed]

Truskey, G.

J. Burmeister, L. Olivier, W. Reichert, and G. Truskey, “Application of total internal reflection fluorescence microscopy to study cell adhesion to biomaterials,” Biomaterials 19, 307-325 (1998).
[CrossRef] [PubMed]

Tsay, J. M.

X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir, and S. Weiss, “Quantum dots for live cells, in vivo imaging, and diagnostics,” Science 307, 538-544 (2005).
[CrossRef] [PubMed]

Tsien, R. Y.

J. Zhang, Y. Ma, S. S. Taylor, and R. Y. Tsien, “Genetically encoded reporters of protein kinase A activity reveal impact of substrate tethering,” Proc. Nat. Acad. Sci. 98, 14997-15002 (2001).
[CrossRef] [PubMed]

Vetrievel, K. S.

K. S. Vetrievel and G. Thinakaran, “Amyloidogenic processing of beta-amyloid precursor protein in intracellular compartments,” Neurology 66, S69-S73 (2006).
[CrossRef]

von Arnim, C.

C. von Arnim, B. von Einem, P. Weber, M. Wagner, D. Schwanzar, R. Spoelgen, W. Strauss, and H. Schneckenburger, “Impact of cholesterol level upon APP and BACE proximity and APP cleavage,” Biochem. Biophys. Res. Comm. 370, 207-212 (2008).
[CrossRef] [PubMed]

von Einem, B.

C. von Arnim, B. von Einem, P. Weber, M. Wagner, D. Schwanzar, R. Spoelgen, W. Strauss, and H. Schneckenburger, “Impact of cholesterol level upon APP and BACE proximity and APP cleavage,” Biochem. Biophys. Res. Comm. 370, 207-212 (2008).
[CrossRef] [PubMed]

Wagner, M.

C. von Arnim, B. von Einem, P. Weber, M. Wagner, D. Schwanzar, R. Spoelgen, W. Strauss, and H. Schneckenburger, “Impact of cholesterol level upon APP and BACE proximity and APP cleavage,” Biochem. Biophys. Res. Comm. 370, 207-212 (2008).
[CrossRef] [PubMed]

H. Schneckenburger, M. Wagner, M. Kretzschmar, W. Strauss, and R. Sailer, “Laser-assisted fluorescence microscopy for measuring cell membrane dynamics,” Photochem. Photobiol. Sci. 3, 817-822 (2004).
[CrossRef] [PubMed]

Wallrabe, H.

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

Webb, S.

Webb, S. E. D.

Weber, P.

C. von Arnim, B. von Einem, P. Weber, M. Wagner, D. Schwanzar, R. Spoelgen, W. Strauss, and H. Schneckenburger, “Impact of cholesterol level upon APP and BACE proximity and APP cleavage,” Biochem. Biophys. Res. Comm. 370, 207-212 (2008).
[CrossRef] [PubMed]

Weiss, S.

X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir, and S. Weiss, “Quantum dots for live cells, in vivo imaging, and diagnostics,” Science 307, 538-544 (2005).
[CrossRef] [PubMed]

Wilson, T.

Wu, A. M.

X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir, and S. Weiss, “Quantum dots for live cells, in vivo imaging, and diagnostics,” Science 307, 538-544 (2005).
[CrossRef] [PubMed]

Zhang, J.

J. Zhang, Y. Ma, S. S. Taylor, and R. Y. Tsien, “Genetically encoded reporters of protein kinase A activity reveal impact of substrate tethering,” Proc. Nat. Acad. Sci. 98, 14997-15002 (2001).
[CrossRef] [PubMed]

Anal. Chem. (1)

K. K. Sharman, A. Periasamy, H. Ashworth, J. N. Demas, and N. H. Snow, “Error analysis of the rapid lifetime determination method for double-exponential decays and new windowing schemes,” Anal. Chem. 71, 947-952 (1999).
[CrossRef] [PubMed]

Appl. Opt. (1)

Biochem. Biophys. Res. Comm. (1)

C. von Arnim, B. von Einem, P. Weber, M. Wagner, D. Schwanzar, R. Spoelgen, W. Strauss, and H. Schneckenburger, “Impact of cholesterol level upon APP and BACE proximity and APP cleavage,” Biochem. Biophys. Res. Comm. 370, 207-212 (2008).
[CrossRef] [PubMed]

Biomaterials (1)

J. Burmeister, L. Olivier, W. Reichert, and G. Truskey, “Application of total internal reflection fluorescence microscopy to study cell adhesion to biomaterials,” Biomaterials 19, 307-325 (1998).
[CrossRef] [PubMed]

Curr. Opin. Biotechnol. (2)

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

N. Thompson and B. Lagerholm, “Total internal reflection fluorescence: applications in cellular biophysics,” Curr. Opin. Biotechnol. 8, 58-64 (1997).
[CrossRef] [PubMed]

Eur. Biophys. J. (1)

M. Gee, L. Lensun, T. Smith, and C. Scholes, “Time-resolved evanescent wave-induced fluorescence anisotropy for the determination of molecular conformational changes of proteins at an interface,” Eur. Biophys. J. 33, 130-139 (2004).
[CrossRef]

J Biomed. Opt. (2)

D. Axelrod, “Selective imaging of surface fluorescence with very high aperture microscope objectives,” J Biomed. Opt. 6, 6-13 (2001).
[CrossRef] [PubMed]

I. Munro, J. McGinty, N. Galletly, J. Requejo-Isidro, P. M. P. Lanigan, D. S. Elson, C. Dunsby, M. A. A. Neil, M. J. Lever, G. W. H. Stamp, and P. M. W. French, “Toward the clinical application of time domain fluorescence lifetime imaging,” J Biomed. Opt. 10, 051403 (2005).
[CrossRef] [PubMed]

J. Biol. Chem. (1)

S. Scheuermann, B. Hambsch, L. Hesse, J. Stumm, C. Schmidt, D. Beher, T. A. Bayer, K. Beyreuther, and G. Multhaup, “Homodimerization of amyloid precursor protein and its implication in the amyloidogenic pathway of Alzheimer's disease,” J. Biol. Chem. 276, 33923-33929 (2001).
[CrossRef] [PubMed]

J. Cell Biol. (1)

D. Axelrod, “Cell-substrate contacts illuminated by total internal reflection fluorescence,” J. Cell Biol. 89, 141-145 (1981).
[CrossRef] [PubMed]

J. Neurosci. (1)

C. Leterrier, J. Lainé, M. Darmon, H. Boudin, J. Rossier, and Z. Lenkei, “Constitutive activation drives compartment-selective endocytosis and axonal targeting of type 1 cannabinoid receptors,” J. Neurosci. 26, 3141-3153 (2006).
[CrossRef] [PubMed]

J. Opt. Soc. Am. B (1)

Microsc. Res. Tech. (2)

Y. Chen and A. Periasamy, “Characterization of two-photon excitation fluorescence lifetime imaging microscopy for protein localization,” Microsc. Res. Tech. 63, 72-80 (2004).
[CrossRef]

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

Nat. Methods (1)

E. Merzlyak, J. Goedhart, D. Shcherbo, M. Bulina, A. Shcheglov, A. Fradkov, A. Gaintzea, K. Lukyanov, S. Lukyanov, T. Gadella, and D. Chudakov, “Bright monomeric red fluorescent protein with an extended fluorescence lifetime,” Nat. Methods 4, 555-557 (2007).
[CrossRef] [PubMed]

Neurology (1)

K. S. Vetrievel and G. Thinakaran, “Amyloidogenic processing of beta-amyloid precursor protein in intracellular compartments,” Neurology 66, S69-S73 (2006).
[CrossRef]

Opt. Express (1)

Opt. Lett. (4)

Photochem. Photobiol. Sci. (1)

H. Schneckenburger, M. Wagner, M. Kretzschmar, W. Strauss, and R. Sailer, “Laser-assisted fluorescence microscopy for measuring cell membrane dynamics,” Photochem. Photobiol. Sci. 3, 817-822 (2004).
[CrossRef] [PubMed]

Proc. Nat. Acad. Sci. (1)

J. Zhang, Y. Ma, S. S. Taylor, and R. Y. Tsien, “Genetically encoded reporters of protein kinase A activity reveal impact of substrate tethering,” Proc. Nat. Acad. Sci. 98, 14997-15002 (2001).
[CrossRef] [PubMed]

Science (1)

X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir, and S. Weiss, “Quantum dots for live cells, in vivo imaging, and diagnostics,” Science 307, 538-544 (2005).
[CrossRef] [PubMed]

Traffic (1)

D. Axelrod, “Total internal reflection fluorescence microscopy in cell biology,” Traffic 2, 764-774 (2001).
[CrossRef] [PubMed]

Other (1)

P. Herman, H. J. Lin, and J. R. Lakowicz, “Lifetime based imaging,” in Biomedical Photonics Handbook (CRC Press, 2003).
[CrossRef]

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Figures (7)

Fig. 1
Fig. 1

Experimental setup. M1, M2, plane broadband dielectric mirrors; OD, adjustable optical density; F, filter; L1, f = 35 mm ; L2 and L3, f = 300 mm ; RM, rotating plane mirror; LS, dichroic beam splitter; L4, f = 50 mm ; L5, f = 150 mm ; camera objectives, f = 50 mm .

Fig. 2
Fig. 2

(a), (c) Intensity images and (b), (d)FLIM maps of a HEK-293 cell transfected with APP-CFP. Comparison of results obtained in (c), (d) TIRF and in (a), (b) classical epifluorescence. The average lifetimes for the entire cell obtained are (b)  2180 ± 68 ps and (d)  2100 ± 168 ps .

Fig. 3
Fig. 3

(a) Fluorescence decay for one pixel of the TIRFLIM map reported Fig. 2d, fitted with a monoexponential model, and (b) histogram of the lifetimes calculated for the entire map.

Fig. 4
Fig. 4

(a) TIRF intensity image and (b) TIRFLIM map of a HEK-293 cell expressing green fluorescent protein (GFP) tagged CB1 receptor. The average lifetime obtained is 2430 ± 100 ps .

Fig. 5
Fig. 5

(a) TIRF intensity image and (b) TIRFLIM map of the YFP in a HEK-293 cell cotransfected with mCherry-CB1R and YFP-APP. The average lifetime obtained is 2592 ± 159 ps .

Fig. 6
Fig. 6

(a) TIRF intensity image and (b) TIRFLIM map of the mCherry in the same cell as Fig. 5. The average lifetime obtained is 1358 ± 57 ps .

Fig. 7
Fig. 7

(a) TIRF intensity image and (b) TIRFLIM map of CFP in a HEK-293 cell transfected with a FRET indicator couple. The average lifetime obtained is 1650 ± 110 ps .

Tables (1)

Tables Icon

Table 1 Results Obtained for Various Fluorophores Transfected in HEK-293 Cells a

Equations (1)

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d = λ exc 4 π ( n 1 2 sin 2 ( θ inc ) n 2 2 ) 1 2 ,

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