Abstract

We present a time domain optically sectioned fluorescence lifetime imaging (FLIM) microscope developed for high-speed live cell imaging. This single photon excited system combines wide field parallel pixel detection with confocal sectioning utilizing spinning Nipkow disc microscopy. It can acquire fluorescence lifetime images of live cells at up to 10 frames per second (fps), permitting high-speed FLIM of cell dynamics and protein interactions with potential for high throughput cell imaging and screening applications. We demonstrate the application of this FLIM microscope to real-time monitoring of changes in lipid order in cell membranes following cholesterol depletion using cyclodextrin and to the activation of the small GTP-ase Ras in live cells using FRET.

© 2007 Optical Society of America

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  23. C. Dunsby, P. M. P. Lanigan, J. McGinty, D. S. Elson, J. Requejo-Isidro, I. Munro, N. Galletly, F. McCann, B. Treanor, B. Önfelt, D. M. Davis, M. A. A. Neil, and P. M. W. French "An electronically tunable ultrafast laser source applied to fluorescence imaging and fluorescence lifetime imaging microscopy," J. Phys. D: Appl. Phys. 37, 3296-3303 (2004).
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  25. A. I. Magee, N. Pirinen, J. Adler, S. N. Pagakis, and I. Parmryd, "Lipid rafts: cell surface platforms for T cell signaling," Biol. Res. 35, 127-131 (2002).
    [CrossRef] [PubMed]
  26. G. van Meer and Q. Lisman, "Sphingolipid transport: rafts and translocators," J. Biol. Chem. 277, 25855-25858 (2002).
    [CrossRef] [PubMed]
  27. K. Simons and R. Ehehalt, "Cholesterol, lipid rafts, and disease," J. Clin. Invest. Sep  110, 597-603 (2002).
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  29. D. M. Owen, P. M. P. Lanigan, C. Dunsby, I. Munro, D. M. Grant, M. A. A. Neil, P. M. W. French, and A. I. Magee, "Fluorescence lifetime imaging provides enhanced contrast when imaging the phase-sensitive dye di-4-ANEPPDHQ in model membranes and live cells," Biophys. J. 90, L80-L82 (2006).
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  30. R. R. Kellner, C. J. Baier, K. I. Willig, S. W. Hell, and F. J. Barrantes, "Nanoscale organization of nicotinic acetylcholine receptors revealed by stimulated emission depletion microscopy," Neuroscience 144, 135-143 (2007).
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  31. N. Tanimura, M. Nagafuku, Y. Minaki, Y. Umeda, F. Hayashi, J. Sakakura, A. Kato, D. R. Liddicoat, M. Ogata, T. Hamaoka, and A. Kosugi, "Dynamic changes in the mobility of LAT in aggregated lipid rafts upon T cell activation," J. Cell Biol. 160, 125-135 (2003).
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  32. K. Gaus, E. Gratton, E. P. Kable, A. S. Jones, I. Gelissen, L. Kritharides, and W. Jessup, "Visualizing lipid structure and raft domains in living cells with two photon microscopy," Proc. Natl. Acad. Sci. U S A 100, 15554-15559 (2003).
    [CrossRef] [PubMed]
  33. A. Miyawaki, J. Llopis, R. Heim, J. M. McCaffery, J. A. Adams, M. Ikura, and R. Y. Tsien, "Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin," Nature 388, 882-887 (1997).
    [CrossRef] [PubMed]
  34. T. Nakamura, K. Aoki, and M. Matsuda, "Monitoring spatio-temporal regulation of Ras and Rho GTPase with GFP-based FRET probes," Methods 37, 146-153 (2005).
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  35. O. Rocks, A. Peyker, M. Kahms, P. J. Verveer, C. Koerner, M. Lumbierres, J. Kuhlmann, H. Waldmann, A. Wittinghofer, and P. I. Bastiaens, "An acylation cycle regulates localization and activity of palmitoylated Ras isoforms," Science 307, 1746-1752 (2005).
    [CrossRef] [PubMed]
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  37. M. Augsten, R. Pusch, C. Biskup, K. Rennert, U. Wittig, K. Beyer, A. Blume, R. Wetzker, K. Friedrich, and I. Rubio, "Live-cell imaging of endogenous Ras-GTP illustrates predominant Ras activation at the plasma membrane," EMBO Rep. 7, 46-51 (2006).
    [CrossRef]
  38. R. Yasuda, C. D. Harvey, H. Zhong, A. Sobczyk, L. van Aelst, and K. Svoboda, "Supersensitive Ras activation in dendrites and spines revealed by two-photon fluorescence lifetime imaging," Nat. Neurosci. 9, 283-291 (2006).
    [CrossRef] [PubMed]
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    [CrossRef]
  40. A. V. Agronskaia, L. Tertoolen, and H. C. Gerritsen, "High frame rate fluorescence lifetime imaging," J. Phys. D. 36, 1655-1662 (2003).
    [CrossRef]
  41. R. M. Ballew and J. N. Demas, "An error analysis of the rapid lifetime determination method for the evaluation of single exponential decays," Anal. Chem. 61, 30-33 (1989).
    [CrossRef]
  42. 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. Stamp, and P. M. W. French "Toward the clinical application of time-domain fluorescence lifetime imaging,"J. Biomed. Opt. 10, 051403 (2005).
    [CrossRef] [PubMed]
  43. A. Esposito, C. P. Dohm, M. Bahr, and F. S. Wouters, "Unsupervised fluorescence lifetime imaging microscopy for high content and high throughput screening," Mol. Cell Proteomics 6, 1446-54 (2007).
    [CrossRef] [PubMed]

2007 (3)

E. B. van Munster, J. Goedhart, G. J. Kremers, E. M. Manders, and T. W. Gadella, "Combination of a spinning disc confocal unit with frequency-domain fluorescence lifetime imaging microscopy," Cytometry A. Apr  71, 207-214 (2007).
[CrossRef]

R. R. Kellner, C. J. Baier, K. I. Willig, S. W. Hell, and F. J. Barrantes, "Nanoscale organization of nicotinic acetylcholine receptors revealed by stimulated emission depletion microscopy," Neuroscience 144, 135-143 (2007).
[CrossRef]

A. Esposito, C. P. Dohm, M. Bahr, and F. S. Wouters, "Unsupervised fluorescence lifetime imaging microscopy for high content and high throughput screening," Mol. Cell Proteomics 6, 1446-54 (2007).
[CrossRef] [PubMed]

2006 (7)

L. Jin, A. C. Millard, J. P. Wuskell, X. Dong, D. Wu, H. A. Clark, and L. M. Loew, "Characterization and application of a new optical probe for membrane lipid domains," Biophys. J. 90, 2563-2575 (2006).
[CrossRef] [PubMed]

D. M. Owen, P. M. P. Lanigan, C. Dunsby, I. Munro, D. M. Grant, M. A. A. Neil, P. M. W. French, and A. I. Magee, "Fluorescence lifetime imaging provides enhanced contrast when imaging the phase-sensitive dye di-4-ANEPPDHQ in model membranes and live cells," Biophys. J. 90, L80-L82 (2006).
[CrossRef] [PubMed]

M. Augsten, R. Pusch, C. Biskup, K. Rennert, U. Wittig, K. Beyer, A. Blume, R. Wetzker, K. Friedrich, and I. Rubio, "Live-cell imaging of endogenous Ras-GTP illustrates predominant Ras activation at the plasma membrane," EMBO Rep. 7, 46-51 (2006).
[CrossRef]

R. Yasuda, C. D. Harvey, H. Zhong, A. Sobczyk, L. van Aelst, and K. Svoboda, "Supersensitive Ras activation in dendrites and spines revealed by two-photon fluorescence lifetime imaging," Nat. Neurosci. 9, 283-291 (2006).
[CrossRef] [PubMed]

E. A. Jares-Erijman and T. M. Jovin, "Imaging molecular interactions in living cells by FRET microscopy," Curr. Opin. Chem. Biol. Oct  10(5), 409-416 (2006).
[CrossRef]

B. N. Giepmans, S. R. Adams, M. H. Ellisman, and R. Y. Tsien, "The fluorescence toolbox for assessing protein location and function," Science Apr  312,217-224 (2006).

R. K. Benninger, Y. Koc, O. Hofmann, J. Requejo-Isidro, M. A. A. Neil, P. M. W. French, and A. J. DeMello, "Quantitative 3D mapping of fluidic temperatures within microchannel networks using fluorescence lifetime imaging," Anal. Chem. 78, 2272-2278 (2006).
[CrossRef] [PubMed]

2005 (6)

E. Wang, C. M. Babbey, and K. W. Dunn, "Performance comparison between the high-speed Yokogawa spinning disc confocal system and single-point scanning confocal systems," J. Microsc. 218,148-159 (2005).
[CrossRef] [PubMed]

D. M. Grant, D. S. Elson, D. Schimpf, C. Dunsby, J. Requejo-Isidro, E. Auksorius, I. Munro, M. A. A. Neil, P. M. W. French, E. Nye, G. W. Stamp, and P. Courtney "Optically sectioned fluorescence lifetime imaging using a Nipkow disc microscope and a tunable ultrafast continuum excitation source," Opt. Lett. 30,3353-3355 (2005).
[CrossRef]

T. Nakamura, K. Aoki, and M. Matsuda, "Monitoring spatio-temporal regulation of Ras and Rho GTPase with GFP-based FRET probes," Methods 37, 146-153 (2005).
[CrossRef] [PubMed]

O. Rocks, A. Peyker, M. Kahms, P. J. Verveer, C. Koerner, M. Lumbierres, J. Kuhlmann, H. Waldmann, A. Wittinghofer, and P. I. Bastiaens, "An acylation cycle regulates localization and activity of palmitoylated Ras isoforms," Science 307, 1746-1752 (2005).
[CrossRef] [PubMed]

A. Peyker, O. Rocks, and P. I. Bastiaenes, "Imaging activation of two Ras isoforms simultaneously in a single cell," Chembiochem. 6, 78-85 (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. 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)

D. S. Elson,  et al, "Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier," New J. Phys. 6, 1367-2630 (2004).
[CrossRef]

G. McConnell, "Confocal laser scanning fluorescence microscopy with a visible continuum source," Opt. Express 12, 2844-2850 (2004).
[CrossRef] [PubMed]

C. Dunsby, P. M. P. Lanigan, J. McGinty, D. S. Elson, J. Requejo-Isidro, I. Munro, N. Galletly, F. McCann, B. Treanor, B. Önfelt, D. M. Davis, M. A. A. Neil, and P. M. W. French "An electronically tunable ultrafast laser source applied to fluorescence imaging and fluorescence lifetime imaging microscopy," J. Phys. D: Appl. Phys. 37, 3296-3303 (2004).
[CrossRef]

S. Lévêque-Fort, M. P. Fontaine-Aupart, G. Roger, and P. Georges, "Fluorescence-lifetime imaging with a multifocal two-photon microscope," Opt. Lett. 29, 2884-2886 (2004).
[CrossRef]

J. Requejo-Isidro, J. McGinty, I. Munro, D. S. Elson, N. P. Galletly, M. J. Lever, M. A. A. Neil, G. W. H. Stamp, P. M. W. French, P. A. Kellett, J. D. Hares, and A. K. L. Dymoke-Bradshaw, "High-speed wide-field time-gated endoscopic fluorescence- lifetime imaging," Opt. Lett. 29, 2249-2251 (2004).
[CrossRef] [PubMed]

2003 (6)

D. S. Lidke, P. Nagy, B. G. Barisas, R. Heintzmann, J. N. Post, K. A. Lidke, A. H. Clayton, D. J. Arndt-Jovin, and T. M. Jovin, "Imaging molecular interactions in cells by dynamic and static fluorescence anisotropy (rFLIM and emFRET)," Biochem. Soc. Trans. 31, 1020-1027 (2003).
[CrossRef] [PubMed]

R. M. Clegg, O. Holub, and C. Gohlke, "Fluorescence lifetime-resolved imaging: measuring lifetimes in an image," Methods Enzymol. 360, 509-42 (2003).
[CrossRef] [PubMed]

N. Tanimura, M. Nagafuku, Y. Minaki, Y. Umeda, F. Hayashi, J. Sakakura, A. Kato, D. R. Liddicoat, M. Ogata, T. Hamaoka, and A. Kosugi, "Dynamic changes in the mobility of LAT in aggregated lipid rafts upon T cell activation," J. Cell Biol. 160, 125-135 (2003).
[CrossRef] [PubMed]

K. Gaus, E. Gratton, E. P. Kable, A. S. Jones, I. Gelissen, L. Kritharides, and W. Jessup, "Visualizing lipid structure and raft domains in living cells with two photon microscopy," Proc. Natl. Acad. Sci. U S A 100, 15554-15559 (2003).
[CrossRef] [PubMed]

A. V. Agronskaia, L. Tertoolen, and H. C. Gerritsen, "High frame rate fluorescence lifetime imaging," J. Phys. D. 36, 1655-1662 (2003).
[CrossRef]

P. J. Verveer and P. I. Bastiaens, "Evaluation of global analysis algorithms for single frequency fluorescence lifetime imaging microscopy data," J. Microsc. Jan  209, 1-7 (2003).
[CrossRef]

2002 (4)

A. I. Magee, N. Pirinen, J. Adler, S. N. Pagakis, and I. Parmryd, "Lipid rafts: cell surface platforms for T cell signaling," Biol. Res. 35, 127-131 (2002).
[CrossRef] [PubMed]

G. van Meer and Q. Lisman, "Sphingolipid transport: rafts and translocators," J. Biol. Chem. 277, 25855-25858 (2002).
[CrossRef] [PubMed]

K. Simons and R. Ehehalt, "Cholesterol, lipid rafts, and disease," J. Clin. Invest. Sep  110, 597-603 (2002).

H. C. Gerritsen, M. A. H. Asselbergs, A. V. Agronskala, and W. G. J. H. M van Sark, "Fluorescence lifetime imaging in scanning microscopes: acquisition speed, photon economy and lifetime resolution," J. Microsc. 206, 218-224 (2002).
[CrossRef] [PubMed]

2001 (2)

F. S. Wouters, P. J. Verveer, and P. I. Bastiaens, "Imaging biochemistry inside cells," Trends Cell Biol. May  11, 203-211 (2001).
[CrossRef]

M. J. Cole, J. Siegel, S. E. Webb, R. Jones, K. Dowling, M. J. Dayel, D. Parsons-Karavassilis, P. M. W. French, M. J. Lever, L. O. Sucharov, M. A. A. Neil, R. Juskaitis, and T. Wilson, "Time domain whole-field fluorescence lifetime imaging with optical sectioning," J. Microsc. 203,246-57 (2001).
[CrossRef] [PubMed]

1998 (1)

M. Straub and S. W. Hell, "Fluorescence lifetime three-dimensional microscopy with picosecond precision using a multifocal multiphoton microscope," Appl. Phys. Lett. 73, 1769-1771 (1998).
[CrossRef]

1997 (3)

M. A. A. Neil, R. Juskaitis, and T. Wilson, "Method of obtaining optical sectioning by using structured light in a conventional microscope," Opt. Lett. 22, 1905-1907 (1997).
[CrossRef]

K. Simons and E. Ikonen, "Functional rafts in cell membranes," Nature 387, 569-572 (1997)
[CrossRef] [PubMed]

A. Miyawaki, J. Llopis, R. Heim, J. M. McCaffery, J. A. Adams, M. Ikura, and R. Y. Tsien, "Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin," Nature 388, 882-887 (1997).
[CrossRef] [PubMed]

1992 (1)

M. Kollner and J. Wolfrum, "How many photons are necessary for fluorescence-lifetime measurements?Chem. Phys. Lett. 200, 199-204 (1992).
[CrossRef]

1989 (1)

R. M. Ballew and J. N. Demas, "An error analysis of the rapid lifetime determination method for the evaluation of single exponential decays," Anal. Chem. 61, 30-33 (1989).
[CrossRef]

Anal. Chem. (2)

R. K. Benninger, Y. Koc, O. Hofmann, J. Requejo-Isidro, M. A. A. Neil, P. M. W. French, and A. J. DeMello, "Quantitative 3D mapping of fluidic temperatures within microchannel networks using fluorescence lifetime imaging," Anal. Chem. 78, 2272-2278 (2006).
[CrossRef] [PubMed]

R. M. Ballew and J. N. Demas, "An error analysis of the rapid lifetime determination method for the evaluation of single exponential decays," Anal. Chem. 61, 30-33 (1989).
[CrossRef]

Appl. Phys. Lett. (1)

M. Straub and S. W. Hell, "Fluorescence lifetime three-dimensional microscopy with picosecond precision using a multifocal multiphoton microscope," Appl. Phys. Lett. 73, 1769-1771 (1998).
[CrossRef]

Biochem. Soc. Trans. (1)

D. S. Lidke, P. Nagy, B. G. Barisas, R. Heintzmann, J. N. Post, K. A. Lidke, A. H. Clayton, D. J. Arndt-Jovin, and T. M. Jovin, "Imaging molecular interactions in cells by dynamic and static fluorescence anisotropy (rFLIM and emFRET)," Biochem. Soc. Trans. 31, 1020-1027 (2003).
[CrossRef] [PubMed]

Biol. Res. (1)

A. I. Magee, N. Pirinen, J. Adler, S. N. Pagakis, and I. Parmryd, "Lipid rafts: cell surface platforms for T cell signaling," Biol. Res. 35, 127-131 (2002).
[CrossRef] [PubMed]

Biophys. J. (2)

L. Jin, A. C. Millard, J. P. Wuskell, X. Dong, D. Wu, H. A. Clark, and L. M. Loew, "Characterization and application of a new optical probe for membrane lipid domains," Biophys. J. 90, 2563-2575 (2006).
[CrossRef] [PubMed]

D. M. Owen, P. M. P. Lanigan, C. Dunsby, I. Munro, D. M. Grant, M. A. A. Neil, P. M. W. French, and A. I. Magee, "Fluorescence lifetime imaging provides enhanced contrast when imaging the phase-sensitive dye di-4-ANEPPDHQ in model membranes and live cells," Biophys. J. 90, L80-L82 (2006).
[CrossRef] [PubMed]

Chem. Phys. Lett. (1)

M. Kollner and J. Wolfrum, "How many photons are necessary for fluorescence-lifetime measurements?Chem. Phys. Lett. 200, 199-204 (1992).
[CrossRef]

Chembiochem. (1)

A. Peyker, O. Rocks, and P. I. Bastiaenes, "Imaging activation of two Ras isoforms simultaneously in a single cell," Chembiochem. 6, 78-85 (2005).
[CrossRef] [PubMed]

Curr. Opin. Chem. Biol. (1)

E. A. Jares-Erijman and T. M. Jovin, "Imaging molecular interactions in living cells by FRET microscopy," Curr. Opin. Chem. Biol. Oct  10(5), 409-416 (2006).
[CrossRef]

Cytometry A. (1)

E. B. van Munster, J. Goedhart, G. J. Kremers, E. M. Manders, and T. W. Gadella, "Combination of a spinning disc confocal unit with frequency-domain fluorescence lifetime imaging microscopy," Cytometry A. Apr  71, 207-214 (2007).
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EMBO Rep. (1)

M. Augsten, R. Pusch, C. Biskup, K. Rennert, U. Wittig, K. Beyer, A. Blume, R. Wetzker, K. Friedrich, and I. Rubio, "Live-cell imaging of endogenous Ras-GTP illustrates predominant Ras activation at the plasma membrane," EMBO Rep. 7, 46-51 (2006).
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J. Biol. Chem. (1)

G. van Meer and Q. Lisman, "Sphingolipid transport: rafts and translocators," J. Biol. Chem. 277, 25855-25858 (2002).
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J. Biomed. Opt. (1)

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. Stamp, and P. M. W. French "Toward the clinical application of time-domain fluorescence lifetime imaging,"J. Biomed. Opt. 10, 051403 (2005).
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J. Cell Biol. (1)

N. Tanimura, M. Nagafuku, Y. Minaki, Y. Umeda, F. Hayashi, J. Sakakura, A. Kato, D. R. Liddicoat, M. Ogata, T. Hamaoka, and A. Kosugi, "Dynamic changes in the mobility of LAT in aggregated lipid rafts upon T cell activation," J. Cell Biol. 160, 125-135 (2003).
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J. Clin. Invest. (1)

K. Simons and R. Ehehalt, "Cholesterol, lipid rafts, and disease," J. Clin. Invest. Sep  110, 597-603 (2002).

J. Microsc. (4)

M. J. Cole, J. Siegel, S. E. Webb, R. Jones, K. Dowling, M. J. Dayel, D. Parsons-Karavassilis, P. M. W. French, M. J. Lever, L. O. Sucharov, M. A. A. Neil, R. Juskaitis, and T. Wilson, "Time domain whole-field fluorescence lifetime imaging with optical sectioning," J. Microsc. 203,246-57 (2001).
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H. C. Gerritsen, M. A. H. Asselbergs, A. V. Agronskala, and W. G. J. H. M van Sark, "Fluorescence lifetime imaging in scanning microscopes: acquisition speed, photon economy and lifetime resolution," J. Microsc. 206, 218-224 (2002).
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P. J. Verveer and P. I. Bastiaens, "Evaluation of global analysis algorithms for single frequency fluorescence lifetime imaging microscopy data," J. Microsc. Jan  209, 1-7 (2003).
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E. Wang, C. M. Babbey, and K. W. Dunn, "Performance comparison between the high-speed Yokogawa spinning disc confocal system and single-point scanning confocal systems," J. Microsc. 218,148-159 (2005).
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J. Phys. D. (1)

A. V. Agronskaia, L. Tertoolen, and H. C. Gerritsen, "High frame rate fluorescence lifetime imaging," J. Phys. D. 36, 1655-1662 (2003).
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J. Phys. D: Appl. Phys. (1)

C. Dunsby, P. M. P. Lanigan, J. McGinty, D. S. Elson, J. Requejo-Isidro, I. Munro, N. Galletly, F. McCann, B. Treanor, B. Önfelt, D. M. Davis, M. A. A. Neil, and P. M. W. French "An electronically tunable ultrafast laser source applied to fluorescence imaging and fluorescence lifetime imaging microscopy," J. Phys. D: Appl. Phys. 37, 3296-3303 (2004).
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Methods (1)

T. Nakamura, K. Aoki, and M. Matsuda, "Monitoring spatio-temporal regulation of Ras and Rho GTPase with GFP-based FRET probes," Methods 37, 146-153 (2005).
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Methods Enzymol. (1)

R. M. Clegg, O. Holub, and C. Gohlke, "Fluorescence lifetime-resolved imaging: measuring lifetimes in an image," Methods Enzymol. 360, 509-42 (2003).
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Mol. Cell Proteomics (1)

A. Esposito, C. P. Dohm, M. Bahr, and F. S. Wouters, "Unsupervised fluorescence lifetime imaging microscopy for high content and high throughput screening," Mol. Cell Proteomics 6, 1446-54 (2007).
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Nat. Neurosci. (1)

R. Yasuda, C. D. Harvey, H. Zhong, A. Sobczyk, L. van Aelst, and K. Svoboda, "Supersensitive Ras activation in dendrites and spines revealed by two-photon fluorescence lifetime imaging," Nat. Neurosci. 9, 283-291 (2006).
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Nature (2)

A. Miyawaki, J. Llopis, R. Heim, J. M. McCaffery, J. A. Adams, M. Ikura, and R. Y. Tsien, "Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin," Nature 388, 882-887 (1997).
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Neuroscience (1)

R. R. Kellner, C. J. Baier, K. I. Willig, S. W. Hell, and F. J. Barrantes, "Nanoscale organization of nicotinic acetylcholine receptors revealed by stimulated emission depletion microscopy," Neuroscience 144, 135-143 (2007).
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New J. Phys. (1)

D. S. Elson,  et al, "Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier," New J. Phys. 6, 1367-2630 (2004).
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Opt. Express (1)

Opt. Lett. (4)

Proc. Natl. Acad. Sci. U S A (1)

K. Gaus, E. Gratton, E. P. Kable, A. S. Jones, I. Gelissen, L. Kritharides, and W. Jessup, "Visualizing lipid structure and raft domains in living cells with two photon microscopy," Proc. Natl. Acad. Sci. U S A 100, 15554-15559 (2003).
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Science (2)

B. N. Giepmans, S. R. Adams, M. H. Ellisman, and R. Y. Tsien, "The fluorescence toolbox for assessing protein location and function," Science Apr  312,217-224 (2006).

O. Rocks, A. Peyker, M. Kahms, P. J. Verveer, C. Koerner, M. Lumbierres, J. Kuhlmann, H. Waldmann, A. Wittinghofer, and P. I. Bastiaens, "An acylation cycle regulates localization and activity of palmitoylated Ras isoforms," Science 307, 1746-1752 (2005).
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F. S. Wouters, P. J. Verveer, and P. I. Bastiaens, "Imaging biochemistry inside cells," Trends Cell Biol. May  11, 203-211 (2001).
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D. S. Elson,  et al, Reviews in Fluorescence 2006 (Springer, US, 2006), Chap 22.

J. R. Lakowicz, Principles of Fluorescence Spectroscopy, 3rd Edition (Springer, US, 2006).
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Figures (9)

Fig. 1.
Fig. 1.

Experimental set up

Fig. 2.
Fig. 2.

(a). Representative images of EGFP expressing cells acquired on the Nipkow disc microscope (left column) and confocal system (right column) with different acquisition times. The white pixels are those where an (erroneous) lifetime has been calculated that lies beyond the bounds of the color scale. (Scale bars =10 µm)

Fig. 2.
Fig. 2.

(b). Plots of the mean fluorescence lifetime and standard deviation measured across cells expressing EGFP using confocal TCSPC and time gated Nipkow disc microscopy.

Fig. 3.
Fig. 3.

Accuracy in lifetime as a function of acquisition time for three cases: i) confocal time correlated single photon counting with a count rate of 106s-1; ii) confocal time correlated single photon counting with a count rate of 105s-1; iii) the Nipkow disc system, assuming a flux per pixel equal to that calculated from cells expressing EGFP. (Lines drawn here correspond to raw, unsmoothed image data).

Fig. 4.
Fig. 4.

Fluorescence lifetime images of HEK 293 cells stained with Di-4-ANEPPDHQ (a) prior to and (b) 9 minutes after addition of 7mM methyl-β-cyclodextrin; (c) change in the mean fluorescence lifetime in the plasma membrane at intervals following addition of methyl-β-cyclodextrin. Lifetime values and error bars are the mean and standard deviation calculated from a region of interest around the plasma membrane, averaged across several cells (Scale bar =10 µm)

Fig. 5.
Fig. 5.

Monitoring Ras activation by FRET: coexpression of mRFP-labeled H-Ras, together with an EGFP-labeled Ras binding domain from C-Raf Kinase (Raf-RBD) permits Ras activation to be studied by observing FRET between the two fluorophores. Prior to activation, Ras is in a GDP-bound state but in response to upstream signaling, dissociates from GDP and binds to GTP, resulting in recruitment of Raf-RBD to the membrane and ensuing FRET signal.

Fig. 6.
Fig. 6.

Time lapse fluorescence lifetime imaging of Raf-RBD-EGFP interacting with H-Ras-mRFP at the cell membrane in MDCK cells. Left column: Donor fluorescence lifetime (continuous scale); middle column: donor fluorescence lifetime (binary scale, thresholded at 2400ps); right column: merged fluorescence lifetime with intensity; bottom: H-Ras-mRFP localization. White arrows indicate regions of lifetime shortening in the plasma membrane (Scale bar =10µm). Each image was acquired in 6s.

Fig. 7.
Fig. 7.

Sectioned fluorescence lifetime image stack through a COS 7 cell expressing H-RasmRFP and Raf-RBD-EGFP, displaying FRET at the plasma membrane following stimulation by EGF. Each image was recorded in 6s, with a 100s total acquisition time (Scale bar =10 µm).

Fig. 8.
Fig. 8.

(a). Images of live MDCK cells (with Mercury lamp excitation) expressing either EGFP or a tandem construct of EGFP-mRFP; (b) fluorescence lifetime images of the same field of view, captured at frame rates of 1 fps (top row), 5 fps (middle row) and 10 fps (bottom row). Also shown are the lifetime histograms corresponding to each image. (Scale bar =10µm).

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