Abstract

A time-resolved fluorescence anisotropy imaging method for studying nanoscale clustering of proteins or lipids was developed and evaluated. It is based on FRET between the identical fluorophores (homo-FRET), which results in a rapid depolarization of the fluorescence. The method employs the time-resolved fluorescence anisotropy decays recorded in a confocal microscope equipped with pulsed excitation and time-gated detection. From the decay the limiting anisotropy r inf was derived, which is a direct measure for the number of fluorophores per cluster. The method was evaluated by imaging GPI-GFP, a lipid raft marker. Small clusters were observed in the plasma membrane while the cytoplasm and the Golgi contained predominantly monomers.

© 2007 Optical Society of America

Full Article  |  PDF Article

References

  • View by:
  • |
  • |
  • |

  1. A. Periasamy, Methods in Cellular Imaging, 1st ed. (Oxford University Press, Oxford, 2001).
  2. B. Valeur, Molecular Fluorescence. Principles and Applications (Wiley-VCH, Weinheim, 2002).
  3. V. E. Centonze, M. Sun, A. Masuda, H. Gerritsen, and B. Herman, "Fluorescence resonance energy transfer imaging microscopy," Method. Enzymol. 360, pp. 542-560 (2003).
    [CrossRef]
  4. E. A. Jares-Erijman and T. M. Jovin, "FRET imaging," Nat. Biotechnol. 21, 1387-1395 (2003).
    [CrossRef] [PubMed]
  5. A. Esposito, H. C. Gerritsen, and F. S. Wouters, "Fluorescence lifetime heterogeneity resolution in the frequency domain by lifetime moments analysis," Biophys. J. 89, 4286-4299 (2005).
    [CrossRef] [PubMed]
  6. J. Sytsma, J. M. Vroom, C. J. De Grauw, and H. C. Gerritsen, "Time-gated fluorescence lifetime imaging and microvolume spectroscopy using two-photon excitation," J. Microsc. 191, 39-51 (1998).
    [CrossRef]
  7. P. Sharma, R. Varma, R. C. Sarasij, Ira, K. Gousset, G. Krishnamoorthy, M. Rao, and S. Mayor, "Nanoscale organization of multiple GPI-anchored proteins in living cell membranes," Cell 116, 577-589 (2004).
    [CrossRef] [PubMed]
  8. I. Gautier, M. Tramier, C. Durieux, J. Coppey, R. B. Pansu, J. C. Nicolas, K. Kemnitz, and M. Coppey-Moisan, "Homo-FRET microscopy in living cells to measure monomer-dimer transition of GFP-tagged proteins," Biophys. J. 80, 3000-3008 (2001).
    [CrossRef] [PubMed]
  9. D. S. Lidke, P. Nagy, B. G. Barisas, R. Heintzmann, J. N. Post, K. A. Lidke, A. H. A. 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. T. 31, 1020-1027 (2003).
    [CrossRef]
  10. R. Varma and S. Mayor, "GPI-anchored proteins are organized in submicron domains at the cell surface," Nature 394, 798-801 (1998).
    [CrossRef] [PubMed]
  11. A. Squire, P. J. Verveer, O. Rocks, and P. I. H. Bastiaens, "Red-edge anisotropy microscopy enables dynamic imaging of homo-FRET between green fluorescent proteins in cells," J. Struct. Biol. 147, 62-69 (2004).
    [CrossRef] [PubMed]
  12. G. Krauss, Biochemistry of Signal Transduction and Regulation, 2nd ed. (Wiley-VCH, Weinheim, 2001).
    [CrossRef]
  13. R. G. W. Anderson and K. Jacobson, "Cell biology - A role for lipid shells in targeting proteins to caveolae, rafts, and other lipid domains," Science 296, 1821-1825 (2002).
    [CrossRef] [PubMed]
  14. A. N. Bader, E. G. Hofman, P. van Bergen en Henegouwen, and H. C. Gerritsen, "Confocal time-resolved fluorescence anisotropy imaging," Proc. SPIE 6441, art. no. 64410C (2007).
    [CrossRef]
  15. C. J. de Grauw and H. C. Gerritsen, "Multiple time-gate module for fluorescence lifetime imaging," Appl. Spectrosc. 55, 670-678 (2001).
    [CrossRef]
  16. A. H. A. Clayton, Q. S. Hanley, D. J. Arndt-Jovin, V. Subramaniam, and T. M. Jovin, "Dynamic fluorescence anisotropy imaging microscopy in the frequency domain (rFLIM)," Biophys. J. 83, 1631-1649 (2002).
    [CrossRef] [PubMed]
  17. K. Suhling, J. Siegel, P. M. P. Lanigan, S. Leveque-Fort, S. E. D. Webb, D. Phillips, D. M. Davis, and P. M. W. French, "Time-resolved fluorescence anisotropy imaging applied to live cells," Opt. Lett. 29, 584-586 (2004).
    [CrossRef] [PubMed]
  18. J. Siegel, K. Suhling, S. Leveque-Fort, S. E. D. Webb, D. M. Davis, D. Phillips, Y. Sabharwal, and P. M. W. French, "Wide-field time-resolved fluorescence anisotropy imaging (TR-FAIM): Imaging the rotational mobility of a fluorophore," Rev. Sci. Instrum. 74, 182-192 (2003).
    [CrossRef]
  19. D. A. Brown and E. London, "Functions of lipid rafts in biological membranes," Annu. Rev. Cell Dev. Biol. 14, 111-136 (1998).
    [CrossRef]
  20. F. Tanaka and N. Mataga, "Theory of time-dependent photo-selection in interacting fixed systems," Photochem. and Photobiol. 29, 1091-1097 (1979).
    [CrossRef]
  21. L. W. Runnels and S. F. Scarlata, "Theory and Application of Fluorescence Homotransfer to Melittin Oligomerization," Biophys. J. 69, 1569-1583 (1995).
    [CrossRef] [PubMed]
  22. V. M. Arganovich and M. D. Galanin, Electronic excitation energy transfer in condensed matter (North-Holland Publishing, Amsterdam, 1982).
  23. K. A. Lidke, B. Rieger, D. S. Lidke, and T. M. Jovin, "The Role of Photon Statistics in Fluorescence Anisotropy Imaging," IEEE T. Image Process. 14, 1237 (2005).
    [CrossRef]
  24. J. D. Bancroft and A. Stevens, Theory and practice of histological techniques (Churchill Livingstone, 1982).
  25. D. Axelrod, "Fluorescence Polarization Microscopy," Method. Cell Biol. 30, 333-352 (1989).
    [CrossRef]

2005 (2)

A. Esposito, H. C. Gerritsen, and F. S. Wouters, "Fluorescence lifetime heterogeneity resolution in the frequency domain by lifetime moments analysis," Biophys. J. 89, 4286-4299 (2005).
[CrossRef] [PubMed]

K. A. Lidke, B. Rieger, D. S. Lidke, and T. M. Jovin, "The Role of Photon Statistics in Fluorescence Anisotropy Imaging," IEEE T. Image Process. 14, 1237 (2005).
[CrossRef]

2004 (3)

A. Squire, P. J. Verveer, O. Rocks, and P. I. H. Bastiaens, "Red-edge anisotropy microscopy enables dynamic imaging of homo-FRET between green fluorescent proteins in cells," J. Struct. Biol. 147, 62-69 (2004).
[CrossRef] [PubMed]

P. Sharma, R. Varma, R. C. Sarasij, Ira, K. Gousset, G. Krishnamoorthy, M. Rao, and S. Mayor, "Nanoscale organization of multiple GPI-anchored proteins in living cell membranes," Cell 116, 577-589 (2004).
[CrossRef] [PubMed]

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

2003 (4)

D. S. Lidke, P. Nagy, B. G. Barisas, R. Heintzmann, J. N. Post, K. A. Lidke, A. H. A. 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. T. 31, 1020-1027 (2003).
[CrossRef]

V. E. Centonze, M. Sun, A. Masuda, H. Gerritsen, and B. Herman, "Fluorescence resonance energy transfer imaging microscopy," Method. Enzymol. 360, pp. 542-560 (2003).
[CrossRef]

E. A. Jares-Erijman and T. M. Jovin, "FRET imaging," Nat. Biotechnol. 21, 1387-1395 (2003).
[CrossRef] [PubMed]

J. Siegel, K. Suhling, S. Leveque-Fort, S. E. D. Webb, D. M. Davis, D. Phillips, Y. Sabharwal, and P. M. W. French, "Wide-field time-resolved fluorescence anisotropy imaging (TR-FAIM): Imaging the rotational mobility of a fluorophore," Rev. Sci. Instrum. 74, 182-192 (2003).
[CrossRef]

2002 (2)

R. G. W. Anderson and K. Jacobson, "Cell biology - A role for lipid shells in targeting proteins to caveolae, rafts, and other lipid domains," Science 296, 1821-1825 (2002).
[CrossRef] [PubMed]

A. H. A. Clayton, Q. S. Hanley, D. J. Arndt-Jovin, V. Subramaniam, and T. M. Jovin, "Dynamic fluorescence anisotropy imaging microscopy in the frequency domain (rFLIM)," Biophys. J. 83, 1631-1649 (2002).
[CrossRef] [PubMed]

2001 (2)

I. Gautier, M. Tramier, C. Durieux, J. Coppey, R. B. Pansu, J. C. Nicolas, K. Kemnitz, and M. Coppey-Moisan, "Homo-FRET microscopy in living cells to measure monomer-dimer transition of GFP-tagged proteins," Biophys. J. 80, 3000-3008 (2001).
[CrossRef] [PubMed]

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

1998 (3)

R. Varma and S. Mayor, "GPI-anchored proteins are organized in submicron domains at the cell surface," Nature 394, 798-801 (1998).
[CrossRef] [PubMed]

J. Sytsma, J. M. Vroom, C. J. De Grauw, and H. C. Gerritsen, "Time-gated fluorescence lifetime imaging and microvolume spectroscopy using two-photon excitation," J. Microsc. 191, 39-51 (1998).
[CrossRef]

D. A. Brown and E. London, "Functions of lipid rafts in biological membranes," Annu. Rev. Cell Dev. Biol. 14, 111-136 (1998).
[CrossRef]

1995 (1)

L. W. Runnels and S. F. Scarlata, "Theory and Application of Fluorescence Homotransfer to Melittin Oligomerization," Biophys. J. 69, 1569-1583 (1995).
[CrossRef] [PubMed]

1989 (1)

D. Axelrod, "Fluorescence Polarization Microscopy," Method. Cell Biol. 30, 333-352 (1989).
[CrossRef]

1979 (1)

F. Tanaka and N. Mataga, "Theory of time-dependent photo-selection in interacting fixed systems," Photochem. and Photobiol. 29, 1091-1097 (1979).
[CrossRef]

Anderson, R. G. W.

R. G. W. Anderson and K. Jacobson, "Cell biology - A role for lipid shells in targeting proteins to caveolae, rafts, and other lipid domains," Science 296, 1821-1825 (2002).
[CrossRef] [PubMed]

Arndt-Jovin, D. J.

D. S. Lidke, P. Nagy, B. G. Barisas, R. Heintzmann, J. N. Post, K. A. Lidke, A. H. A. 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. T. 31, 1020-1027 (2003).
[CrossRef]

A. H. A. Clayton, Q. S. Hanley, D. J. Arndt-Jovin, V. Subramaniam, and T. M. Jovin, "Dynamic fluorescence anisotropy imaging microscopy in the frequency domain (rFLIM)," Biophys. J. 83, 1631-1649 (2002).
[CrossRef] [PubMed]

Axelrod, D.

D. Axelrod, "Fluorescence Polarization Microscopy," Method. Cell Biol. 30, 333-352 (1989).
[CrossRef]

Barisas, B. G.

D. S. Lidke, P. Nagy, B. G. Barisas, R. Heintzmann, J. N. Post, K. A. Lidke, A. H. A. 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. T. 31, 1020-1027 (2003).
[CrossRef]

Bastiaens, P. I. H.

A. Squire, P. J. Verveer, O. Rocks, and P. I. H. Bastiaens, "Red-edge anisotropy microscopy enables dynamic imaging of homo-FRET between green fluorescent proteins in cells," J. Struct. Biol. 147, 62-69 (2004).
[CrossRef] [PubMed]

Brown, D. A.

D. A. Brown and E. London, "Functions of lipid rafts in biological membranes," Annu. Rev. Cell Dev. Biol. 14, 111-136 (1998).
[CrossRef]

Centonze, V. E.

V. E. Centonze, M. Sun, A. Masuda, H. Gerritsen, and B. Herman, "Fluorescence resonance energy transfer imaging microscopy," Method. Enzymol. 360, pp. 542-560 (2003).
[CrossRef]

Clayton, A. H. A.

D. S. Lidke, P. Nagy, B. G. Barisas, R. Heintzmann, J. N. Post, K. A. Lidke, A. H. A. 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. T. 31, 1020-1027 (2003).
[CrossRef]

A. H. A. Clayton, Q. S. Hanley, D. J. Arndt-Jovin, V. Subramaniam, and T. M. Jovin, "Dynamic fluorescence anisotropy imaging microscopy in the frequency domain (rFLIM)," Biophys. J. 83, 1631-1649 (2002).
[CrossRef] [PubMed]

Coppey, J.

I. Gautier, M. Tramier, C. Durieux, J. Coppey, R. B. Pansu, J. C. Nicolas, K. Kemnitz, and M. Coppey-Moisan, "Homo-FRET microscopy in living cells to measure monomer-dimer transition of GFP-tagged proteins," Biophys. J. 80, 3000-3008 (2001).
[CrossRef] [PubMed]

Coppey-Moisan, M.

I. Gautier, M. Tramier, C. Durieux, J. Coppey, R. B. Pansu, J. C. Nicolas, K. Kemnitz, and M. Coppey-Moisan, "Homo-FRET microscopy in living cells to measure monomer-dimer transition of GFP-tagged proteins," Biophys. J. 80, 3000-3008 (2001).
[CrossRef] [PubMed]

Davis, D. M.

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

J. Siegel, K. Suhling, S. Leveque-Fort, S. E. D. Webb, D. M. Davis, D. Phillips, Y. Sabharwal, and P. M. W. French, "Wide-field time-resolved fluorescence anisotropy imaging (TR-FAIM): Imaging the rotational mobility of a fluorophore," Rev. Sci. Instrum. 74, 182-192 (2003).
[CrossRef]

de Grauw, C. J.

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

J. Sytsma, J. M. Vroom, C. J. De Grauw, and H. C. Gerritsen, "Time-gated fluorescence lifetime imaging and microvolume spectroscopy using two-photon excitation," J. Microsc. 191, 39-51 (1998).
[CrossRef]

Durieux, C.

I. Gautier, M. Tramier, C. Durieux, J. Coppey, R. B. Pansu, J. C. Nicolas, K. Kemnitz, and M. Coppey-Moisan, "Homo-FRET microscopy in living cells to measure monomer-dimer transition of GFP-tagged proteins," Biophys. J. 80, 3000-3008 (2001).
[CrossRef] [PubMed]

Esposito, A.

A. Esposito, H. C. Gerritsen, and F. S. Wouters, "Fluorescence lifetime heterogeneity resolution in the frequency domain by lifetime moments analysis," Biophys. J. 89, 4286-4299 (2005).
[CrossRef] [PubMed]

French, P. M. W.

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

J. Siegel, K. Suhling, S. Leveque-Fort, S. E. D. Webb, D. M. Davis, D. Phillips, Y. Sabharwal, and P. M. W. French, "Wide-field time-resolved fluorescence anisotropy imaging (TR-FAIM): Imaging the rotational mobility of a fluorophore," Rev. Sci. Instrum. 74, 182-192 (2003).
[CrossRef]

Gautier, I.

I. Gautier, M. Tramier, C. Durieux, J. Coppey, R. B. Pansu, J. C. Nicolas, K. Kemnitz, and M. Coppey-Moisan, "Homo-FRET microscopy in living cells to measure monomer-dimer transition of GFP-tagged proteins," Biophys. J. 80, 3000-3008 (2001).
[CrossRef] [PubMed]

Gerritsen, H.

V. E. Centonze, M. Sun, A. Masuda, H. Gerritsen, and B. Herman, "Fluorescence resonance energy transfer imaging microscopy," Method. Enzymol. 360, pp. 542-560 (2003).
[CrossRef]

Gerritsen, H. C.

A. Esposito, H. C. Gerritsen, and F. S. Wouters, "Fluorescence lifetime heterogeneity resolution in the frequency domain by lifetime moments analysis," Biophys. J. 89, 4286-4299 (2005).
[CrossRef] [PubMed]

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

J. Sytsma, J. M. Vroom, C. J. De Grauw, and H. C. Gerritsen, "Time-gated fluorescence lifetime imaging and microvolume spectroscopy using two-photon excitation," J. Microsc. 191, 39-51 (1998).
[CrossRef]

Hanley, Q. S.

A. H. A. Clayton, Q. S. Hanley, D. J. Arndt-Jovin, V. Subramaniam, and T. M. Jovin, "Dynamic fluorescence anisotropy imaging microscopy in the frequency domain (rFLIM)," Biophys. J. 83, 1631-1649 (2002).
[CrossRef] [PubMed]

Heintzmann, R.

D. S. Lidke, P. Nagy, B. G. Barisas, R. Heintzmann, J. N. Post, K. A. Lidke, A. H. A. 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. T. 31, 1020-1027 (2003).
[CrossRef]

Herman, B.

V. E. Centonze, M. Sun, A. Masuda, H. Gerritsen, and B. Herman, "Fluorescence resonance energy transfer imaging microscopy," Method. Enzymol. 360, pp. 542-560 (2003).
[CrossRef]

Ira, R. C.

P. Sharma, R. Varma, R. C. Sarasij, Ira, K. Gousset, G. Krishnamoorthy, M. Rao, and S. Mayor, "Nanoscale organization of multiple GPI-anchored proteins in living cell membranes," Cell 116, 577-589 (2004).
[CrossRef] [PubMed]

Jacobson, K.

R. G. W. Anderson and K. Jacobson, "Cell biology - A role for lipid shells in targeting proteins to caveolae, rafts, and other lipid domains," Science 296, 1821-1825 (2002).
[CrossRef] [PubMed]

Jares-Erijman, E. A.

E. A. Jares-Erijman and T. M. Jovin, "FRET imaging," Nat. Biotechnol. 21, 1387-1395 (2003).
[CrossRef] [PubMed]

Jovin, T. M.

K. A. Lidke, B. Rieger, D. S. Lidke, and T. M. Jovin, "The Role of Photon Statistics in Fluorescence Anisotropy Imaging," IEEE T. Image Process. 14, 1237 (2005).
[CrossRef]

E. A. Jares-Erijman and T. M. Jovin, "FRET imaging," Nat. Biotechnol. 21, 1387-1395 (2003).
[CrossRef] [PubMed]

D. S. Lidke, P. Nagy, B. G. Barisas, R. Heintzmann, J. N. Post, K. A. Lidke, A. H. A. 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. T. 31, 1020-1027 (2003).
[CrossRef]

A. H. A. Clayton, Q. S. Hanley, D. J. Arndt-Jovin, V. Subramaniam, and T. M. Jovin, "Dynamic fluorescence anisotropy imaging microscopy in the frequency domain (rFLIM)," Biophys. J. 83, 1631-1649 (2002).
[CrossRef] [PubMed]

Kemnitz, K.

I. Gautier, M. Tramier, C. Durieux, J. Coppey, R. B. Pansu, J. C. Nicolas, K. Kemnitz, and M. Coppey-Moisan, "Homo-FRET microscopy in living cells to measure monomer-dimer transition of GFP-tagged proteins," Biophys. J. 80, 3000-3008 (2001).
[CrossRef] [PubMed]

Lanigan, P. M. P.

Leveque-Fort, S.

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

J. Siegel, K. Suhling, S. Leveque-Fort, S. E. D. Webb, D. M. Davis, D. Phillips, Y. Sabharwal, and P. M. W. French, "Wide-field time-resolved fluorescence anisotropy imaging (TR-FAIM): Imaging the rotational mobility of a fluorophore," Rev. Sci. Instrum. 74, 182-192 (2003).
[CrossRef]

Lidke, D. S.

K. A. Lidke, B. Rieger, D. S. Lidke, and T. M. Jovin, "The Role of Photon Statistics in Fluorescence Anisotropy Imaging," IEEE T. Image Process. 14, 1237 (2005).
[CrossRef]

D. S. Lidke, P. Nagy, B. G. Barisas, R. Heintzmann, J. N. Post, K. A. Lidke, A. H. A. 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. T. 31, 1020-1027 (2003).
[CrossRef]

Lidke, K. A.

K. A. Lidke, B. Rieger, D. S. Lidke, and T. M. Jovin, "The Role of Photon Statistics in Fluorescence Anisotropy Imaging," IEEE T. Image Process. 14, 1237 (2005).
[CrossRef]

D. S. Lidke, P. Nagy, B. G. Barisas, R. Heintzmann, J. N. Post, K. A. Lidke, A. H. A. 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. T. 31, 1020-1027 (2003).
[CrossRef]

London, E.

D. A. Brown and E. London, "Functions of lipid rafts in biological membranes," Annu. Rev. Cell Dev. Biol. 14, 111-136 (1998).
[CrossRef]

Masuda, A.

V. E. Centonze, M. Sun, A. Masuda, H. Gerritsen, and B. Herman, "Fluorescence resonance energy transfer imaging microscopy," Method. Enzymol. 360, pp. 542-560 (2003).
[CrossRef]

Mataga, N.

F. Tanaka and N. Mataga, "Theory of time-dependent photo-selection in interacting fixed systems," Photochem. and Photobiol. 29, 1091-1097 (1979).
[CrossRef]

Mayor, S.

R. Varma and S. Mayor, "GPI-anchored proteins are organized in submicron domains at the cell surface," Nature 394, 798-801 (1998).
[CrossRef] [PubMed]

Nagy, P.

D. S. Lidke, P. Nagy, B. G. Barisas, R. Heintzmann, J. N. Post, K. A. Lidke, A. H. A. 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. T. 31, 1020-1027 (2003).
[CrossRef]

Nicolas, J. C.

I. Gautier, M. Tramier, C. Durieux, J. Coppey, R. B. Pansu, J. C. Nicolas, K. Kemnitz, and M. Coppey-Moisan, "Homo-FRET microscopy in living cells to measure monomer-dimer transition of GFP-tagged proteins," Biophys. J. 80, 3000-3008 (2001).
[CrossRef] [PubMed]

Pansu, R. B.

I. Gautier, M. Tramier, C. Durieux, J. Coppey, R. B. Pansu, J. C. Nicolas, K. Kemnitz, and M. Coppey-Moisan, "Homo-FRET microscopy in living cells to measure monomer-dimer transition of GFP-tagged proteins," Biophys. J. 80, 3000-3008 (2001).
[CrossRef] [PubMed]

Phillips, D.

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

J. Siegel, K. Suhling, S. Leveque-Fort, S. E. D. Webb, D. M. Davis, D. Phillips, Y. Sabharwal, and P. M. W. French, "Wide-field time-resolved fluorescence anisotropy imaging (TR-FAIM): Imaging the rotational mobility of a fluorophore," Rev. Sci. Instrum. 74, 182-192 (2003).
[CrossRef]

Post, J. N.

D. S. Lidke, P. Nagy, B. G. Barisas, R. Heintzmann, J. N. Post, K. A. Lidke, A. H. A. 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. T. 31, 1020-1027 (2003).
[CrossRef]

Rieger, B.

K. A. Lidke, B. Rieger, D. S. Lidke, and T. M. Jovin, "The Role of Photon Statistics in Fluorescence Anisotropy Imaging," IEEE T. Image Process. 14, 1237 (2005).
[CrossRef]

Rocks, O.

A. Squire, P. J. Verveer, O. Rocks, and P. I. H. Bastiaens, "Red-edge anisotropy microscopy enables dynamic imaging of homo-FRET between green fluorescent proteins in cells," J. Struct. Biol. 147, 62-69 (2004).
[CrossRef] [PubMed]

Runnels, L. W.

L. W. Runnels and S. F. Scarlata, "Theory and Application of Fluorescence Homotransfer to Melittin Oligomerization," Biophys. J. 69, 1569-1583 (1995).
[CrossRef] [PubMed]

Sabharwal, Y.

J. Siegel, K. Suhling, S. Leveque-Fort, S. E. D. Webb, D. M. Davis, D. Phillips, Y. Sabharwal, and P. M. W. French, "Wide-field time-resolved fluorescence anisotropy imaging (TR-FAIM): Imaging the rotational mobility of a fluorophore," Rev. Sci. Instrum. 74, 182-192 (2003).
[CrossRef]

Sarasij, R. C.

P. Sharma, R. Varma, R. C. Sarasij, Ira, K. Gousset, G. Krishnamoorthy, M. Rao, and S. Mayor, "Nanoscale organization of multiple GPI-anchored proteins in living cell membranes," Cell 116, 577-589 (2004).
[CrossRef] [PubMed]

Scarlata, S. F.

L. W. Runnels and S. F. Scarlata, "Theory and Application of Fluorescence Homotransfer to Melittin Oligomerization," Biophys. J. 69, 1569-1583 (1995).
[CrossRef] [PubMed]

Sharma, P.

P. Sharma, R. Varma, R. C. Sarasij, Ira, K. Gousset, G. Krishnamoorthy, M. Rao, and S. Mayor, "Nanoscale organization of multiple GPI-anchored proteins in living cell membranes," Cell 116, 577-589 (2004).
[CrossRef] [PubMed]

Siegel, J.

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

J. Siegel, K. Suhling, S. Leveque-Fort, S. E. D. Webb, D. M. Davis, D. Phillips, Y. Sabharwal, and P. M. W. French, "Wide-field time-resolved fluorescence anisotropy imaging (TR-FAIM): Imaging the rotational mobility of a fluorophore," Rev. Sci. Instrum. 74, 182-192 (2003).
[CrossRef]

Squire, A.

A. Squire, P. J. Verveer, O. Rocks, and P. I. H. Bastiaens, "Red-edge anisotropy microscopy enables dynamic imaging of homo-FRET between green fluorescent proteins in cells," J. Struct. Biol. 147, 62-69 (2004).
[CrossRef] [PubMed]

Subramaniam, V.

A. H. A. Clayton, Q. S. Hanley, D. J. Arndt-Jovin, V. Subramaniam, and T. M. Jovin, "Dynamic fluorescence anisotropy imaging microscopy in the frequency domain (rFLIM)," Biophys. J. 83, 1631-1649 (2002).
[CrossRef] [PubMed]

Suhling, K.

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

J. Siegel, K. Suhling, S. Leveque-Fort, S. E. D. Webb, D. M. Davis, D. Phillips, Y. Sabharwal, and P. M. W. French, "Wide-field time-resolved fluorescence anisotropy imaging (TR-FAIM): Imaging the rotational mobility of a fluorophore," Rev. Sci. Instrum. 74, 182-192 (2003).
[CrossRef]

Sun, M.

V. E. Centonze, M. Sun, A. Masuda, H. Gerritsen, and B. Herman, "Fluorescence resonance energy transfer imaging microscopy," Method. Enzymol. 360, pp. 542-560 (2003).
[CrossRef]

Sytsma, J.

J. Sytsma, J. M. Vroom, C. J. De Grauw, and H. C. Gerritsen, "Time-gated fluorescence lifetime imaging and microvolume spectroscopy using two-photon excitation," J. Microsc. 191, 39-51 (1998).
[CrossRef]

Tanaka, F.

F. Tanaka and N. Mataga, "Theory of time-dependent photo-selection in interacting fixed systems," Photochem. and Photobiol. 29, 1091-1097 (1979).
[CrossRef]

Tramier, M.

I. Gautier, M. Tramier, C. Durieux, J. Coppey, R. B. Pansu, J. C. Nicolas, K. Kemnitz, and M. Coppey-Moisan, "Homo-FRET microscopy in living cells to measure monomer-dimer transition of GFP-tagged proteins," Biophys. J. 80, 3000-3008 (2001).
[CrossRef] [PubMed]

Varma, R.

P. Sharma, R. Varma, R. C. Sarasij, Ira, K. Gousset, G. Krishnamoorthy, M. Rao, and S. Mayor, "Nanoscale organization of multiple GPI-anchored proteins in living cell membranes," Cell 116, 577-589 (2004).
[CrossRef] [PubMed]

R. Varma and S. Mayor, "GPI-anchored proteins are organized in submicron domains at the cell surface," Nature 394, 798-801 (1998).
[CrossRef] [PubMed]

Verveer, P. J.

A. Squire, P. J. Verveer, O. Rocks, and P. I. H. Bastiaens, "Red-edge anisotropy microscopy enables dynamic imaging of homo-FRET between green fluorescent proteins in cells," J. Struct. Biol. 147, 62-69 (2004).
[CrossRef] [PubMed]

Vroom, J. M.

J. Sytsma, J. M. Vroom, C. J. De Grauw, and H. C. Gerritsen, "Time-gated fluorescence lifetime imaging and microvolume spectroscopy using two-photon excitation," J. Microsc. 191, 39-51 (1998).
[CrossRef]

Webb, S. E. D.

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

J. Siegel, K. Suhling, S. Leveque-Fort, S. E. D. Webb, D. M. Davis, D. Phillips, Y. Sabharwal, and P. M. W. French, "Wide-field time-resolved fluorescence anisotropy imaging (TR-FAIM): Imaging the rotational mobility of a fluorophore," Rev. Sci. Instrum. 74, 182-192 (2003).
[CrossRef]

Wouters, F. S.

A. Esposito, H. C. Gerritsen, and F. S. Wouters, "Fluorescence lifetime heterogeneity resolution in the frequency domain by lifetime moments analysis," Biophys. J. 89, 4286-4299 (2005).
[CrossRef] [PubMed]

Annu. Rev. Cell Dev. Biol. (1)

D. A. Brown and E. London, "Functions of lipid rafts in biological membranes," Annu. Rev. Cell Dev. Biol. 14, 111-136 (1998).
[CrossRef]

Appl. Spectrosc. (1)

Biochem. Soc. T. (1)

D. S. Lidke, P. Nagy, B. G. Barisas, R. Heintzmann, J. N. Post, K. A. Lidke, A. H. A. 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. T. 31, 1020-1027 (2003).
[CrossRef]

Biophys. J. (4)

A. H. A. Clayton, Q. S. Hanley, D. J. Arndt-Jovin, V. Subramaniam, and T. M. Jovin, "Dynamic fluorescence anisotropy imaging microscopy in the frequency domain (rFLIM)," Biophys. J. 83, 1631-1649 (2002).
[CrossRef] [PubMed]

A. Esposito, H. C. Gerritsen, and F. S. Wouters, "Fluorescence lifetime heterogeneity resolution in the frequency domain by lifetime moments analysis," Biophys. J. 89, 4286-4299 (2005).
[CrossRef] [PubMed]

I. Gautier, M. Tramier, C. Durieux, J. Coppey, R. B. Pansu, J. C. Nicolas, K. Kemnitz, and M. Coppey-Moisan, "Homo-FRET microscopy in living cells to measure monomer-dimer transition of GFP-tagged proteins," Biophys. J. 80, 3000-3008 (2001).
[CrossRef] [PubMed]

L. W. Runnels and S. F. Scarlata, "Theory and Application of Fluorescence Homotransfer to Melittin Oligomerization," Biophys. J. 69, 1569-1583 (1995).
[CrossRef] [PubMed]

Cell (1)

P. Sharma, R. Varma, R. C. Sarasij, Ira, K. Gousset, G. Krishnamoorthy, M. Rao, and S. Mayor, "Nanoscale organization of multiple GPI-anchored proteins in living cell membranes," Cell 116, 577-589 (2004).
[CrossRef] [PubMed]

IEEE T. Image Process. (1)

K. A. Lidke, B. Rieger, D. S. Lidke, and T. M. Jovin, "The Role of Photon Statistics in Fluorescence Anisotropy Imaging," IEEE T. Image Process. 14, 1237 (2005).
[CrossRef]

J. Microsc. (1)

J. Sytsma, J. M. Vroom, C. J. De Grauw, and H. C. Gerritsen, "Time-gated fluorescence lifetime imaging and microvolume spectroscopy using two-photon excitation," J. Microsc. 191, 39-51 (1998).
[CrossRef]

J. Struct. Biol. (1)

A. Squire, P. J. Verveer, O. Rocks, and P. I. H. Bastiaens, "Red-edge anisotropy microscopy enables dynamic imaging of homo-FRET between green fluorescent proteins in cells," J. Struct. Biol. 147, 62-69 (2004).
[CrossRef] [PubMed]

Method. Cell Biol. (1)

D. Axelrod, "Fluorescence Polarization Microscopy," Method. Cell Biol. 30, 333-352 (1989).
[CrossRef]

Method. Enzymol. (1)

V. E. Centonze, M. Sun, A. Masuda, H. Gerritsen, and B. Herman, "Fluorescence resonance energy transfer imaging microscopy," Method. Enzymol. 360, pp. 542-560 (2003).
[CrossRef]

Nat. Biotechnol. (1)

E. A. Jares-Erijman and T. M. Jovin, "FRET imaging," Nat. Biotechnol. 21, 1387-1395 (2003).
[CrossRef] [PubMed]

Nature (1)

R. Varma and S. Mayor, "GPI-anchored proteins are organized in submicron domains at the cell surface," Nature 394, 798-801 (1998).
[CrossRef] [PubMed]

Opt. Lett. (1)

Photochem. and Photobiol. (1)

F. Tanaka and N. Mataga, "Theory of time-dependent photo-selection in interacting fixed systems," Photochem. and Photobiol. 29, 1091-1097 (1979).
[CrossRef]

Rev. Sci. Instrum. (1)

J. Siegel, K. Suhling, S. Leveque-Fort, S. E. D. Webb, D. M. Davis, D. Phillips, Y. Sabharwal, and P. M. W. French, "Wide-field time-resolved fluorescence anisotropy imaging (TR-FAIM): Imaging the rotational mobility of a fluorophore," Rev. Sci. Instrum. 74, 182-192 (2003).
[CrossRef]

Science (1)

R. G. W. Anderson and K. Jacobson, "Cell biology - A role for lipid shells in targeting proteins to caveolae, rafts, and other lipid domains," Science 296, 1821-1825 (2002).
[CrossRef] [PubMed]

Other (6)

A. N. Bader, E. G. Hofman, P. van Bergen en Henegouwen, and H. C. Gerritsen, "Confocal time-resolved fluorescence anisotropy imaging," Proc. SPIE 6441, art. no. 64410C (2007).
[CrossRef]

J. D. Bancroft and A. Stevens, Theory and practice of histological techniques (Churchill Livingstone, 1982).

V. M. Arganovich and M. D. Galanin, Electronic excitation energy transfer in condensed matter (North-Holland Publishing, Amsterdam, 1982).

G. Krauss, Biochemistry of Signal Transduction and Regulation, 2nd ed. (Wiley-VCH, Weinheim, 2001).
[CrossRef]

A. Periasamy, Methods in Cellular Imaging, 1st ed. (Oxford University Press, Oxford, 2001).

B. Valeur, Molecular Fluorescence. Principles and Applications (Wiley-VCH, Weinheim, 2002).

Cited By

OSA participates in CrossRef's Cited-By Linking service. Citing articles from OSA journals and other participating publishers are listed here.

Alert me when this article is cited.


Figures (7)

Fig. 1.
Fig. 1.

(top) Schematic diagram of energy transfer in a dimer. (bottom) Graphs of the cluster size N as a function of the measured anisotropy for various values of efficiency E (based on Eq. (6), with ωτ = E/(1-E)).

Fig. 2.
Fig. 2.

(A) Graph of the steady state anisotropy of a dimer as a function of the homo-FRET efficiency (using Eq. (6), r mono = 0.4). (B) For three values of E, the time resolved anisotropy decays are plotted. The steady state anisotropy levels are indicated by the grey bar.

Fig. 3.
Fig. 3.

Time-resolved anisotropy decays of fluorescein in glycerol/water (v/v) solutions. The black lines represent the decays obtained using time-correlated single photon counting; the bars are the result of time gated anisotropy imaging, averaged over 160×160 pixels.

Fig. 4.
Fig. 4.

(A, B, C) Intensity, anisotropy (r inf) and cluster size image of a NIH 3T3 cell expressing GPI-GFP. (D) Time-gated anisotropy decays of the ruffle region and the Golgi apparatus (yellow bars are the decay of GFP monomers in 50/50 glycerol/buffer). The dashed lines indicate the r inf levels for the given cluster sizes. (E) Plots of the average anisotropy (over 50 pixels of similar intensity) vs total counts (I par + 2*I per) of the ruffle region and the Golgi apparatus. The error bars indicate the standard deviation of anisotropy values per intensity range.

Fig. 5.
Fig. 5.

Intensity and cluster size image of a NIH 3T3 cell expressing GPI-GFP.

Fig. 6.
Fig. 6.

3D confocal intensity and cluster size images of NIH 3T3 cells expressing GPI-GFP. Each image is 33 μm2 (80 by 80 pixels); the step size in the Z-direction is 1 μm.

Fig. 7.
Fig. 7.

(A) Intensity and cluster size image of the 3t3cell expressing GPI-GFP. For the two regions of interest (membrane and ruffles, highlighted in red), the decrease in cluster size was plotted versus the fraction of the GFPs that is bleached (B, red dots). For three models distributions (C), the theoretical bleaching curves are plotted in (B) as well. The arrows in (C) indicate how these models are tuned to match the initial experimental cluster size for the two regions of interest.

Equations (8)

Equations on this page are rendered with MathJax. Learn more.

r = I par I per I par + 2 I per
r ( t ) = r 0 e t Φ
r ( t ) = ( r 0 r inf ) e 2 ω t + r inf
ω = ( R 0 R ) 6 τ 1
E = ω ( τ 1 + ω )
r N = r mono N
r N = r mono 1 + ω . τ 1 + N . ω . τ + r et ( N 1 ) . ω . τ 1 + N . ω . τ
r N , E = 1 = r inf = r mono N

Metrics