Abstract

Fluorescence lifetime imaging microscopy (FLIM) provides a promising, robust method of detecting molecular interactions in vivo via fluorescence/Förster resonance energy transfer (FRET), by monitoring the variation of donor fluorescence lifetime, which is insensitive to many artifacts influencing convential intensity-based measurements, e.g. fluorophore concentration, photobleaching, and spectral bleed-through. As proof of principle, we demonstrate the capability of a novel picosecondresolution FLIM system to detect molecular interactions in a wellestablished FRET assay. We then apply the FLIM system to detect the molecular interaction of a transforming oncogene RhoC with a binding partner RhoGDIγ in vivo, which is critical to understand and interfere with Rho signaling for cancer therapeutics.

© 2007 Optical Society of America

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  1. P. Legrain and L. Selig, "Genome-wide protein interaction maps using two-hybrid systems," FEBS Lett. 480, 32-36 (2000).
    [CrossRef] [PubMed]
  2. S. Fields, "Proteomics in genomeland," Science 291, 1221-1224 (2001).
    [CrossRef] [PubMed]
  3. E. Golemis, ed., Protein-protein interactions: A molecular cloning manual, 1st ed. (Cold Spring Harbor Laboratory Press, Woodbury, 2001), p. 682.
  4. Y. Chen and J. D. Mills, "Protein localization in living cells and tissues using FRET and FLIM," Differentiation 71, 528-541 (2003).
    [CrossRef] [PubMed]
  5. K. L. van Golen, Z.-F. Wu, X. T. Qiao, L. W. Bao, and S. D. Merajver, "RhoC GTPase, a novel transforming oncogene for human mammary epithelial cells that partially recapitulates the inflammatory breast cancer phenotype," Cancer Res. 60, 5832-5838 (2000).
    [PubMed]
  6. H. Suwa, G. Ohshio, T. Imamura, G. Watanabe, S. Arii, M. Imamura, S. Narumiya, H. Hiai, and M. Fukumoto, "Overexpression of the RhoC gene correlates with progression of ductal adenocarcinoma of the pancreas," Br. J. Cancer 77, 147-152 (1998).
    [CrossRef] [PubMed]
  7. E. A. Clark, T. R. Golub, E. S. Lander, and R. O. Hynes, "Genomic analysis of metastasis reveals an essential role for RhoC," Nature 406, 532-535 (2000).
    [CrossRef] [PubMed]
  8. G. Fritz, C. Brachetti, F. Bahlmann, M. Schmidt, and B. Kaina, "Rho GTPases in human breast tumours: expression and mutation analyses and correlation with clinical parameters," Br. J. Cancer 87, 635-644 (2002).
    [CrossRef] [PubMed]
  9. W. B. Zhong, C. Y. Wang, T. C. Chang, and W. S. Lee, "Lovastatin induces apoptosis of anaplastic thyroid cancer cells via inhibition of protein geranylgeranylation and de novo protein synthesis," Endocrinology 144, 3852-3859 (2003).
    [CrossRef] [PubMed]
  10. C. Marionnet, C. Lalou, K. Mollier, M. Chazal, G. Delestaing, D. Compan, O. Verola, C. Vilmer, J. Cuminet, L. Dubertret, and N. Basset-Seguin, "Differential molecular profiling between skin carcinomas reveals four newly reported genes potentially implicated in squamous cell carcinoma development," Oncogene 22, 3500-3505 (2003).
    [CrossRef] [PubMed]
  11. W. Wang, L. Y. Yang, G. W. Huang, and W. Q. Lu, "Expression and significance of RhoC gene in hepatocellular carcinoma," World Journal of Gastroenterology 9, 1950-1953 (2003).
    [PubMed]
  12. R. Y. Tsien, "The green fluorescent protein," Annual Reviews of Biochemistry 67, 509-544 (1998).
    [CrossRef]
  13. A. Periasamy, "Fluorescence resonance energy transfer microscopy: a mini review," J. Biomed. Opt. 6, 287-291 (2001).
    [CrossRef] [PubMed]
  14. F. J. M. van Kuppeveld, W. J. G. Melchers, P. H. G. M. Willems, and T. W. J. Gadella, Jr., "Homomultimerization of the coxsackievirus 2B protein in living cells visualized by fluorescence resonance energy transfer microscopy," J. Virol. 76, 9446-9456 (2002).
    [CrossRef] [PubMed]
  15. M. Elangovan, R. N. Day, and A. Periasamy, "Nanosecond fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy to localize the protein interactions in a single living cell," J. Microsc. 205, 3-14 (2002).
    [CrossRef] [PubMed]
  16. A. Tsuji, Y. Sato, M. Hirano, T. Suga, H. Koshimoto, T. Taguchi, and S. Ohsuka, "Development of a time-resolved fluorometric method for observing hybridization in living cells using fluorescence resonance energy transfer," Biophys. J. 81, 501-515 (2001).
    [CrossRef] [PubMed]
  17. J. R. Lakowicz, Principles of Fluorescence Spectroscopy, 2nd ed. (Kluwer Academic/Plenum, New York, 1999), p. 698.
  18. P. I. H. Bastiaens and A. Squire, "Fluorescence lifetime imaging microscopy: spatial resolution of biochemical processes in the cell," Trends Cell Biol. 9, 48-52 (1999).
    [CrossRef] [PubMed]
  19. J. A. Schmid and H. H. Sitte, "Fluorescence resonance energy transfer in the study of cancer pathways," Curr. Opin. Oncol. 15, 55-64 (2003).
    [CrossRef]
  20. P. K. Urayama and M.-A. Mycek, "Fluorescence lifetime imaging microscopy of endogenous biological fluorescence," in Handbook of Biomedical Fluorescence, M.-A. Mycek and B. W. Pogue, eds. (Marcel-Dekker Inc., New York, New York, 2003), pp. 211-236.
  21. J. Lippincott-Schwartz, E. Snapp, and A. Kenworthy, "Studying protein dynamics in living cells," Nat. Rev. Mol. Cell Biol. 2, 444-456 (2001).
    [CrossRef] [PubMed]
  22. W. Zhong, P. Urayama, and M.-A. Mycek, "Imaging fluorescence lifetime modulation of a ruthenium-based dye in living cells: the potential for oxygen sensing," J. Phys. D: Appl. Phys. 36, 1689-1695 (2003).
    [CrossRef]
  23. C. W. Chang, D. Sud, and M. A. Mycek, "Fluorescence Lifetime Imaging Microscopy," in Methods in Cell Biology, Vol. 81 - Digital Microscopy, 3rd Edition, D. E. Wolf and G. Sluder, eds. (Academic Press, San Diego, 2007), pp. 495-524.
  24. I. Bugiel, K. König, and H. Wabnitz, "Investigation of cell by fluorescence laser scanning microscopy with subnanosecond time resolution," Lasers in the Life Sciences 3, 47-53 (1989).
  25. X. F. Wang, A. Periasamy, B. Herman, and D. Coleman, "Fluorescence lifetime imaging microscopy (FLIM): Instrumentation and applications," Crit. Rev. Anal. Chem. 23, 369-395 (1992).
    [CrossRef]
  26. T. French, P. T. C. So, C. Y. Dong, K. M. Berland, and E. Gratton, "Fluorescence lifetime imaging techniques for microscopy," Methods Cell Biol. 56, 277-304 (1998).
    [CrossRef] [PubMed]
  27. T. W. J. Gadella, Jr., "Fluorescence lifetime imaging microscopy (FLIM): instrumentation and application," in Fluorescent and luminescent probes for biological activity, W. T. Mason, ed. (Academic Press, San Diego, 1999), pp. 467-479.
    [CrossRef]
  28. P. J. Tadrous, "Methods for imaging the structure and function of living tissues and cells: 2. fluorescence lifetime imaging," J. Pathol. 191, 229-234 (2000).
    [CrossRef] [PubMed]
  29. R. Cubeddu, D. Comelli, C. D'Andrea, P. Taroni, and G. Valentini, "Time-resolved fluorescence imaging in biology and medicine," J. Phys. D: Appl. Phys. 35, R61-R76 (2002).
    [CrossRef]
  30. P. K. Urayama, W. Zhong, J. A. Beamish, F. K. Minn, R. D. Sloboda, K. H. Dragnev, E. Dmitrovsky, and M.-A. Mycek, "A UV-visible fluorescence lifetime imaging microscope for laser-based biological sensing with picosecond resolution," Applied Physics B: Lasers and Optics 76, 483-496 (2003).
    [CrossRef]
  31. H. J. Lin, P. Herman, and J. R. Lakowicz, "Fluorescence lifetime-resolved pH imaging of living cells," Cytometry A 52, 77-89 (2003).
    [CrossRef] [PubMed]
  32. P. K. Urayama, J. A. Beamish, F. K. Minn, E. A. Hamon, and M.-A. Mycek, "A UV fluorescence lifetime imaging microscope to probe endogenous cellular fluorescence," presented at the Conference on Lasers and Electro-Optics, 2002.
  33. X. F. Wang, T. Uchida, D. M. Coleman, and S. Minami, "A two-dimensional fluorescence lifetime imaging system using a gated image intensifier," Appl. Spectrosc. 45, 360-366 (1991).
    [CrossRef]
  34. 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]
  35. M.-A. Mycek, P. K. Urayama, K. Heyman, and M. Bussey, "Using POPOP's viscosity dependent lifetime as a picosecond resolution standard in near-UV fluorescence lifetime imaging microscopy," Proc. SPIE 4962, 143-150 (2003).
    [CrossRef]
  36. C. J. Grauw and H. C. Gerritsen, "Multiple time-gate module for fluorescence lifetime imaging," Appl. Spectrosc. 55, 670-678 (2001).
    [CrossRef]
  37. H. C. Gerritsen, M. A. H. Asselbergs, A. V. Agronskaia, and W. G. J. H. M. Van Sark, "Fluorescence lifetime imaging in scanning microscopes: acquisistion speed, photon economy and lifetime resolution," J. Microsc. 206, 218-224 (2002).
    [CrossRef] [PubMed]
  38. M. Tramier, I. Gautier, T. Piolot, S. Ravalet, K. Kemnitz, J. Coppey, C. Durieux, V. Mignotte, and M. Coppey-Moisan, "Picosecond-hetero-FRET microscopy to probe protein-protein interactions in live cells," Biophys. J. 83, 3570-3577 (2002).
    [CrossRef] [PubMed]
  39. M. A. Rizzo, G. H. Springer, B. Granada, and D. W. Piston, "An improved cyan fluorescenct protein variant useful for FRET," Nat. Biotechnol. 22, 445-449 (2004).
    [CrossRef] [PubMed]
  40. R. Rose, M. Weyand, M. Lammers, T. Ishizaki, M. R. Ahmadian, and A. Wittinghofer, "Structural and mechanistic insights into the interaction between Rho and mammalian Dia," Nature 435, 513-518 (2005).
    [CrossRef] [PubMed]
  41. P. Madaule, T. Furuyashiki, T. Reid, T. Ishizaki, G. Watanabe, N. Morii, and S. Narumiya, "A novel partner for the GTP-bound forms of rho and rac," FEBS Lett. 377, 243-248 (1995).
    [CrossRef] [PubMed]
  42. A. K. Hadjantonakis and A. Nagy, "The color of mice: in the light of GFP-variant reporters," Histochem. Cell Biol. 115, 49-58 (2001).
    [PubMed]
  43. G. Feng, R. H. Mellor, M. Bernstein, C. Keller-Peck, Q. T. Nguyen, M. Wallace, J. M. Nerbonne, J. W. Lichtman, and J. R. Sanes, "Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP," Neuron 28, 41-51 (2000).
    [CrossRef] [PubMed]
  44. J. A. Brewer, B. P. Sleckman, W. Swat, and L. J. Muglia, "Green fluorescent protein-glucocorticoid receptor knockin mice reveal dynamic receptor modulation during thymocyte development," J. Immunol. 169, 1309-1318 (2002).
    [PubMed]
  45. F. Schaufele, I. Demarco, and R. N. Day, "FRET imaging in the wide-field microscope," in Molecular imaging: FRET microscopy and spectroscopy, A. Periasamy and R. N. Day, eds. (Oxford University Press, New York, 2005), pp. 72-94.
  46. J. Zhang, R. E. Campbell, A. Y. Ting, and R. Y. Tsien, "Creating new fluorescent probes for cell biology," Nat. Rev. Mol. Cell Biol. 3, 906-918 (2002).
    [CrossRef] [PubMed]
  47. G. Valentin, C. Verheggen, T. Piolot, H. Neel, M. Coppey-Moisan, and E. Bertrand, "Photoconversion of YFP into a CFP-like species during acceptor photobleaching FRET experiments," Nat Methods 2, 801 (2005).
    [CrossRef] [PubMed]
  48. C. Thaler, S. S. Vogel, S. R. Ikeda, and H. Chen, "Photobleaching of YFP does not produce a CFP-like species that affects FRET measurements," Nat Methods 3, 491; author reply 492-493 (2006).
    [CrossRef]
  49. S. E. Verrier and H. D. Soling, "Photobleaching of YFP does not produce a CFP-like species that affects FRET measurements," Nat Methods 3, 491-492; author reply 492-493 (2006).
    [CrossRef] [PubMed]
  50. R. Reddel, Y. K. Ke, V. Gerwin, M. McMenamin, J. Lechner, R. Su, D. Brash, J.-B. Park, J. Limb Rhim, and C. Harris, "Transformation of human bronchial epithelial cells by infections by SV40 or adenovius-12 SV40 hybrid virus, or transfection via strontium phosphate coprecipitation with the plasmid containing SV40 early region genes," Cancer Res. 48, 1904-1909 (1988).
    [PubMed]
  51. A. W. Nguyen and P. S. Daugherty, "Evolutionary optimization of fluorescent proteins for intracellular FRET," Nat. Biotechnol. 23, 355-360 (2005).
    [CrossRef] [PubMed]
  52. O. Griesbeck, G. S. Baird, R. E. Campbell, D. A. Zacharias, and R. Y. Tsien, "Reducing the environmental sensitivity of yellow fluorescent protein. Mechanism and applications," J. Biol. Chem. 276, 29188-29194 (2001).
    [CrossRef] [PubMed]

2005 (3)

R. Rose, M. Weyand, M. Lammers, T. Ishizaki, M. R. Ahmadian, and A. Wittinghofer, "Structural and mechanistic insights into the interaction between Rho and mammalian Dia," Nature 435, 513-518 (2005).
[CrossRef] [PubMed]

G. Valentin, C. Verheggen, T. Piolot, H. Neel, M. Coppey-Moisan, and E. Bertrand, "Photoconversion of YFP into a CFP-like species during acceptor photobleaching FRET experiments," Nat Methods 2, 801 (2005).
[CrossRef] [PubMed]

A. W. Nguyen and P. S. Daugherty, "Evolutionary optimization of fluorescent proteins for intracellular FRET," Nat. Biotechnol. 23, 355-360 (2005).
[CrossRef] [PubMed]

2004 (1)

M. A. Rizzo, G. H. Springer, B. Granada, and D. W. Piston, "An improved cyan fluorescenct protein variant useful for FRET," Nat. Biotechnol. 22, 445-449 (2004).
[CrossRef] [PubMed]

2003 (9)

M.-A. Mycek, P. K. Urayama, K. Heyman, and M. Bussey, "Using POPOP's viscosity dependent lifetime as a picosecond resolution standard in near-UV fluorescence lifetime imaging microscopy," Proc. SPIE 4962, 143-150 (2003).
[CrossRef]

W. Zhong, P. Urayama, and M.-A. Mycek, "Imaging fluorescence lifetime modulation of a ruthenium-based dye in living cells: the potential for oxygen sensing," J. Phys. D: Appl. Phys. 36, 1689-1695 (2003).
[CrossRef]

P. K. Urayama, W. Zhong, J. A. Beamish, F. K. Minn, R. D. Sloboda, K. H. Dragnev, E. Dmitrovsky, and M.-A. Mycek, "A UV-visible fluorescence lifetime imaging microscope for laser-based biological sensing with picosecond resolution," Applied Physics B: Lasers and Optics 76, 483-496 (2003).
[CrossRef]

H. J. Lin, P. Herman, and J. R. Lakowicz, "Fluorescence lifetime-resolved pH imaging of living cells," Cytometry A 52, 77-89 (2003).
[CrossRef] [PubMed]

Y. Chen and J. D. Mills, "Protein localization in living cells and tissues using FRET and FLIM," Differentiation 71, 528-541 (2003).
[CrossRef] [PubMed]

W. B. Zhong, C. Y. Wang, T. C. Chang, and W. S. Lee, "Lovastatin induces apoptosis of anaplastic thyroid cancer cells via inhibition of protein geranylgeranylation and de novo protein synthesis," Endocrinology 144, 3852-3859 (2003).
[CrossRef] [PubMed]

C. Marionnet, C. Lalou, K. Mollier, M. Chazal, G. Delestaing, D. Compan, O. Verola, C. Vilmer, J. Cuminet, L. Dubertret, and N. Basset-Seguin, "Differential molecular profiling between skin carcinomas reveals four newly reported genes potentially implicated in squamous cell carcinoma development," Oncogene 22, 3500-3505 (2003).
[CrossRef] [PubMed]

W. Wang, L. Y. Yang, G. W. Huang, and W. Q. Lu, "Expression and significance of RhoC gene in hepatocellular carcinoma," World Journal of Gastroenterology 9, 1950-1953 (2003).
[PubMed]

J. A. Schmid and H. H. Sitte, "Fluorescence resonance energy transfer in the study of cancer pathways," Curr. Opin. Oncol. 15, 55-64 (2003).
[CrossRef]

2002 (8)

G. Fritz, C. Brachetti, F. Bahlmann, M. Schmidt, and B. Kaina, "Rho GTPases in human breast tumours: expression and mutation analyses and correlation with clinical parameters," Br. J. Cancer 87, 635-644 (2002).
[CrossRef] [PubMed]

F. J. M. van Kuppeveld, W. J. G. Melchers, P. H. G. M. Willems, and T. W. J. Gadella, Jr., "Homomultimerization of the coxsackievirus 2B protein in living cells visualized by fluorescence resonance energy transfer microscopy," J. Virol. 76, 9446-9456 (2002).
[CrossRef] [PubMed]

M. Elangovan, R. N. Day, and A. Periasamy, "Nanosecond fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy to localize the protein interactions in a single living cell," J. Microsc. 205, 3-14 (2002).
[CrossRef] [PubMed]

R. Cubeddu, D. Comelli, C. D'Andrea, P. Taroni, and G. Valentini, "Time-resolved fluorescence imaging in biology and medicine," J. Phys. D: Appl. Phys. 35, R61-R76 (2002).
[CrossRef]

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

M. Tramier, I. Gautier, T. Piolot, S. Ravalet, K. Kemnitz, J. Coppey, C. Durieux, V. Mignotte, and M. Coppey-Moisan, "Picosecond-hetero-FRET microscopy to probe protein-protein interactions in live cells," Biophys. J. 83, 3570-3577 (2002).
[CrossRef] [PubMed]

J. A. Brewer, B. P. Sleckman, W. Swat, and L. J. Muglia, "Green fluorescent protein-glucocorticoid receptor knockin mice reveal dynamic receptor modulation during thymocyte development," J. Immunol. 169, 1309-1318 (2002).
[PubMed]

J. Zhang, R. E. Campbell, A. Y. Ting, and R. Y. Tsien, "Creating new fluorescent probes for cell biology," Nat. Rev. Mol. Cell Biol. 3, 906-918 (2002).
[CrossRef] [PubMed]

2001 (7)

A. K. Hadjantonakis and A. Nagy, "The color of mice: in the light of GFP-variant reporters," Histochem. Cell Biol. 115, 49-58 (2001).
[PubMed]

O. Griesbeck, G. S. Baird, R. E. Campbell, D. A. Zacharias, and R. Y. Tsien, "Reducing the environmental sensitivity of yellow fluorescent protein. Mechanism and applications," J. Biol. Chem. 276, 29188-29194 (2001).
[CrossRef] [PubMed]

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

A. Periasamy, "Fluorescence resonance energy transfer microscopy: a mini review," J. Biomed. Opt. 6, 287-291 (2001).
[CrossRef] [PubMed]

A. Tsuji, Y. Sato, M. Hirano, T. Suga, H. Koshimoto, T. Taguchi, and S. Ohsuka, "Development of a time-resolved fluorometric method for observing hybridization in living cells using fluorescence resonance energy transfer," Biophys. J. 81, 501-515 (2001).
[CrossRef] [PubMed]

J. Lippincott-Schwartz, E. Snapp, and A. Kenworthy, "Studying protein dynamics in living cells," Nat. Rev. Mol. Cell Biol. 2, 444-456 (2001).
[CrossRef] [PubMed]

S. Fields, "Proteomics in genomeland," Science 291, 1221-1224 (2001).
[CrossRef] [PubMed]

2000 (5)

P. Legrain and L. Selig, "Genome-wide protein interaction maps using two-hybrid systems," FEBS Lett. 480, 32-36 (2000).
[CrossRef] [PubMed]

K. L. van Golen, Z.-F. Wu, X. T. Qiao, L. W. Bao, and S. D. Merajver, "RhoC GTPase, a novel transforming oncogene for human mammary epithelial cells that partially recapitulates the inflammatory breast cancer phenotype," Cancer Res. 60, 5832-5838 (2000).
[PubMed]

E. A. Clark, T. R. Golub, E. S. Lander, and R. O. Hynes, "Genomic analysis of metastasis reveals an essential role for RhoC," Nature 406, 532-535 (2000).
[CrossRef] [PubMed]

P. J. Tadrous, "Methods for imaging the structure and function of living tissues and cells: 2. fluorescence lifetime imaging," J. Pathol. 191, 229-234 (2000).
[CrossRef] [PubMed]

G. Feng, R. H. Mellor, M. Bernstein, C. Keller-Peck, Q. T. Nguyen, M. Wallace, J. M. Nerbonne, J. W. Lichtman, and J. R. Sanes, "Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP," Neuron 28, 41-51 (2000).
[CrossRef] [PubMed]

1999 (2)

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]

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

1998 (3)

H. Suwa, G. Ohshio, T. Imamura, G. Watanabe, S. Arii, M. Imamura, S. Narumiya, H. Hiai, and M. Fukumoto, "Overexpression of the RhoC gene correlates with progression of ductal adenocarcinoma of the pancreas," Br. J. Cancer 77, 147-152 (1998).
[CrossRef] [PubMed]

R. Y. Tsien, "The green fluorescent protein," Annual Reviews of Biochemistry 67, 509-544 (1998).
[CrossRef]

T. French, P. T. C. So, C. Y. Dong, K. M. Berland, and E. Gratton, "Fluorescence lifetime imaging techniques for microscopy," Methods Cell Biol. 56, 277-304 (1998).
[CrossRef] [PubMed]

1995 (1)

P. Madaule, T. Furuyashiki, T. Reid, T. Ishizaki, G. Watanabe, N. Morii, and S. Narumiya, "A novel partner for the GTP-bound forms of rho and rac," FEBS Lett. 377, 243-248 (1995).
[CrossRef] [PubMed]

1992 (1)

X. F. Wang, A. Periasamy, B. Herman, and D. Coleman, "Fluorescence lifetime imaging microscopy (FLIM): Instrumentation and applications," Crit. Rev. Anal. Chem. 23, 369-395 (1992).
[CrossRef]

1991 (1)

1989 (1)

I. Bugiel, K. König, and H. Wabnitz, "Investigation of cell by fluorescence laser scanning microscopy with subnanosecond time resolution," Lasers in the Life Sciences 3, 47-53 (1989).

1988 (1)

R. Reddel, Y. K. Ke, V. Gerwin, M. McMenamin, J. Lechner, R. Su, D. Brash, J.-B. Park, J. Limb Rhim, and C. Harris, "Transformation of human bronchial epithelial cells by infections by SV40 or adenovius-12 SV40 hybrid virus, or transfection via strontium phosphate coprecipitation with the plasmid containing SV40 early region genes," Cancer Res. 48, 1904-1909 (1988).
[PubMed]

Agronskaia, A. V.

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

Ahmadian, M. R.

R. Rose, M. Weyand, M. Lammers, T. Ishizaki, M. R. Ahmadian, and A. Wittinghofer, "Structural and mechanistic insights into the interaction between Rho and mammalian Dia," Nature 435, 513-518 (2005).
[CrossRef] [PubMed]

Arii, S.

H. Suwa, G. Ohshio, T. Imamura, G. Watanabe, S. Arii, M. Imamura, S. Narumiya, H. Hiai, and M. Fukumoto, "Overexpression of the RhoC gene correlates with progression of ductal adenocarcinoma of the pancreas," Br. J. Cancer 77, 147-152 (1998).
[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]

Asselbergs, M. A. H.

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

Bahlmann, F.

G. Fritz, C. Brachetti, F. Bahlmann, M. Schmidt, and B. Kaina, "Rho GTPases in human breast tumours: expression and mutation analyses and correlation with clinical parameters," Br. J. Cancer 87, 635-644 (2002).
[CrossRef] [PubMed]

Baird, G. S.

O. Griesbeck, G. S. Baird, R. E. Campbell, D. A. Zacharias, and R. Y. Tsien, "Reducing the environmental sensitivity of yellow fluorescent protein. Mechanism and applications," J. Biol. Chem. 276, 29188-29194 (2001).
[CrossRef] [PubMed]

Bao, L. W.

K. L. van Golen, Z.-F. Wu, X. T. Qiao, L. W. Bao, and S. D. Merajver, "RhoC GTPase, a novel transforming oncogene for human mammary epithelial cells that partially recapitulates the inflammatory breast cancer phenotype," Cancer Res. 60, 5832-5838 (2000).
[PubMed]

Basset-Seguin, N.

C. Marionnet, C. Lalou, K. Mollier, M. Chazal, G. Delestaing, D. Compan, O. Verola, C. Vilmer, J. Cuminet, L. Dubertret, and N. Basset-Seguin, "Differential molecular profiling between skin carcinomas reveals four newly reported genes potentially implicated in squamous cell carcinoma development," Oncogene 22, 3500-3505 (2003).
[CrossRef] [PubMed]

Bastiaens, P. I. H.

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

Beamish, J. A.

P. K. Urayama, W. Zhong, J. A. Beamish, F. K. Minn, R. D. Sloboda, K. H. Dragnev, E. Dmitrovsky, and M.-A. Mycek, "A UV-visible fluorescence lifetime imaging microscope for laser-based biological sensing with picosecond resolution," Applied Physics B: Lasers and Optics 76, 483-496 (2003).
[CrossRef]

Berland, K. M.

T. French, P. T. C. So, C. Y. Dong, K. M. Berland, and E. Gratton, "Fluorescence lifetime imaging techniques for microscopy," Methods Cell Biol. 56, 277-304 (1998).
[CrossRef] [PubMed]

Bernstein, M.

G. Feng, R. H. Mellor, M. Bernstein, C. Keller-Peck, Q. T. Nguyen, M. Wallace, J. M. Nerbonne, J. W. Lichtman, and J. R. Sanes, "Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP," Neuron 28, 41-51 (2000).
[CrossRef] [PubMed]

Bertrand, E.

G. Valentin, C. Verheggen, T. Piolot, H. Neel, M. Coppey-Moisan, and E. Bertrand, "Photoconversion of YFP into a CFP-like species during acceptor photobleaching FRET experiments," Nat Methods 2, 801 (2005).
[CrossRef] [PubMed]

Brachetti, C.

G. Fritz, C. Brachetti, F. Bahlmann, M. Schmidt, and B. Kaina, "Rho GTPases in human breast tumours: expression and mutation analyses and correlation with clinical parameters," Br. J. Cancer 87, 635-644 (2002).
[CrossRef] [PubMed]

Brash, D.

R. Reddel, Y. K. Ke, V. Gerwin, M. McMenamin, J. Lechner, R. Su, D. Brash, J.-B. Park, J. Limb Rhim, and C. Harris, "Transformation of human bronchial epithelial cells by infections by SV40 or adenovius-12 SV40 hybrid virus, or transfection via strontium phosphate coprecipitation with the plasmid containing SV40 early region genes," Cancer Res. 48, 1904-1909 (1988).
[PubMed]

Brewer, J. A.

J. A. Brewer, B. P. Sleckman, W. Swat, and L. J. Muglia, "Green fluorescent protein-glucocorticoid receptor knockin mice reveal dynamic receptor modulation during thymocyte development," J. Immunol. 169, 1309-1318 (2002).
[PubMed]

Bugiel, I.

I. Bugiel, K. König, and H. Wabnitz, "Investigation of cell by fluorescence laser scanning microscopy with subnanosecond time resolution," Lasers in the Life Sciences 3, 47-53 (1989).

Bussey, M.

M.-A. Mycek, P. K. Urayama, K. Heyman, and M. Bussey, "Using POPOP's viscosity dependent lifetime as a picosecond resolution standard in near-UV fluorescence lifetime imaging microscopy," Proc. SPIE 4962, 143-150 (2003).
[CrossRef]

Campbell, R. E.

J. Zhang, R. E. Campbell, A. Y. Ting, and R. Y. Tsien, "Creating new fluorescent probes for cell biology," Nat. Rev. Mol. Cell Biol. 3, 906-918 (2002).
[CrossRef] [PubMed]

O. Griesbeck, G. S. Baird, R. E. Campbell, D. A. Zacharias, and R. Y. Tsien, "Reducing the environmental sensitivity of yellow fluorescent protein. Mechanism and applications," J. Biol. Chem. 276, 29188-29194 (2001).
[CrossRef] [PubMed]

Chang, T. C.

W. B. Zhong, C. Y. Wang, T. C. Chang, and W. S. Lee, "Lovastatin induces apoptosis of anaplastic thyroid cancer cells via inhibition of protein geranylgeranylation and de novo protein synthesis," Endocrinology 144, 3852-3859 (2003).
[CrossRef] [PubMed]

Chazal, M.

C. Marionnet, C. Lalou, K. Mollier, M. Chazal, G. Delestaing, D. Compan, O. Verola, C. Vilmer, J. Cuminet, L. Dubertret, and N. Basset-Seguin, "Differential molecular profiling between skin carcinomas reveals four newly reported genes potentially implicated in squamous cell carcinoma development," Oncogene 22, 3500-3505 (2003).
[CrossRef] [PubMed]

Chen, Y.

Y. Chen and J. D. Mills, "Protein localization in living cells and tissues using FRET and FLIM," Differentiation 71, 528-541 (2003).
[CrossRef] [PubMed]

Clark, E. A.

E. A. Clark, T. R. Golub, E. S. Lander, and R. O. Hynes, "Genomic analysis of metastasis reveals an essential role for RhoC," Nature 406, 532-535 (2000).
[CrossRef] [PubMed]

Coleman, D.

X. F. Wang, A. Periasamy, B. Herman, and D. Coleman, "Fluorescence lifetime imaging microscopy (FLIM): Instrumentation and applications," Crit. Rev. Anal. Chem. 23, 369-395 (1992).
[CrossRef]

Coleman, D. M.

Comelli, D.

R. Cubeddu, D. Comelli, C. D'Andrea, P. Taroni, and G. Valentini, "Time-resolved fluorescence imaging in biology and medicine," J. Phys. D: Appl. Phys. 35, R61-R76 (2002).
[CrossRef]

Compan, D.

C. Marionnet, C. Lalou, K. Mollier, M. Chazal, G. Delestaing, D. Compan, O. Verola, C. Vilmer, J. Cuminet, L. Dubertret, and N. Basset-Seguin, "Differential molecular profiling between skin carcinomas reveals four newly reported genes potentially implicated in squamous cell carcinoma development," Oncogene 22, 3500-3505 (2003).
[CrossRef] [PubMed]

Coppey, J.

M. Tramier, I. Gautier, T. Piolot, S. Ravalet, K. Kemnitz, J. Coppey, C. Durieux, V. Mignotte, and M. Coppey-Moisan, "Picosecond-hetero-FRET microscopy to probe protein-protein interactions in live cells," Biophys. J. 83, 3570-3577 (2002).
[CrossRef] [PubMed]

Coppey-Moisan, M.

G. Valentin, C. Verheggen, T. Piolot, H. Neel, M. Coppey-Moisan, and E. Bertrand, "Photoconversion of YFP into a CFP-like species during acceptor photobleaching FRET experiments," Nat Methods 2, 801 (2005).
[CrossRef] [PubMed]

M. Tramier, I. Gautier, T. Piolot, S. Ravalet, K. Kemnitz, J. Coppey, C. Durieux, V. Mignotte, and M. Coppey-Moisan, "Picosecond-hetero-FRET microscopy to probe protein-protein interactions in live cells," Biophys. J. 83, 3570-3577 (2002).
[CrossRef] [PubMed]

Cubeddu, R.

R. Cubeddu, D. Comelli, C. D'Andrea, P. Taroni, and G. Valentini, "Time-resolved fluorescence imaging in biology and medicine," J. Phys. D: Appl. Phys. 35, R61-R76 (2002).
[CrossRef]

Cuminet, J.

C. Marionnet, C. Lalou, K. Mollier, M. Chazal, G. Delestaing, D. Compan, O. Verola, C. Vilmer, J. Cuminet, L. Dubertret, and N. Basset-Seguin, "Differential molecular profiling between skin carcinomas reveals four newly reported genes potentially implicated in squamous cell carcinoma development," Oncogene 22, 3500-3505 (2003).
[CrossRef] [PubMed]

D'Andrea, C.

R. Cubeddu, D. Comelli, C. D'Andrea, P. Taroni, and G. Valentini, "Time-resolved fluorescence imaging in biology and medicine," J. Phys. D: Appl. Phys. 35, R61-R76 (2002).
[CrossRef]

Daugherty, P. S.

A. W. Nguyen and P. S. Daugherty, "Evolutionary optimization of fluorescent proteins for intracellular FRET," Nat. Biotechnol. 23, 355-360 (2005).
[CrossRef] [PubMed]

Day, R. N.

M. Elangovan, R. N. Day, and A. Periasamy, "Nanosecond fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy to localize the protein interactions in a single living cell," J. Microsc. 205, 3-14 (2002).
[CrossRef] [PubMed]

Delestaing, G.

C. Marionnet, C. Lalou, K. Mollier, M. Chazal, G. Delestaing, D. Compan, O. Verola, C. Vilmer, J. Cuminet, L. Dubertret, and N. Basset-Seguin, "Differential molecular profiling between skin carcinomas reveals four newly reported genes potentially implicated in squamous cell carcinoma development," Oncogene 22, 3500-3505 (2003).
[CrossRef] [PubMed]

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]

Dmitrovsky, E.

P. K. Urayama, W. Zhong, J. A. Beamish, F. K. Minn, R. D. Sloboda, K. H. Dragnev, E. Dmitrovsky, and M.-A. Mycek, "A UV-visible fluorescence lifetime imaging microscope for laser-based biological sensing with picosecond resolution," Applied Physics B: Lasers and Optics 76, 483-496 (2003).
[CrossRef]

Dong, C. Y.

T. French, P. T. C. So, C. Y. Dong, K. M. Berland, and E. Gratton, "Fluorescence lifetime imaging techniques for microscopy," Methods Cell Biol. 56, 277-304 (1998).
[CrossRef] [PubMed]

Dragnev, K. H.

P. K. Urayama, W. Zhong, J. A. Beamish, F. K. Minn, R. D. Sloboda, K. H. Dragnev, E. Dmitrovsky, and M.-A. Mycek, "A UV-visible fluorescence lifetime imaging microscope for laser-based biological sensing with picosecond resolution," Applied Physics B: Lasers and Optics 76, 483-496 (2003).
[CrossRef]

Dubertret, L.

C. Marionnet, C. Lalou, K. Mollier, M. Chazal, G. Delestaing, D. Compan, O. Verola, C. Vilmer, J. Cuminet, L. Dubertret, and N. Basset-Seguin, "Differential molecular profiling between skin carcinomas reveals four newly reported genes potentially implicated in squamous cell carcinoma development," Oncogene 22, 3500-3505 (2003).
[CrossRef] [PubMed]

Durieux, C.

M. Tramier, I. Gautier, T. Piolot, S. Ravalet, K. Kemnitz, J. Coppey, C. Durieux, V. Mignotte, and M. Coppey-Moisan, "Picosecond-hetero-FRET microscopy to probe protein-protein interactions in live cells," Biophys. J. 83, 3570-3577 (2002).
[CrossRef] [PubMed]

Elangovan, M.

M. Elangovan, R. N. Day, and A. Periasamy, "Nanosecond fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy to localize the protein interactions in a single living cell," J. Microsc. 205, 3-14 (2002).
[CrossRef] [PubMed]

Feng, G.

G. Feng, R. H. Mellor, M. Bernstein, C. Keller-Peck, Q. T. Nguyen, M. Wallace, J. M. Nerbonne, J. W. Lichtman, and J. R. Sanes, "Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP," Neuron 28, 41-51 (2000).
[CrossRef] [PubMed]

Fields, S.

S. Fields, "Proteomics in genomeland," Science 291, 1221-1224 (2001).
[CrossRef] [PubMed]

French, T.

T. French, P. T. C. So, C. Y. Dong, K. M. Berland, and E. Gratton, "Fluorescence lifetime imaging techniques for microscopy," Methods Cell Biol. 56, 277-304 (1998).
[CrossRef] [PubMed]

Fritz, G.

G. Fritz, C. Brachetti, F. Bahlmann, M. Schmidt, and B. Kaina, "Rho GTPases in human breast tumours: expression and mutation analyses and correlation with clinical parameters," Br. J. Cancer 87, 635-644 (2002).
[CrossRef] [PubMed]

Fukumoto, M.

H. Suwa, G. Ohshio, T. Imamura, G. Watanabe, S. Arii, M. Imamura, S. Narumiya, H. Hiai, and M. Fukumoto, "Overexpression of the RhoC gene correlates with progression of ductal adenocarcinoma of the pancreas," Br. J. Cancer 77, 147-152 (1998).
[CrossRef] [PubMed]

Furuyashiki, T.

P. Madaule, T. Furuyashiki, T. Reid, T. Ishizaki, G. Watanabe, N. Morii, and S. Narumiya, "A novel partner for the GTP-bound forms of rho and rac," FEBS Lett. 377, 243-248 (1995).
[CrossRef] [PubMed]

Gadella, T. W. J.

F. J. M. van Kuppeveld, W. J. G. Melchers, P. H. G. M. Willems, and T. W. J. Gadella, Jr., "Homomultimerization of the coxsackievirus 2B protein in living cells visualized by fluorescence resonance energy transfer microscopy," J. Virol. 76, 9446-9456 (2002).
[CrossRef] [PubMed]

Gautier, I.

M. Tramier, I. Gautier, T. Piolot, S. Ravalet, K. Kemnitz, J. Coppey, C. Durieux, V. Mignotte, and M. Coppey-Moisan, "Picosecond-hetero-FRET microscopy to probe protein-protein interactions in live cells," Biophys. J. 83, 3570-3577 (2002).
[CrossRef] [PubMed]

Gerritsen, H. C.

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

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

Gerwin, V.

R. Reddel, Y. K. Ke, V. Gerwin, M. McMenamin, J. Lechner, R. Su, D. Brash, J.-B. Park, J. Limb Rhim, and C. Harris, "Transformation of human bronchial epithelial cells by infections by SV40 or adenovius-12 SV40 hybrid virus, or transfection via strontium phosphate coprecipitation with the plasmid containing SV40 early region genes," Cancer Res. 48, 1904-1909 (1988).
[PubMed]

Golub, T. R.

E. A. Clark, T. R. Golub, E. S. Lander, and R. O. Hynes, "Genomic analysis of metastasis reveals an essential role for RhoC," Nature 406, 532-535 (2000).
[CrossRef] [PubMed]

Granada, B.

M. A. Rizzo, G. H. Springer, B. Granada, and D. W. Piston, "An improved cyan fluorescenct protein variant useful for FRET," Nat. Biotechnol. 22, 445-449 (2004).
[CrossRef] [PubMed]

Gratton, E.

T. French, P. T. C. So, C. Y. Dong, K. M. Berland, and E. Gratton, "Fluorescence lifetime imaging techniques for microscopy," Methods Cell Biol. 56, 277-304 (1998).
[CrossRef] [PubMed]

Grauw, C. J.

Griesbeck, O.

O. Griesbeck, G. S. Baird, R. E. Campbell, D. A. Zacharias, and R. Y. Tsien, "Reducing the environmental sensitivity of yellow fluorescent protein. Mechanism and applications," J. Biol. Chem. 276, 29188-29194 (2001).
[CrossRef] [PubMed]

Hadjantonakis, A. K.

A. K. Hadjantonakis and A. Nagy, "The color of mice: in the light of GFP-variant reporters," Histochem. Cell Biol. 115, 49-58 (2001).
[PubMed]

Harris, C.

R. Reddel, Y. K. Ke, V. Gerwin, M. McMenamin, J. Lechner, R. Su, D. Brash, J.-B. Park, J. Limb Rhim, and C. Harris, "Transformation of human bronchial epithelial cells by infections by SV40 or adenovius-12 SV40 hybrid virus, or transfection via strontium phosphate coprecipitation with the plasmid containing SV40 early region genes," Cancer Res. 48, 1904-1909 (1988).
[PubMed]

Herman, B.

X. F. Wang, A. Periasamy, B. Herman, and D. Coleman, "Fluorescence lifetime imaging microscopy (FLIM): Instrumentation and applications," Crit. Rev. Anal. Chem. 23, 369-395 (1992).
[CrossRef]

Herman, P.

H. J. Lin, P. Herman, and J. R. Lakowicz, "Fluorescence lifetime-resolved pH imaging of living cells," Cytometry A 52, 77-89 (2003).
[CrossRef] [PubMed]

Heyman, K.

M.-A. Mycek, P. K. Urayama, K. Heyman, and M. Bussey, "Using POPOP's viscosity dependent lifetime as a picosecond resolution standard in near-UV fluorescence lifetime imaging microscopy," Proc. SPIE 4962, 143-150 (2003).
[CrossRef]

Hiai, H.

H. Suwa, G. Ohshio, T. Imamura, G. Watanabe, S. Arii, M. Imamura, S. Narumiya, H. Hiai, and M. Fukumoto, "Overexpression of the RhoC gene correlates with progression of ductal adenocarcinoma of the pancreas," Br. J. Cancer 77, 147-152 (1998).
[CrossRef] [PubMed]

Hirano, M.

A. Tsuji, Y. Sato, M. Hirano, T. Suga, H. Koshimoto, T. Taguchi, and S. Ohsuka, "Development of a time-resolved fluorometric method for observing hybridization in living cells using fluorescence resonance energy transfer," Biophys. J. 81, 501-515 (2001).
[CrossRef] [PubMed]

Huang, G. W.

W. Wang, L. Y. Yang, G. W. Huang, and W. Q. Lu, "Expression and significance of RhoC gene in hepatocellular carcinoma," World Journal of Gastroenterology 9, 1950-1953 (2003).
[PubMed]

Hynes, R. O.

E. A. Clark, T. R. Golub, E. S. Lander, and R. O. Hynes, "Genomic analysis of metastasis reveals an essential role for RhoC," Nature 406, 532-535 (2000).
[CrossRef] [PubMed]

Imamura, M.

H. Suwa, G. Ohshio, T. Imamura, G. Watanabe, S. Arii, M. Imamura, S. Narumiya, H. Hiai, and M. Fukumoto, "Overexpression of the RhoC gene correlates with progression of ductal adenocarcinoma of the pancreas," Br. J. Cancer 77, 147-152 (1998).
[CrossRef] [PubMed]

Imamura, T.

H. Suwa, G. Ohshio, T. Imamura, G. Watanabe, S. Arii, M. Imamura, S. Narumiya, H. Hiai, and M. Fukumoto, "Overexpression of the RhoC gene correlates with progression of ductal adenocarcinoma of the pancreas," Br. J. Cancer 77, 147-152 (1998).
[CrossRef] [PubMed]

Ishizaki, T.

R. Rose, M. Weyand, M. Lammers, T. Ishizaki, M. R. Ahmadian, and A. Wittinghofer, "Structural and mechanistic insights into the interaction between Rho and mammalian Dia," Nature 435, 513-518 (2005).
[CrossRef] [PubMed]

P. Madaule, T. Furuyashiki, T. Reid, T. Ishizaki, G. Watanabe, N. Morii, and S. Narumiya, "A novel partner for the GTP-bound forms of rho and rac," FEBS Lett. 377, 243-248 (1995).
[CrossRef] [PubMed]

Kaina, B.

G. Fritz, C. Brachetti, F. Bahlmann, M. Schmidt, and B. Kaina, "Rho GTPases in human breast tumours: expression and mutation analyses and correlation with clinical parameters," Br. J. Cancer 87, 635-644 (2002).
[CrossRef] [PubMed]

Ke, Y. K.

R. Reddel, Y. K. Ke, V. Gerwin, M. McMenamin, J. Lechner, R. Su, D. Brash, J.-B. Park, J. Limb Rhim, and C. Harris, "Transformation of human bronchial epithelial cells by infections by SV40 or adenovius-12 SV40 hybrid virus, or transfection via strontium phosphate coprecipitation with the plasmid containing SV40 early region genes," Cancer Res. 48, 1904-1909 (1988).
[PubMed]

Keller-Peck, C.

G. Feng, R. H. Mellor, M. Bernstein, C. Keller-Peck, Q. T. Nguyen, M. Wallace, J. M. Nerbonne, J. W. Lichtman, and J. R. Sanes, "Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP," Neuron 28, 41-51 (2000).
[CrossRef] [PubMed]

Kemnitz, K.

M. Tramier, I. Gautier, T. Piolot, S. Ravalet, K. Kemnitz, J. Coppey, C. Durieux, V. Mignotte, and M. Coppey-Moisan, "Picosecond-hetero-FRET microscopy to probe protein-protein interactions in live cells," Biophys. J. 83, 3570-3577 (2002).
[CrossRef] [PubMed]

Kenworthy, A.

J. Lippincott-Schwartz, E. Snapp, and A. Kenworthy, "Studying protein dynamics in living cells," Nat. Rev. Mol. Cell Biol. 2, 444-456 (2001).
[CrossRef] [PubMed]

König, K.

I. Bugiel, K. König, and H. Wabnitz, "Investigation of cell by fluorescence laser scanning microscopy with subnanosecond time resolution," Lasers in the Life Sciences 3, 47-53 (1989).

Koshimoto, H.

A. Tsuji, Y. Sato, M. Hirano, T. Suga, H. Koshimoto, T. Taguchi, and S. Ohsuka, "Development of a time-resolved fluorometric method for observing hybridization in living cells using fluorescence resonance energy transfer," Biophys. J. 81, 501-515 (2001).
[CrossRef] [PubMed]

Lakowicz, J. R.

H. J. Lin, P. Herman, and J. R. Lakowicz, "Fluorescence lifetime-resolved pH imaging of living cells," Cytometry A 52, 77-89 (2003).
[CrossRef] [PubMed]

Lalou, C.

C. Marionnet, C. Lalou, K. Mollier, M. Chazal, G. Delestaing, D. Compan, O. Verola, C. Vilmer, J. Cuminet, L. Dubertret, and N. Basset-Seguin, "Differential molecular profiling between skin carcinomas reveals four newly reported genes potentially implicated in squamous cell carcinoma development," Oncogene 22, 3500-3505 (2003).
[CrossRef] [PubMed]

Lammers, M.

R. Rose, M. Weyand, M. Lammers, T. Ishizaki, M. R. Ahmadian, and A. Wittinghofer, "Structural and mechanistic insights into the interaction between Rho and mammalian Dia," Nature 435, 513-518 (2005).
[CrossRef] [PubMed]

Lander, E. S.

E. A. Clark, T. R. Golub, E. S. Lander, and R. O. Hynes, "Genomic analysis of metastasis reveals an essential role for RhoC," Nature 406, 532-535 (2000).
[CrossRef] [PubMed]

Lechner, J.

R. Reddel, Y. K. Ke, V. Gerwin, M. McMenamin, J. Lechner, R. Su, D. Brash, J.-B. Park, J. Limb Rhim, and C. Harris, "Transformation of human bronchial epithelial cells by infections by SV40 or adenovius-12 SV40 hybrid virus, or transfection via strontium phosphate coprecipitation with the plasmid containing SV40 early region genes," Cancer Res. 48, 1904-1909 (1988).
[PubMed]

Lee, W. S.

W. B. Zhong, C. Y. Wang, T. C. Chang, and W. S. Lee, "Lovastatin induces apoptosis of anaplastic thyroid cancer cells via inhibition of protein geranylgeranylation and de novo protein synthesis," Endocrinology 144, 3852-3859 (2003).
[CrossRef] [PubMed]

Legrain, P.

P. Legrain and L. Selig, "Genome-wide protein interaction maps using two-hybrid systems," FEBS Lett. 480, 32-36 (2000).
[CrossRef] [PubMed]

Lichtman, J. W.

G. Feng, R. H. Mellor, M. Bernstein, C. Keller-Peck, Q. T. Nguyen, M. Wallace, J. M. Nerbonne, J. W. Lichtman, and J. R. Sanes, "Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP," Neuron 28, 41-51 (2000).
[CrossRef] [PubMed]

Limb Rhim, J.

R. Reddel, Y. K. Ke, V. Gerwin, M. McMenamin, J. Lechner, R. Su, D. Brash, J.-B. Park, J. Limb Rhim, and C. Harris, "Transformation of human bronchial epithelial cells by infections by SV40 or adenovius-12 SV40 hybrid virus, or transfection via strontium phosphate coprecipitation with the plasmid containing SV40 early region genes," Cancer Res. 48, 1904-1909 (1988).
[PubMed]

Lin, H. J.

H. J. Lin, P. Herman, and J. R. Lakowicz, "Fluorescence lifetime-resolved pH imaging of living cells," Cytometry A 52, 77-89 (2003).
[CrossRef] [PubMed]

Lippincott-Schwartz, J.

J. Lippincott-Schwartz, E. Snapp, and A. Kenworthy, "Studying protein dynamics in living cells," Nat. Rev. Mol. Cell Biol. 2, 444-456 (2001).
[CrossRef] [PubMed]

Lu, W. Q.

W. Wang, L. Y. Yang, G. W. Huang, and W. Q. Lu, "Expression and significance of RhoC gene in hepatocellular carcinoma," World Journal of Gastroenterology 9, 1950-1953 (2003).
[PubMed]

Madaule, P.

P. Madaule, T. Furuyashiki, T. Reid, T. Ishizaki, G. Watanabe, N. Morii, and S. Narumiya, "A novel partner for the GTP-bound forms of rho and rac," FEBS Lett. 377, 243-248 (1995).
[CrossRef] [PubMed]

Marionnet, C.

C. Marionnet, C. Lalou, K. Mollier, M. Chazal, G. Delestaing, D. Compan, O. Verola, C. Vilmer, J. Cuminet, L. Dubertret, and N. Basset-Seguin, "Differential molecular profiling between skin carcinomas reveals four newly reported genes potentially implicated in squamous cell carcinoma development," Oncogene 22, 3500-3505 (2003).
[CrossRef] [PubMed]

McMenamin, M.

R. Reddel, Y. K. Ke, V. Gerwin, M. McMenamin, J. Lechner, R. Su, D. Brash, J.-B. Park, J. Limb Rhim, and C. Harris, "Transformation of human bronchial epithelial cells by infections by SV40 or adenovius-12 SV40 hybrid virus, or transfection via strontium phosphate coprecipitation with the plasmid containing SV40 early region genes," Cancer Res. 48, 1904-1909 (1988).
[PubMed]

Melchers, W. J. G.

F. J. M. van Kuppeveld, W. J. G. Melchers, P. H. G. M. Willems, and T. W. J. Gadella, Jr., "Homomultimerization of the coxsackievirus 2B protein in living cells visualized by fluorescence resonance energy transfer microscopy," J. Virol. 76, 9446-9456 (2002).
[CrossRef] [PubMed]

Mellor, R. H.

G. Feng, R. H. Mellor, M. Bernstein, C. Keller-Peck, Q. T. Nguyen, M. Wallace, J. M. Nerbonne, J. W. Lichtman, and J. R. Sanes, "Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP," Neuron 28, 41-51 (2000).
[CrossRef] [PubMed]

Merajver, S. D.

K. L. van Golen, Z.-F. Wu, X. T. Qiao, L. W. Bao, and S. D. Merajver, "RhoC GTPase, a novel transforming oncogene for human mammary epithelial cells that partially recapitulates the inflammatory breast cancer phenotype," Cancer Res. 60, 5832-5838 (2000).
[PubMed]

Mignotte, V.

M. Tramier, I. Gautier, T. Piolot, S. Ravalet, K. Kemnitz, J. Coppey, C. Durieux, V. Mignotte, and M. Coppey-Moisan, "Picosecond-hetero-FRET microscopy to probe protein-protein interactions in live cells," Biophys. J. 83, 3570-3577 (2002).
[CrossRef] [PubMed]

Mills, J. D.

Y. Chen and J. D. Mills, "Protein localization in living cells and tissues using FRET and FLIM," Differentiation 71, 528-541 (2003).
[CrossRef] [PubMed]

Minami, S.

Minn, F. K.

P. K. Urayama, W. Zhong, J. A. Beamish, F. K. Minn, R. D. Sloboda, K. H. Dragnev, E. Dmitrovsky, and M.-A. Mycek, "A UV-visible fluorescence lifetime imaging microscope for laser-based biological sensing with picosecond resolution," Applied Physics B: Lasers and Optics 76, 483-496 (2003).
[CrossRef]

Mollier, K.

C. Marionnet, C. Lalou, K. Mollier, M. Chazal, G. Delestaing, D. Compan, O. Verola, C. Vilmer, J. Cuminet, L. Dubertret, and N. Basset-Seguin, "Differential molecular profiling between skin carcinomas reveals four newly reported genes potentially implicated in squamous cell carcinoma development," Oncogene 22, 3500-3505 (2003).
[CrossRef] [PubMed]

Morii, N.

P. Madaule, T. Furuyashiki, T. Reid, T. Ishizaki, G. Watanabe, N. Morii, and S. Narumiya, "A novel partner for the GTP-bound forms of rho and rac," FEBS Lett. 377, 243-248 (1995).
[CrossRef] [PubMed]

Muglia, L. J.

J. A. Brewer, B. P. Sleckman, W. Swat, and L. J. Muglia, "Green fluorescent protein-glucocorticoid receptor knockin mice reveal dynamic receptor modulation during thymocyte development," J. Immunol. 169, 1309-1318 (2002).
[PubMed]

Mycek, M.-A.

P. K. Urayama, W. Zhong, J. A. Beamish, F. K. Minn, R. D. Sloboda, K. H. Dragnev, E. Dmitrovsky, and M.-A. Mycek, "A UV-visible fluorescence lifetime imaging microscope for laser-based biological sensing with picosecond resolution," Applied Physics B: Lasers and Optics 76, 483-496 (2003).
[CrossRef]

W. Zhong, P. Urayama, and M.-A. Mycek, "Imaging fluorescence lifetime modulation of a ruthenium-based dye in living cells: the potential for oxygen sensing," J. Phys. D: Appl. Phys. 36, 1689-1695 (2003).
[CrossRef]

M.-A. Mycek, P. K. Urayama, K. Heyman, and M. Bussey, "Using POPOP's viscosity dependent lifetime as a picosecond resolution standard in near-UV fluorescence lifetime imaging microscopy," Proc. SPIE 4962, 143-150 (2003).
[CrossRef]

Nagy, A.

A. K. Hadjantonakis and A. Nagy, "The color of mice: in the light of GFP-variant reporters," Histochem. Cell Biol. 115, 49-58 (2001).
[PubMed]

Narumiya, S.

H. Suwa, G. Ohshio, T. Imamura, G. Watanabe, S. Arii, M. Imamura, S. Narumiya, H. Hiai, and M. Fukumoto, "Overexpression of the RhoC gene correlates with progression of ductal adenocarcinoma of the pancreas," Br. J. Cancer 77, 147-152 (1998).
[CrossRef] [PubMed]

P. Madaule, T. Furuyashiki, T. Reid, T. Ishizaki, G. Watanabe, N. Morii, and S. Narumiya, "A novel partner for the GTP-bound forms of rho and rac," FEBS Lett. 377, 243-248 (1995).
[CrossRef] [PubMed]

Neel, H.

G. Valentin, C. Verheggen, T. Piolot, H. Neel, M. Coppey-Moisan, and E. Bertrand, "Photoconversion of YFP into a CFP-like species during acceptor photobleaching FRET experiments," Nat Methods 2, 801 (2005).
[CrossRef] [PubMed]

Nerbonne, J. M.

G. Feng, R. H. Mellor, M. Bernstein, C. Keller-Peck, Q. T. Nguyen, M. Wallace, J. M. Nerbonne, J. W. Lichtman, and J. R. Sanes, "Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP," Neuron 28, 41-51 (2000).
[CrossRef] [PubMed]

Nguyen, A. W.

A. W. Nguyen and P. S. Daugherty, "Evolutionary optimization of fluorescent proteins for intracellular FRET," Nat. Biotechnol. 23, 355-360 (2005).
[CrossRef] [PubMed]

Nguyen, Q. T.

G. Feng, R. H. Mellor, M. Bernstein, C. Keller-Peck, Q. T. Nguyen, M. Wallace, J. M. Nerbonne, J. W. Lichtman, and J. R. Sanes, "Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP," Neuron 28, 41-51 (2000).
[CrossRef] [PubMed]

Ohshio, G.

H. Suwa, G. Ohshio, T. Imamura, G. Watanabe, S. Arii, M. Imamura, S. Narumiya, H. Hiai, and M. Fukumoto, "Overexpression of the RhoC gene correlates with progression of ductal adenocarcinoma of the pancreas," Br. J. Cancer 77, 147-152 (1998).
[CrossRef] [PubMed]

Ohsuka, S.

A. Tsuji, Y. Sato, M. Hirano, T. Suga, H. Koshimoto, T. Taguchi, and S. Ohsuka, "Development of a time-resolved fluorometric method for observing hybridization in living cells using fluorescence resonance energy transfer," Biophys. J. 81, 501-515 (2001).
[CrossRef] [PubMed]

Park, J.-B.

R. Reddel, Y. K. Ke, V. Gerwin, M. McMenamin, J. Lechner, R. Su, D. Brash, J.-B. Park, J. Limb Rhim, and C. Harris, "Transformation of human bronchial epithelial cells by infections by SV40 or adenovius-12 SV40 hybrid virus, or transfection via strontium phosphate coprecipitation with the plasmid containing SV40 early region genes," Cancer Res. 48, 1904-1909 (1988).
[PubMed]

Periasamy, A.

M. Elangovan, R. N. Day, and A. Periasamy, "Nanosecond fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy to localize the protein interactions in a single living cell," J. Microsc. 205, 3-14 (2002).
[CrossRef] [PubMed]

A. Periasamy, "Fluorescence resonance energy transfer microscopy: a mini review," J. Biomed. Opt. 6, 287-291 (2001).
[CrossRef] [PubMed]

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]

X. F. Wang, A. Periasamy, B. Herman, and D. Coleman, "Fluorescence lifetime imaging microscopy (FLIM): Instrumentation and applications," Crit. Rev. Anal. Chem. 23, 369-395 (1992).
[CrossRef]

Piolot, T.

G. Valentin, C. Verheggen, T. Piolot, H. Neel, M. Coppey-Moisan, and E. Bertrand, "Photoconversion of YFP into a CFP-like species during acceptor photobleaching FRET experiments," Nat Methods 2, 801 (2005).
[CrossRef] [PubMed]

M. Tramier, I. Gautier, T. Piolot, S. Ravalet, K. Kemnitz, J. Coppey, C. Durieux, V. Mignotte, and M. Coppey-Moisan, "Picosecond-hetero-FRET microscopy to probe protein-protein interactions in live cells," Biophys. J. 83, 3570-3577 (2002).
[CrossRef] [PubMed]

Piston, D. W.

M. A. Rizzo, G. H. Springer, B. Granada, and D. W. Piston, "An improved cyan fluorescenct protein variant useful for FRET," Nat. Biotechnol. 22, 445-449 (2004).
[CrossRef] [PubMed]

Qiao, X. T.

K. L. van Golen, Z.-F. Wu, X. T. Qiao, L. W. Bao, and S. D. Merajver, "RhoC GTPase, a novel transforming oncogene for human mammary epithelial cells that partially recapitulates the inflammatory breast cancer phenotype," Cancer Res. 60, 5832-5838 (2000).
[PubMed]

Ravalet, S.

M. Tramier, I. Gautier, T. Piolot, S. Ravalet, K. Kemnitz, J. Coppey, C. Durieux, V. Mignotte, and M. Coppey-Moisan, "Picosecond-hetero-FRET microscopy to probe protein-protein interactions in live cells," Biophys. J. 83, 3570-3577 (2002).
[CrossRef] [PubMed]

Reddel, R.

R. Reddel, Y. K. Ke, V. Gerwin, M. McMenamin, J. Lechner, R. Su, D. Brash, J.-B. Park, J. Limb Rhim, and C. Harris, "Transformation of human bronchial epithelial cells by infections by SV40 or adenovius-12 SV40 hybrid virus, or transfection via strontium phosphate coprecipitation with the plasmid containing SV40 early region genes," Cancer Res. 48, 1904-1909 (1988).
[PubMed]

Reid, T.

P. Madaule, T. Furuyashiki, T. Reid, T. Ishizaki, G. Watanabe, N. Morii, and S. Narumiya, "A novel partner for the GTP-bound forms of rho and rac," FEBS Lett. 377, 243-248 (1995).
[CrossRef] [PubMed]

Rizzo, M. A.

M. A. Rizzo, G. H. Springer, B. Granada, and D. W. Piston, "An improved cyan fluorescenct protein variant useful for FRET," Nat. Biotechnol. 22, 445-449 (2004).
[CrossRef] [PubMed]

Rose, R.

R. Rose, M. Weyand, M. Lammers, T. Ishizaki, M. R. Ahmadian, and A. Wittinghofer, "Structural and mechanistic insights into the interaction between Rho and mammalian Dia," Nature 435, 513-518 (2005).
[CrossRef] [PubMed]

Sanes, J. R.

G. Feng, R. H. Mellor, M. Bernstein, C. Keller-Peck, Q. T. Nguyen, M. Wallace, J. M. Nerbonne, J. W. Lichtman, and J. R. Sanes, "Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP," Neuron 28, 41-51 (2000).
[CrossRef] [PubMed]

Sato, Y.

A. Tsuji, Y. Sato, M. Hirano, T. Suga, H. Koshimoto, T. Taguchi, and S. Ohsuka, "Development of a time-resolved fluorometric method for observing hybridization in living cells using fluorescence resonance energy transfer," Biophys. J. 81, 501-515 (2001).
[CrossRef] [PubMed]

Schmid, J. A.

J. A. Schmid and H. H. Sitte, "Fluorescence resonance energy transfer in the study of cancer pathways," Curr. Opin. Oncol. 15, 55-64 (2003).
[CrossRef]

Schmidt, M.

G. Fritz, C. Brachetti, F. Bahlmann, M. Schmidt, and B. Kaina, "Rho GTPases in human breast tumours: expression and mutation analyses and correlation with clinical parameters," Br. J. Cancer 87, 635-644 (2002).
[CrossRef] [PubMed]

Selig, L.

P. Legrain and L. Selig, "Genome-wide protein interaction maps using two-hybrid systems," FEBS Lett. 480, 32-36 (2000).
[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]

Sitte, H. H.

J. A. Schmid and H. H. Sitte, "Fluorescence resonance energy transfer in the study of cancer pathways," Curr. Opin. Oncol. 15, 55-64 (2003).
[CrossRef]

Sleckman, B. P.

J. A. Brewer, B. P. Sleckman, W. Swat, and L. J. Muglia, "Green fluorescent protein-glucocorticoid receptor knockin mice reveal dynamic receptor modulation during thymocyte development," J. Immunol. 169, 1309-1318 (2002).
[PubMed]

Sloboda, R. D.

P. K. Urayama, W. Zhong, J. A. Beamish, F. K. Minn, R. D. Sloboda, K. H. Dragnev, E. Dmitrovsky, and M.-A. Mycek, "A UV-visible fluorescence lifetime imaging microscope for laser-based biological sensing with picosecond resolution," Applied Physics B: Lasers and Optics 76, 483-496 (2003).
[CrossRef]

Snapp, E.

J. Lippincott-Schwartz, E. Snapp, and A. Kenworthy, "Studying protein dynamics in living cells," Nat. Rev. Mol. Cell Biol. 2, 444-456 (2001).
[CrossRef] [PubMed]

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]

So, P. T. C.

T. French, P. T. C. So, C. Y. Dong, K. M. Berland, and E. Gratton, "Fluorescence lifetime imaging techniques for microscopy," Methods Cell Biol. 56, 277-304 (1998).
[CrossRef] [PubMed]

Springer, G. H.

M. A. Rizzo, G. H. Springer, B. Granada, and D. W. Piston, "An improved cyan fluorescenct protein variant useful for FRET," Nat. Biotechnol. 22, 445-449 (2004).
[CrossRef] [PubMed]

Squire, A.

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

Su, R.

R. Reddel, Y. K. Ke, V. Gerwin, M. McMenamin, J. Lechner, R. Su, D. Brash, J.-B. Park, J. Limb Rhim, and C. Harris, "Transformation of human bronchial epithelial cells by infections by SV40 or adenovius-12 SV40 hybrid virus, or transfection via strontium phosphate coprecipitation with the plasmid containing SV40 early region genes," Cancer Res. 48, 1904-1909 (1988).
[PubMed]

Suga, T.

A. Tsuji, Y. Sato, M. Hirano, T. Suga, H. Koshimoto, T. Taguchi, and S. Ohsuka, "Development of a time-resolved fluorometric method for observing hybridization in living cells using fluorescence resonance energy transfer," Biophys. J. 81, 501-515 (2001).
[CrossRef] [PubMed]

Suwa, H.

H. Suwa, G. Ohshio, T. Imamura, G. Watanabe, S. Arii, M. Imamura, S. Narumiya, H. Hiai, and M. Fukumoto, "Overexpression of the RhoC gene correlates with progression of ductal adenocarcinoma of the pancreas," Br. J. Cancer 77, 147-152 (1998).
[CrossRef] [PubMed]

Swat, W.

J. A. Brewer, B. P. Sleckman, W. Swat, and L. J. Muglia, "Green fluorescent protein-glucocorticoid receptor knockin mice reveal dynamic receptor modulation during thymocyte development," J. Immunol. 169, 1309-1318 (2002).
[PubMed]

Tadrous, P. J.

P. J. Tadrous, "Methods for imaging the structure and function of living tissues and cells: 2. fluorescence lifetime imaging," J. Pathol. 191, 229-234 (2000).
[CrossRef] [PubMed]

Taguchi, T.

A. Tsuji, Y. Sato, M. Hirano, T. Suga, H. Koshimoto, T. Taguchi, and S. Ohsuka, "Development of a time-resolved fluorometric method for observing hybridization in living cells using fluorescence resonance energy transfer," Biophys. J. 81, 501-515 (2001).
[CrossRef] [PubMed]

Taroni, P.

R. Cubeddu, D. Comelli, C. D'Andrea, P. Taroni, and G. Valentini, "Time-resolved fluorescence imaging in biology and medicine," J. Phys. D: Appl. Phys. 35, R61-R76 (2002).
[CrossRef]

Ting, A. Y.

J. Zhang, R. E. Campbell, A. Y. Ting, and R. Y. Tsien, "Creating new fluorescent probes for cell biology," Nat. Rev. Mol. Cell Biol. 3, 906-918 (2002).
[CrossRef] [PubMed]

Tramier, M.

M. Tramier, I. Gautier, T. Piolot, S. Ravalet, K. Kemnitz, J. Coppey, C. Durieux, V. Mignotte, and M. Coppey-Moisan, "Picosecond-hetero-FRET microscopy to probe protein-protein interactions in live cells," Biophys. J. 83, 3570-3577 (2002).
[CrossRef] [PubMed]

Tsien, R. Y.

J. Zhang, R. E. Campbell, A. Y. Ting, and R. Y. Tsien, "Creating new fluorescent probes for cell biology," Nat. Rev. Mol. Cell Biol. 3, 906-918 (2002).
[CrossRef] [PubMed]

O. Griesbeck, G. S. Baird, R. E. Campbell, D. A. Zacharias, and R. Y. Tsien, "Reducing the environmental sensitivity of yellow fluorescent protein. Mechanism and applications," J. Biol. Chem. 276, 29188-29194 (2001).
[CrossRef] [PubMed]

R. Y. Tsien, "The green fluorescent protein," Annual Reviews of Biochemistry 67, 509-544 (1998).
[CrossRef]

Tsuji, A.

A. Tsuji, Y. Sato, M. Hirano, T. Suga, H. Koshimoto, T. Taguchi, and S. Ohsuka, "Development of a time-resolved fluorometric method for observing hybridization in living cells using fluorescence resonance energy transfer," Biophys. J. 81, 501-515 (2001).
[CrossRef] [PubMed]

Uchida, T.

Urayama, P.

W. Zhong, P. Urayama, and M.-A. Mycek, "Imaging fluorescence lifetime modulation of a ruthenium-based dye in living cells: the potential for oxygen sensing," J. Phys. D: Appl. Phys. 36, 1689-1695 (2003).
[CrossRef]

Urayama, P. K.

P. K. Urayama, W. Zhong, J. A. Beamish, F. K. Minn, R. D. Sloboda, K. H. Dragnev, E. Dmitrovsky, and M.-A. Mycek, "A UV-visible fluorescence lifetime imaging microscope for laser-based biological sensing with picosecond resolution," Applied Physics B: Lasers and Optics 76, 483-496 (2003).
[CrossRef]

M.-A. Mycek, P. K. Urayama, K. Heyman, and M. Bussey, "Using POPOP's viscosity dependent lifetime as a picosecond resolution standard in near-UV fluorescence lifetime imaging microscopy," Proc. SPIE 4962, 143-150 (2003).
[CrossRef]

Valentin, G.

G. Valentin, C. Verheggen, T. Piolot, H. Neel, M. Coppey-Moisan, and E. Bertrand, "Photoconversion of YFP into a CFP-like species during acceptor photobleaching FRET experiments," Nat Methods 2, 801 (2005).
[CrossRef] [PubMed]

Valentini, G.

R. Cubeddu, D. Comelli, C. D'Andrea, P. Taroni, and G. Valentini, "Time-resolved fluorescence imaging in biology and medicine," J. Phys. D: Appl. Phys. 35, R61-R76 (2002).
[CrossRef]

van Golen, K. L.

K. L. van Golen, Z.-F. Wu, X. T. Qiao, L. W. Bao, and S. D. Merajver, "RhoC GTPase, a novel transforming oncogene for human mammary epithelial cells that partially recapitulates the inflammatory breast cancer phenotype," Cancer Res. 60, 5832-5838 (2000).
[PubMed]

van Kuppeveld, F. J. M.

F. J. M. van Kuppeveld, W. J. G. Melchers, P. H. G. M. Willems, and T. W. J. Gadella, Jr., "Homomultimerization of the coxsackievirus 2B protein in living cells visualized by fluorescence resonance energy transfer microscopy," J. Virol. 76, 9446-9456 (2002).
[CrossRef] [PubMed]

Van Sark, W. G. J. H. M.

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

Verheggen, C.

G. Valentin, C. Verheggen, T. Piolot, H. Neel, M. Coppey-Moisan, and E. Bertrand, "Photoconversion of YFP into a CFP-like species during acceptor photobleaching FRET experiments," Nat Methods 2, 801 (2005).
[CrossRef] [PubMed]

Verola, O.

C. Marionnet, C. Lalou, K. Mollier, M. Chazal, G. Delestaing, D. Compan, O. Verola, C. Vilmer, J. Cuminet, L. Dubertret, and N. Basset-Seguin, "Differential molecular profiling between skin carcinomas reveals four newly reported genes potentially implicated in squamous cell carcinoma development," Oncogene 22, 3500-3505 (2003).
[CrossRef] [PubMed]

Vilmer, C.

C. Marionnet, C. Lalou, K. Mollier, M. Chazal, G. Delestaing, D. Compan, O. Verola, C. Vilmer, J. Cuminet, L. Dubertret, and N. Basset-Seguin, "Differential molecular profiling between skin carcinomas reveals four newly reported genes potentially implicated in squamous cell carcinoma development," Oncogene 22, 3500-3505 (2003).
[CrossRef] [PubMed]

Wabnitz, H.

I. Bugiel, K. König, and H. Wabnitz, "Investigation of cell by fluorescence laser scanning microscopy with subnanosecond time resolution," Lasers in the Life Sciences 3, 47-53 (1989).

Wallace, M.

G. Feng, R. H. Mellor, M. Bernstein, C. Keller-Peck, Q. T. Nguyen, M. Wallace, J. M. Nerbonne, J. W. Lichtman, and J. R. Sanes, "Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP," Neuron 28, 41-51 (2000).
[CrossRef] [PubMed]

Wang, C. Y.

W. B. Zhong, C. Y. Wang, T. C. Chang, and W. S. Lee, "Lovastatin induces apoptosis of anaplastic thyroid cancer cells via inhibition of protein geranylgeranylation and de novo protein synthesis," Endocrinology 144, 3852-3859 (2003).
[CrossRef] [PubMed]

Wang, W.

W. Wang, L. Y. Yang, G. W. Huang, and W. Q. Lu, "Expression and significance of RhoC gene in hepatocellular carcinoma," World Journal of Gastroenterology 9, 1950-1953 (2003).
[PubMed]

Wang, X. F.

X. F. Wang, A. Periasamy, B. Herman, and D. Coleman, "Fluorescence lifetime imaging microscopy (FLIM): Instrumentation and applications," Crit. Rev. Anal. Chem. 23, 369-395 (1992).
[CrossRef]

X. F. Wang, T. Uchida, D. M. Coleman, and S. Minami, "A two-dimensional fluorescence lifetime imaging system using a gated image intensifier," Appl. Spectrosc. 45, 360-366 (1991).
[CrossRef]

Watanabe, G.

H. Suwa, G. Ohshio, T. Imamura, G. Watanabe, S. Arii, M. Imamura, S. Narumiya, H. Hiai, and M. Fukumoto, "Overexpression of the RhoC gene correlates with progression of ductal adenocarcinoma of the pancreas," Br. J. Cancer 77, 147-152 (1998).
[CrossRef] [PubMed]

P. Madaule, T. Furuyashiki, T. Reid, T. Ishizaki, G. Watanabe, N. Morii, and S. Narumiya, "A novel partner for the GTP-bound forms of rho and rac," FEBS Lett. 377, 243-248 (1995).
[CrossRef] [PubMed]

Weyand, M.

R. Rose, M. Weyand, M. Lammers, T. Ishizaki, M. R. Ahmadian, and A. Wittinghofer, "Structural and mechanistic insights into the interaction between Rho and mammalian Dia," Nature 435, 513-518 (2005).
[CrossRef] [PubMed]

Willems, P. H. G. M.

F. J. M. van Kuppeveld, W. J. G. Melchers, P. H. G. M. Willems, and T. W. J. Gadella, Jr., "Homomultimerization of the coxsackievirus 2B protein in living cells visualized by fluorescence resonance energy transfer microscopy," J. Virol. 76, 9446-9456 (2002).
[CrossRef] [PubMed]

Wittinghofer, A.

R. Rose, M. Weyand, M. Lammers, T. Ishizaki, M. R. Ahmadian, and A. Wittinghofer, "Structural and mechanistic insights into the interaction between Rho and mammalian Dia," Nature 435, 513-518 (2005).
[CrossRef] [PubMed]

Wu, Z.-F.

K. L. van Golen, Z.-F. Wu, X. T. Qiao, L. W. Bao, and S. D. Merajver, "RhoC GTPase, a novel transforming oncogene for human mammary epithelial cells that partially recapitulates the inflammatory breast cancer phenotype," Cancer Res. 60, 5832-5838 (2000).
[PubMed]

Yang, L. Y.

W. Wang, L. Y. Yang, G. W. Huang, and W. Q. Lu, "Expression and significance of RhoC gene in hepatocellular carcinoma," World Journal of Gastroenterology 9, 1950-1953 (2003).
[PubMed]

Zacharias, D. A.

O. Griesbeck, G. S. Baird, R. E. Campbell, D. A. Zacharias, and R. Y. Tsien, "Reducing the environmental sensitivity of yellow fluorescent protein. Mechanism and applications," J. Biol. Chem. 276, 29188-29194 (2001).
[CrossRef] [PubMed]

Zhang, J.

J. Zhang, R. E. Campbell, A. Y. Ting, and R. Y. Tsien, "Creating new fluorescent probes for cell biology," Nat. Rev. Mol. Cell Biol. 3, 906-918 (2002).
[CrossRef] [PubMed]

Zhong, W.

P. K. Urayama, W. Zhong, J. A. Beamish, F. K. Minn, R. D. Sloboda, K. H. Dragnev, E. Dmitrovsky, and M.-A. Mycek, "A UV-visible fluorescence lifetime imaging microscope for laser-based biological sensing with picosecond resolution," Applied Physics B: Lasers and Optics 76, 483-496 (2003).
[CrossRef]

W. Zhong, P. Urayama, and M.-A. Mycek, "Imaging fluorescence lifetime modulation of a ruthenium-based dye in living cells: the potential for oxygen sensing," J. Phys. D: Appl. Phys. 36, 1689-1695 (2003).
[CrossRef]

Zhong, W. B.

W. B. Zhong, C. Y. Wang, T. C. Chang, and W. S. Lee, "Lovastatin induces apoptosis of anaplastic thyroid cancer cells via inhibition of protein geranylgeranylation and de novo protein synthesis," Endocrinology 144, 3852-3859 (2003).
[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]

Annual Reviews of Biochemistry (1)

R. Y. Tsien, "The green fluorescent protein," Annual Reviews of Biochemistry 67, 509-544 (1998).
[CrossRef]

Appl. Spectrosc. (2)

Applied Physics B: Lasers and Optics (1)

P. K. Urayama, W. Zhong, J. A. Beamish, F. K. Minn, R. D. Sloboda, K. H. Dragnev, E. Dmitrovsky, and M.-A. Mycek, "A UV-visible fluorescence lifetime imaging microscope for laser-based biological sensing with picosecond resolution," Applied Physics B: Lasers and Optics 76, 483-496 (2003).
[CrossRef]

Biophys. J. (2)

M. Tramier, I. Gautier, T. Piolot, S. Ravalet, K. Kemnitz, J. Coppey, C. Durieux, V. Mignotte, and M. Coppey-Moisan, "Picosecond-hetero-FRET microscopy to probe protein-protein interactions in live cells," Biophys. J. 83, 3570-3577 (2002).
[CrossRef] [PubMed]

A. Tsuji, Y. Sato, M. Hirano, T. Suga, H. Koshimoto, T. Taguchi, and S. Ohsuka, "Development of a time-resolved fluorometric method for observing hybridization in living cells using fluorescence resonance energy transfer," Biophys. J. 81, 501-515 (2001).
[CrossRef] [PubMed]

Br. J. Cancer (2)

H. Suwa, G. Ohshio, T. Imamura, G. Watanabe, S. Arii, M. Imamura, S. Narumiya, H. Hiai, and M. Fukumoto, "Overexpression of the RhoC gene correlates with progression of ductal adenocarcinoma of the pancreas," Br. J. Cancer 77, 147-152 (1998).
[CrossRef] [PubMed]

G. Fritz, C. Brachetti, F. Bahlmann, M. Schmidt, and B. Kaina, "Rho GTPases in human breast tumours: expression and mutation analyses and correlation with clinical parameters," Br. J. Cancer 87, 635-644 (2002).
[CrossRef] [PubMed]

Cancer Res. (2)

K. L. van Golen, Z.-F. Wu, X. T. Qiao, L. W. Bao, and S. D. Merajver, "RhoC GTPase, a novel transforming oncogene for human mammary epithelial cells that partially recapitulates the inflammatory breast cancer phenotype," Cancer Res. 60, 5832-5838 (2000).
[PubMed]

R. Reddel, Y. K. Ke, V. Gerwin, M. McMenamin, J. Lechner, R. Su, D. Brash, J.-B. Park, J. Limb Rhim, and C. Harris, "Transformation of human bronchial epithelial cells by infections by SV40 or adenovius-12 SV40 hybrid virus, or transfection via strontium phosphate coprecipitation with the plasmid containing SV40 early region genes," Cancer Res. 48, 1904-1909 (1988).
[PubMed]

Crit. Rev. Anal. Chem. (1)

X. F. Wang, A. Periasamy, B. Herman, and D. Coleman, "Fluorescence lifetime imaging microscopy (FLIM): Instrumentation and applications," Crit. Rev. Anal. Chem. 23, 369-395 (1992).
[CrossRef]

Curr. Opin. Oncol. (1)

J. A. Schmid and H. H. Sitte, "Fluorescence resonance energy transfer in the study of cancer pathways," Curr. Opin. Oncol. 15, 55-64 (2003).
[CrossRef]

Cytometry A (1)

H. J. Lin, P. Herman, and J. R. Lakowicz, "Fluorescence lifetime-resolved pH imaging of living cells," Cytometry A 52, 77-89 (2003).
[CrossRef] [PubMed]

Differentiation (1)

Y. Chen and J. D. Mills, "Protein localization in living cells and tissues using FRET and FLIM," Differentiation 71, 528-541 (2003).
[CrossRef] [PubMed]

Endocrinology (1)

W. B. Zhong, C. Y. Wang, T. C. Chang, and W. S. Lee, "Lovastatin induces apoptosis of anaplastic thyroid cancer cells via inhibition of protein geranylgeranylation and de novo protein synthesis," Endocrinology 144, 3852-3859 (2003).
[CrossRef] [PubMed]

FEBS Lett. (2)

P. Legrain and L. Selig, "Genome-wide protein interaction maps using two-hybrid systems," FEBS Lett. 480, 32-36 (2000).
[CrossRef] [PubMed]

P. Madaule, T. Furuyashiki, T. Reid, T. Ishizaki, G. Watanabe, N. Morii, and S. Narumiya, "A novel partner for the GTP-bound forms of rho and rac," FEBS Lett. 377, 243-248 (1995).
[CrossRef] [PubMed]

Histochem. Cell Biol. (1)

A. K. Hadjantonakis and A. Nagy, "The color of mice: in the light of GFP-variant reporters," Histochem. Cell Biol. 115, 49-58 (2001).
[PubMed]

J. Biol. Chem. (1)

O. Griesbeck, G. S. Baird, R. E. Campbell, D. A. Zacharias, and R. Y. Tsien, "Reducing the environmental sensitivity of yellow fluorescent protein. Mechanism and applications," J. Biol. Chem. 276, 29188-29194 (2001).
[CrossRef] [PubMed]

J. Biomed. Opt. (1)

A. Periasamy, "Fluorescence resonance energy transfer microscopy: a mini review," J. Biomed. Opt. 6, 287-291 (2001).
[CrossRef] [PubMed]

J. Immunol. (1)

J. A. Brewer, B. P. Sleckman, W. Swat, and L. J. Muglia, "Green fluorescent protein-glucocorticoid receptor knockin mice reveal dynamic receptor modulation during thymocyte development," J. Immunol. 169, 1309-1318 (2002).
[PubMed]

J. Microsc. (2)

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

M. Elangovan, R. N. Day, and A. Periasamy, "Nanosecond fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy to localize the protein interactions in a single living cell," J. Microsc. 205, 3-14 (2002).
[CrossRef] [PubMed]

J. Pathol. (1)

P. J. Tadrous, "Methods for imaging the structure and function of living tissues and cells: 2. fluorescence lifetime imaging," J. Pathol. 191, 229-234 (2000).
[CrossRef] [PubMed]

J. Phys. D: Appl. Phys. (2)

R. Cubeddu, D. Comelli, C. D'Andrea, P. Taroni, and G. Valentini, "Time-resolved fluorescence imaging in biology and medicine," J. Phys. D: Appl. Phys. 35, R61-R76 (2002).
[CrossRef]

W. Zhong, P. Urayama, and M.-A. Mycek, "Imaging fluorescence lifetime modulation of a ruthenium-based dye in living cells: the potential for oxygen sensing," J. Phys. D: Appl. Phys. 36, 1689-1695 (2003).
[CrossRef]

J. Virol. (1)

F. J. M. van Kuppeveld, W. J. G. Melchers, P. H. G. M. Willems, and T. W. J. Gadella, Jr., "Homomultimerization of the coxsackievirus 2B protein in living cells visualized by fluorescence resonance energy transfer microscopy," J. Virol. 76, 9446-9456 (2002).
[CrossRef] [PubMed]

Lasers in the Life Sciences (1)

I. Bugiel, K. König, and H. Wabnitz, "Investigation of cell by fluorescence laser scanning microscopy with subnanosecond time resolution," Lasers in the Life Sciences 3, 47-53 (1989).

Methods Cell Biol. (1)

T. French, P. T. C. So, C. Y. Dong, K. M. Berland, and E. Gratton, "Fluorescence lifetime imaging techniques for microscopy," Methods Cell Biol. 56, 277-304 (1998).
[CrossRef] [PubMed]

Nat Methods (1)

G. Valentin, C. Verheggen, T. Piolot, H. Neel, M. Coppey-Moisan, and E. Bertrand, "Photoconversion of YFP into a CFP-like species during acceptor photobleaching FRET experiments," Nat Methods 2, 801 (2005).
[CrossRef] [PubMed]

Nat. Biotechnol. (2)

A. W. Nguyen and P. S. Daugherty, "Evolutionary optimization of fluorescent proteins for intracellular FRET," Nat. Biotechnol. 23, 355-360 (2005).
[CrossRef] [PubMed]

M. A. Rizzo, G. H. Springer, B. Granada, and D. W. Piston, "An improved cyan fluorescenct protein variant useful for FRET," Nat. Biotechnol. 22, 445-449 (2004).
[CrossRef] [PubMed]

Nat. Rev. Mol. Cell Biol. (2)

J. Lippincott-Schwartz, E. Snapp, and A. Kenworthy, "Studying protein dynamics in living cells," Nat. Rev. Mol. Cell Biol. 2, 444-456 (2001).
[CrossRef] [PubMed]

J. Zhang, R. E. Campbell, A. Y. Ting, and R. Y. Tsien, "Creating new fluorescent probes for cell biology," Nat. Rev. Mol. Cell Biol. 3, 906-918 (2002).
[CrossRef] [PubMed]

Nature (2)

R. Rose, M. Weyand, M. Lammers, T. Ishizaki, M. R. Ahmadian, and A. Wittinghofer, "Structural and mechanistic insights into the interaction between Rho and mammalian Dia," Nature 435, 513-518 (2005).
[CrossRef] [PubMed]

E. A. Clark, T. R. Golub, E. S. Lander, and R. O. Hynes, "Genomic analysis of metastasis reveals an essential role for RhoC," Nature 406, 532-535 (2000).
[CrossRef] [PubMed]

Neuron (1)

G. Feng, R. H. Mellor, M. Bernstein, C. Keller-Peck, Q. T. Nguyen, M. Wallace, J. M. Nerbonne, J. W. Lichtman, and J. R. Sanes, "Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP," Neuron 28, 41-51 (2000).
[CrossRef] [PubMed]

Oncogene (1)

C. Marionnet, C. Lalou, K. Mollier, M. Chazal, G. Delestaing, D. Compan, O. Verola, C. Vilmer, J. Cuminet, L. Dubertret, and N. Basset-Seguin, "Differential molecular profiling between skin carcinomas reveals four newly reported genes potentially implicated in squamous cell carcinoma development," Oncogene 22, 3500-3505 (2003).
[CrossRef] [PubMed]

Proc. SPIE (1)

M.-A. Mycek, P. K. Urayama, K. Heyman, and M. Bussey, "Using POPOP's viscosity dependent lifetime as a picosecond resolution standard in near-UV fluorescence lifetime imaging microscopy," Proc. SPIE 4962, 143-150 (2003).
[CrossRef]

Science (1)

S. Fields, "Proteomics in genomeland," Science 291, 1221-1224 (2001).
[CrossRef] [PubMed]

Trends Cell Biol. (1)

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

World Journal of Gastroenterology (1)

W. Wang, L. Y. Yang, G. W. Huang, and W. Q. Lu, "Expression and significance of RhoC gene in hepatocellular carcinoma," World Journal of Gastroenterology 9, 1950-1953 (2003).
[PubMed]

Other (9)

E. Golemis, ed., Protein-protein interactions: A molecular cloning manual, 1st ed. (Cold Spring Harbor Laboratory Press, Woodbury, 2001), p. 682.

J. R. Lakowicz, Principles of Fluorescence Spectroscopy, 2nd ed. (Kluwer Academic/Plenum, New York, 1999), p. 698.

P. K. Urayama, J. A. Beamish, F. K. Minn, E. A. Hamon, and M.-A. Mycek, "A UV fluorescence lifetime imaging microscope to probe endogenous cellular fluorescence," presented at the Conference on Lasers and Electro-Optics, 2002.

T. W. J. Gadella, Jr., "Fluorescence lifetime imaging microscopy (FLIM): instrumentation and application," in Fluorescent and luminescent probes for biological activity, W. T. Mason, ed. (Academic Press, San Diego, 1999), pp. 467-479.
[CrossRef]

C. W. Chang, D. Sud, and M. A. Mycek, "Fluorescence Lifetime Imaging Microscopy," in Methods in Cell Biology, Vol. 81 - Digital Microscopy, 3rd Edition, D. E. Wolf and G. Sluder, eds. (Academic Press, San Diego, 2007), pp. 495-524.

P. K. Urayama and M.-A. Mycek, "Fluorescence lifetime imaging microscopy of endogenous biological fluorescence," in Handbook of Biomedical Fluorescence, M.-A. Mycek and B. W. Pogue, eds. (Marcel-Dekker Inc., New York, New York, 2003), pp. 211-236.

F. Schaufele, I. Demarco, and R. N. Day, "FRET imaging in the wide-field microscope," in Molecular imaging: FRET microscopy and spectroscopy, A. Periasamy and R. N. Day, eds. (Oxford University Press, New York, 2005), pp. 72-94.

C. Thaler, S. S. Vogel, S. R. Ikeda, and H. Chen, "Photobleaching of YFP does not produce a CFP-like species that affects FRET measurements," Nat Methods 3, 491; author reply 492-493 (2006).
[CrossRef]

S. E. Verrier and H. D. Soling, "Photobleaching of YFP does not produce a CFP-like species that affects FRET measurements," Nat Methods 3, 491-492; author reply 492-493 (2006).
[CrossRef] [PubMed]

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Figures (7)

Fig. 1.
Fig. 1.

(a). FLIM concept. The system captures fluorescence intensity image at a time tG after the excitation pulse over the interval Δt. Lifetime can be created using intensity images captured at several different tG . (b) FLIM Setup. Excitation light from a nitrogen laser pumping a dye laser (UV-visible-NIR) is delivered by an optical fiber to a microscope. Fluorescence emission is captured by a fast-gated intensified charge-coupled device, which is triggered by a delay generator, using a timing reference provided by a reference pulse split from the excitation light with a beam splitter. Abbreviations: CCD= charge-coupled device; HRI= high rate imager; INT= intensifier; TTL I/O= TTL input/output card; OD= optical discriminator; BS= beam splitter; DC= dichroic mirror; FM= “flippable” mirror; L1, L2, L3, L4= quartz lenses; M= mirror. Thick solid lines= light path; thin solid line= electronic path.

Fig. 2.
Fig. 2.

FRET experiment in solution. (a) Concept of a FRET assay used to monitor cleavage of a peptide substrate consisting of a linked fluorescence donor and a quenching acceptor, (b) FLIM lifetime images of the donor before cleavage (upper) and after cleavage (lower). The scale bar represents 50 µm.

Fig. 3.
Fig. 3.

ECFP fluorescence intensity (a) and lifetime (c) images of living CV1 cells transfected with ECFP-RhoGDIγ+EYFP-RhoC. The intensity image and the corresponding histogram (b) show that the distribution of ECFP fluorescence both within individual cells and within a population was heterogenous, making it difficult to interpret intensity-based FRET experiments. The lifetime image and the corresponding histogram (d), however, exhibit far smaller variability within individual cells and within a population, providing a sensitive method for detecting molecular binding events via FRET. The scale bar represents 20 µm.

Fig. 4.
Fig. 4.

Schematic depiction of FRET studies on RhoC and RhoGDIγ. Interactions between RhoGDIγ and RhoC would cause FRET to occur between fluorescent donor (ECFP) and fluorescent acceptor (EYFP). Excitation and emission maxima of ECFP and EYFP are labeled. Lifetime methods monitor the decrease of donor fluorescence lifetime (τ) to detect FRET.

Fig. 5.
Fig. 5.

ECFP fluorescence intensity ((a) and (b)) and lifetime ((d) and (e)) images of CV1 cells transfected with ECFP-RhoGDIγ+EYFP and ECFP-RhoGDIγ+EYFP-RhoC. The intensity images ((a) and (b)) show large variations in ECFP fluorescence intensity within each cell population, making it difficult to compare fluorescence intensity between different populations (c). The lifetime images of cells transfected with ECFP-RhoGDIγ+EYFP-RhoC (e) appeared redder than cells transfected with ECFP-RhoGDIγ+EYFP (d), suggesting that ECFP lifetime of the former was shorter than that of the latter. Histograms of lifetimes from these two populations of cells (f) confirmed that the mean lifetime of cells in (e) was shorter than that of cells in (d), indicating an interaction between RhoGDIγ and RhoC. The scale bar represents 20 µm.

Fig. 6.
Fig. 6.

ECFP intensity (left) and lifetime (right) values of cells transfected with ECFP (top) and Cerulean (bottom) fusion plasmids. Group 1 (left box plot in (a) and (b)) consisted of 37 cells transfected with ECFP-RhoGDIγ and EYFP. Group 2 (right box plot in (a) and (b)) consisted of 60 cells transfected with ECFP-RhoGDIγ+EYFP-RhoC. Groups 3 and 4 (left and right box plots in both (c) and (d)) consisted of 30 cells transfected with Cerulean-RhoGDIγ+EYFP and 60 cells transfected with Cerulean-RhoGDIγ+EYFP-RhoC, respectively. The first quartile, the median, and the third quartile are shown within the boxes and the 10th/90th percentiles outside the boxes. All the plots are labeled with p-values of the Student’s t-test, together with the mean intensity or lifetime value and standard deviation for each group of cells. (a) and (c) Fluorescence intensities suffered from high variation within the cell population. (b) and (d) The FRET-induced decreases in lifetime (group 2 vs. group 1; group 4 vs. group 3) were statistically significant (p-value<0.05)), indicating RhoGDlr-RhoC molecular interaction.

Fig. 7.
Fig. 7.

Biochemical confirmation of RhoC-RhoGDIγ interaction. Immunoprecipitation of HAtagged RhoC was performed using lysates of CV-1 cells transiently expressing Myc-RhoGDIγ, HA-RhoC or both. Untransfected CV-1 (wildtype) was included as a mock control. The immunoprecipitates (or whole cell lysates) were analyzed by immunoblotting with an anti-Myc tag antibody. The position of Myc-GDIγ is indicated.

Equations (1)

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τ p = N ( Σ t i 2 ) ( Σ t i ) 2 N Σ t i ln I i , p ( Σ t i ) ( Σ ln I i , p ) ,

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