OSA's Digital Library

Optics Express

Optics Express

  • Editor: C. Martijn de Sterke
  • Vol. 15, Iss. 26 — Dec. 24, 2007
  • pp: 18220–18235

Picosecond-resolution fluorescence lifetime imaging microscopy: a useful tool for sensing molecular interactions in vivo via FRET

Wei Zhong, Mei Wu, Ching-Wei Chang, Karl A. Merrick, Sofia D. Merajver, and Mary-Ann Mycek  »View Author Affiliations

Optics Express, Vol. 15, Issue 26, pp. 18220-18235 (2007)

View Full Text Article

Enhanced HTML    Acrobat PDF (1184 KB)

Browse Journals / Lookup Meetings

Browse by Journal and Year


Lookup Conference Papers

Close Browse Journals / Lookup Meetings

Article Tools



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

OCIS Codes
(170.1530) Medical optics and biotechnology : Cell analysis
(170.2520) Medical optics and biotechnology : Fluorescence microscopy
(170.3650) Medical optics and biotechnology : Lifetime-based sensing
(170.4580) Medical optics and biotechnology : Optical diagnostics for medicine
(170.6920) Medical optics and biotechnology : Time-resolved imaging

ToC Category:
Medical Optics and Biotechnology

Original Manuscript: October 19, 2007
Revised Manuscript: December 13, 2007
Manuscript Accepted: December 14, 2007
Published: December 19, 2007

Virtual Issues
Vol. 3, Iss. 1 Virtual Journal for Biomedical Optics

Wei Zhong, Mei Wu, Ching-Wei Chang, Karl A. Merrick, Sofia D. Merajver, and Mary-Ann Mycek, "Picosecond-resolution fluorescence lifetime imaging microscopy: a useful tool for sensing molecular interactions in vivo via FRET," Opt. Express 15, 18220-18235 (2007)

Sort:  Author  |  Year  |  Journal  |  Reset  


  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]

Cited By

Alert me when this paper is cited

OSA is able to provide readers links to articles that cite this paper by participating in CrossRef's Cited-By Linking service. CrossRef includes content from more than 3000 publishers and societies. In addition to listing OSA journal articles that cite this paper, citing articles from other participating publishers will also be listed.

« Previous Article  |  Next Article »

OSA is a member of CrossRef.

CrossCheck Deposited