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Optics Express

Optics Express

  • Editor: C. Martijn de Sterke
  • Vol. 18, Iss. 11 — May. 24, 2010
  • pp: 11148–11158

Fluorescence lifetime endoscopy using TCSPC for the measurement of FRET in live cells

Gilbert O. Fruhwirth, Simon Ameer-Beg, Richard Cook, Timothy Watson, Tony Ng, and Frederic Festy  »View Author Affiliations

Optics Express, Vol. 18, Issue 11, pp. 11148-11158 (2010)

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Development of remote imaging for diagnostic purposes has progressed dramatically since endoscopy began in the 1960’s. The recent advent of a clinically licensed intensity-based fluorescence micro-endoscopic instrument has offered the prospect of real-time cellular resolution imaging. However, interrogating protein-protein interactions deep inside living tissue requires precise fluorescence lifetime measurements to derive the Förster resonance energy transfer between two tagged fluorescent markers. We developed a new instrument combining remote fiber endoscopic cellular-resolution imaging with TCSPC-FLIM technology to interrogate and discriminate mixed fluorochrome labeled beads and expressible GFP/TagRFP tags within live cells. Endoscopic-FLIM (e-FLIM) data was validated by comparison with data acquired via conventional FLIM and e-FLIM was found to be accurate for both bright bead and dim live cell samples. The fiber based micro-endoscope allowed remote imaging of 4 µm and 10 µm beads within a thick Matrigel matrix with confident fluorophore discrimination using lifetime information. More importantly, this new technique enabled us to reliably measure protein-protein interactions in live cells embedded in a 3D matrix, as demonstrated by the dimerization of the fluorescent protein-tagged membrane receptor CXCR4. This cell-based application successfully demonstrated the suitability and great potential of this new technique for in vivo pre-clinical biomedical and possibly human clinical applications.

© 2010 OSA

OCIS Codes
(170.2150) Medical optics and biotechnology : Endoscopic imaging
(170.2520) Medical optics and biotechnology : Fluorescence microscopy

ToC Category:
Medical Optics and Biotechnology

Original Manuscript: February 3, 2010
Revised Manuscript: March 31, 2010
Manuscript Accepted: April 13, 2010
Published: May 12, 2010

Virtual Issues
Vol. 5, Iss. 10 Virtual Journal for Biomedical Optics

Gilbert O. Fruhwirth, Simon Ameer-Beg, Richard Cook, Timothy Watson, Tony Ng, and Frederic Festy, "Fluorescence lifetime endoscopy using TCSPC for the measurement of FRET in live cells," Opt. Express 18, 11148-11158 (2010)

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  1. P. Cotton, and C. Williams, Practical gastrointestinal endoscopy (Blackwell Science, 2003).
  2. K. Suhling, P. M. French, and D. Phillips, “Time-resolved fluorescence microscopy,” Photochem. Photobiol. Sci. 4(1), 13–22 (2005). [CrossRef]
  3. Z. Papagatsia, A. Tappuni, T. F. Watson, and R. J. Cook, “Single wavelength micro-endoscopy in non-surgical vascular lesion diagnosis & characterization,” J. Microsc. 230(2), 203–211 (2008). [CrossRef] [PubMed]
  4. R. El-Gazzar, M. Macluskey, and G. R. Ogden, “Evidence for a field change effect based on angiogenesis in the oral mucosa? A brief report,” Oral Oncol. 41(1), 25–30 (2005). [CrossRef]
  5. F. Festy, S. M. Ameer-Beg, T. Ng, and K. Suhling, “Imaging proteins in vivo using fluorescence lifetime microscopy,” Mol. Biosyst. 3(6), 381–391 (2007). [CrossRef] [PubMed]
  6. G. McConnell, J. M. Girkin, S. M. Ameer-Beg, P. R. Barber, B. Vojnovic, T. Ng, A. Banerjee, T. F. Watson, and R. J. Cook, “Time-correlated single-photon counting fluorescence lifetime confocal imaging of decayed and sound dental structures with a white-light supercontinuum source,” J. Microsc. 225(2), 126–136 (2007). [CrossRef] [PubMed]
  7. W. Becker, H. Hickl, C. Zander, K. H. Drexhage, M. Sauer, S. Siebert, and J. Wolfrum, “Time-resolved detection and identification of single analyte molecules in microcapillaries by time-correlated single photon counting,” Rev. Sci. Instrum. 70(3), 1835–1841 (1999). [CrossRef]
  8. T. W. J. Gadella, T. M. Jovin, and R. M. Clegg, “Fluorescence lifetime imaging microscopy (FLIM): Spatial resolution of microstructures on the nanosecond time scale,” Biophys. Chem. 48(2), 221–239 (1993). [CrossRef]
  9. C. Buranachai, D. Kamiyama, A. Chiba, B. D. Williams, and R. M. Clegg, “Rapid frequency-domain FLIM spinning disk confocal microscope: lifetime resolution, image improvement and wavelet analysis,” J. Fluoresc. 18(5), 929–942 (2008). [CrossRef] [PubMed]
  10. E. A. Jares-Erijman and T. M. Jovin, “FRET imaging,” Nat. Biotechnol. 21(11), 1387–1395 (2003). [CrossRef] [PubMed]
  11. T. Förster, “Intermolecular energy migration and fluorescence,” Ann. Phys. 2, 55 (1948). [CrossRef]
  12. L. Stryer, “Fluorescence energy transfer as a spectroscopic ruler,” Annu. Rev. Biochem. 47(1), 819–846 (1978). [CrossRef] [PubMed]
  13. T. Ng, A. Squire, G. Hansra, F. Bornancin, C. Prevostel, A. Hanby, W. Harris, D. Barnes, S. Schmidt, H. Mellor, P. I. Bastiaens, and P. J. Parker, “Imaging protein kinase Calpha activation in cells,” Science 283(5410), 2085–2089 (1999). [CrossRef] [PubMed]
  14. T. Ng, D. Shima, A. Squire, P. I. H. Bastiaens, S. Gschmeissner, M. J. Humphries, and P. J. Parker, “PKCalpha regulates beta1 integrin-dependent cell motility through association and control of integrin traffic,” EMBO J. 18, 3909–3923 (1999). [CrossRef] [PubMed]
  15. F. S. Wouters and P. I. Bastiaens, “Fluorescence lifetime imaging of receptor tyrosine kinase activity in cells,” Curr. Biol. 9(19), 1127–1130 (1999). [CrossRef] [PubMed]
  16. F. S. Wouters, P. J. Verveer, and P. I. Bastiaens, “Imaging biochemistry inside cells,” Trends Cell Biol. 11(5), 203–211 (2001). [CrossRef] [PubMed]
  17. S. Pelet, M. J. R. Previte, and P. T. So, “Comparing the quantification of Forster resonance energy transfer measurement accuracies based on intensity, spectral, and lifetime imaging,” J. Biomed. Opt. 11(3), 034017 (2006). [CrossRef]
  18. M. Parsons, J. Monypenny, S. M. Ameer-Beg, T. H. Millard, L. M. Machesky, M. Peter, M. D. Keppler, G. Schiavo, R. Watson, J. Chernoff, D. Zicha, B. Vojnovic, and T. Ng, “Spatially distinct binding of Cdc42 to PAK1 and N-WASP in breast carcinoma cells,” Mol. Cell. Biol. 25(5), 1680–1695 (2005). [CrossRef] [PubMed]
  19. M. Peter and S. M. Ameer-Beg, “Imaging molecular interactions by multiphoton FLIM,” Biol. Cell 96(3), 231–236 (2004). [CrossRef] [PubMed]
  20. M. Peter, S. M. Ameer-Beg, M. K. Y. Hughes, M. D. Keppler, S. Prag, M. Marsh, B. Vojnovic, and T. Ng, “Multiphoton-FLIM quantification of the EGFP-mRFP1 FRET pair for localization of membrane receptor-kinase interactions,” Biophys. J. 88(2), 1224–1237 (2005). [CrossRef]
  21. A. Schönle, M. Glatz, and S. W. Hell, “Four-dimensional multiphoton microscopy with time-correlated single-photon counting,” Appl. Opt. 39(34), 6306–6311 (2000). [CrossRef]
  22. R. R. Duncan, A. Bergmann, M. A. Cousin, D. K. Apps, and M. J. Shipston, “Multi-dimensional time-correlated single photon counting (TCSPC) fluorescence lifetime imaging microscopy (FLIM) to detect FRET in cells,” J. Microsc. 215(1), 1–12 (2004). [CrossRef] [PubMed]
  23. V. Calleja, S. M. Ameer-Beg, B. Vojnovic, R. Woscholski, J. Downward, and B. Larijani, “Monitoring conformational changes of proteins in cells by fluorescence lifetime imaging microscopy,” Biochem. J. 372(1), 33–40 (2003). [CrossRef] [PubMed]
  24. J. Requejo-Isidro, J. McGinty, I. Munro, D. S. Elson, N. P. Galletly, M. J. Lever, M. A. A. Neil, G. W. Stamp, P. M. French, P. A. Kellett, J. D. Hares, and A. K. Dymoke-Bradshaw, “High-speed wide-field time-gated endoscopic fluorescence-lifetime imaging,” Opt. Lett. 29(19), 2249–2251 (2004). [CrossRef] [PubMed]
  25. D. Elson, J. Requejo-Isidro, I. Munro, F. Reavell, J. Siegel, K. Suhling, P. Tadrous, R. Benninger, P. Lanigan, J. McGinty, C. Talbot, B. Treanor, S. Webb, A. Sandison, A. Wallace, D. Davis, J. Lever, M. Neil, D. Phillips, G. Stamp, and P. French, “Time-domain fluorescence lifetime imaging applied to biological tissue,” Photochem. Photobiol. Sci. 3(8), 795–801 (2004). [CrossRef] [PubMed]
  26. I. Munro, J. McGinty, N. Galletly, J. Requejo-Isidro, P. M. P. Lanigan, D. S. Elson, C. Dunsby, M. A. Neil, M. J. Lever, G. W. Stamp, and P. M. French, “Toward the clinical application of time-domain fluorescence lifetime imaging,” J. Biomed. Opt. 10(5), 051403 (2005). [CrossRef] [PubMed]
  27. S. Kumar, C. Dunsby, P. A. A. De Beule, D. M. Owen, U. Anand, P. M. P. Lanigan, R. K. P. Benninger, D. M. Davis, M. A. Neil, P. Anand, C. Benham, A. Naylor, and P. M. French, “Multifocal multiphoton excitation and time correlated single photon counting detection for 3-D fluorescence lifetime imaging,” Opt. Express 15(20), 12548–12561 (2007). [CrossRef] [PubMed]
  28. K. Makrogianneli, L. M. Carlin, M. D. Keppler, D. R. Matthews, E. Ofo, A. Coolen, S. M. Ameer-Beg, P. R. Barber, B. Vojnovic, and T. Ng, “Integrating receptor signal inputs that influence small Rho GTPase activation dynamics at the immunological synapse,” Mol. Cell. Biol. 29(11), 2997–3006 (2009). [CrossRef] [PubMed]
  29. S. Prag, M. Parsons, M. D. Keppler, S. M. Ameer-Beg, P. Barber, J. Hunt, A. J. Beavil, R. Calvert, M. Arpin, B. Vojnovic, and T. Ng, “Activated ezrin promotes cell migration through recruitment of the GEF Dbl to lipid rafts and preferential downstream activation of Cdc42,” Mol. Biol. Cell 18(8), 2935–2948 (2007). [CrossRef] [PubMed]
  30. P. R. Barber, S. M. Ameer-Beg, J. Gilbey, L. M. Carlin, M. Keppler, T. Ng, and B. Vojnovic, “Multiphoton time-domain fluorescence lifetime imaging microscopy: practical application to protein–protein interactions using global analysis,” J. R. Soc. Interface 6(0), 93–105 (2009). [CrossRef]
  31. H. Morise, O. Shimomura, F. H. Johnson, and J. Winant, “Intermolecular energy transfer in the bioluminescent system of Aequorea,” Biochemistry 13(12), 2656–2662 (1974). [CrossRef] [PubMed]
  32. E. M. Merzlyak, J. Goedhart, D. Shcherbo, M. E. Bulina, A. S. Shcheglov, A. F. Fradkov, A. Gaintzeva, K. A. Lukyanov, S. Lukyanov, T. W. Gadella, and D. M. Chudakov, “Bright monomeric red fluorescent protein with an extended fluorescence lifetime,” Nat. Methods 4(7), 555–557 (2007). [CrossRef] [PubMed]
  33. A. J. Vila-Coro, J. M. Rodríguez-Frade, A. Martín De Ana, M. C. Moreno-Ortíz, C. Martínez-A, and M. Mellado, “The chemokine SDF-1alpha triggers CXCR4 receptor dimerization and activates the JAK/STAT pathway,” FASEB J. 13(13), 1699–1710 (1999). [PubMed]
  34. G. J. Babcock, M. Farzan, and J. Sodroski, “Ligand-independent dimerization of CXCR4, a principal HIV-1 coreceptor,” J. Biol. Chem. 278(5), 3378–3385 (2003). [CrossRef]
  35. R. Maeda-Mamiya, E. Noiri, H. Isobe, W. Nakanishi, K. Okamoto, K. Doi, T. Sugaya, T. Izumi, T. Homma, and E. Nakamura, “In vivo gene delivery by cationic tetraamino fullerene,” Procs. Nat. Acad. Sci. 107(12), 5339–5344 (2010). [CrossRef]
  36. C. LoPresti, H. Lomas, M. Massignani, T. Smart, and G. Battaglia, “Polymersomes: nature inspired nanometer sized compartments,” J. Mater. Chem. 19(22), 3576–3590 (2009). [CrossRef]
  37. H. Lomas, M. Massignani, K. A. Abdullah, I. Canton, C. Lo Presti, S. MacNeil, J. Du, A. Blanazs, J. Madsen, S. P. Armes, A. L. Lewis, and G. Battaglia, “Non-cytotoxic polymer vesicles for rapid and efficient intracellular delivery,” Faraday Discuss. 139, 143–159, discussion 213–228, 419–420 (2008). [CrossRef] [PubMed]
  38. H. Bar, I. Yacoby, and I. Benhar, “Killing cancer cells by targeted drug-carrying phage nanomedicines,” BMC Biotechnol. 8(1), 37 (2008). [CrossRef] [PubMed]
  39. T. Y. Lee, C. T. Lin, S. Y. Kuo, D. K. Chang, and H. C. Wu, “Peptide-mediated targeting to tumor blood vessels of lung cancer for drug delivery,” Cancer Res. 67(22), 10958–10965 (2007). [CrossRef] [PubMed]
  40. C. C. Fjeld, W. T. Birdsong, and R. H. Goodman, “Differential binding of NAD+ and NADH allows the transcriptional corepressor carboxyl-terminal binding protein to serve as a metabolic sensor,” Proc. Natl. Acad. Sci. U.S.A. 100(16), 9202–9207 (2003). [CrossRef] [PubMed]
  41. A. Cobos-Correa, J. B. Trojanek, S. Diemer, M. A. Mall, and C. Schultz, “Membrane-bound FRET probe visualizes MMP12 activity in pulmonary inflammation,” Nat. Chem. Biol. 5(9), 628–630 (2009). [CrossRef] [PubMed]

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