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Photobleaching-Based Quantitative Analysis of Fluorescence Resonance Energy Transfer inside Single Living Cell

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Abstract

The current advances of fluorescence microscopy and new fluorescent probes make fluorescence resonance energy transfer (FRET) a powerful technique for studying protein-protein interactions inside living cells. It is very hard to quantitatively analyze FRET efficiency using intensity-based FRET imaging microscopy due to the presence of autofluorescence and spectral crosstalks. In this study, we for the first time developed a novel photobleaching-based method to quantitatively detect FRET efficiency (Pb-FRET) by selectively photobleaching acceptor. The Pb-FRET method requires two fluorescence detection channels: a donor channel (CH 1 ) to selectively detect the fluorescence from donor, and a FRET channel (CH 2 ) which normally includes the fluorescence from both acceptor and donor due to emission spectral crosstalk. We used the Pb-FRET method to quantitatively measure the FRET efficiency of SCAT3, a caspase-3 indicator based on FRET, inside single living cells stably expressing SCAT3 during STS-induced apoptosis. At 0, 6 and 12 h after STS treatment, the FRET efficiency of SCAT3 obtained by Pb-FRET inside living cells was verified by two-photon excitation (TPE) fluorescence lifetime imaging microscopy (FLIM). The temporal resolution of Pb-FRET method is in second time-scale for ROI photobleaching, even in microsecond time-scale for spot photobleaching. Our results demonstrate that the Pb-FRET method is independent of photobleaching degree, and is very useful for quantitatively monitoring protein-protein interactions inside single living cell.

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References

  1. Lakowicz JR (1999) Principles of fluorescence spectroscopy. Plenum, NewYork

    Google Scholar 

  2. Förster T (1948) Intermolecular energy migration and fluorescence. Ann Phys 6:55–75. doi:10.1002/andp. 19484370105

    Article  Google Scholar 

  3. Stryer L, Haugland R (1967) Energy transfer: a spectroscopic ruler. Proc Natl Acad Sci USA 58:719–726. doi:10.1073/pnas.58.2.719

    Article  CAS  PubMed  Google Scholar 

  4. Gordon GW, Berry G, Liang XH, Levine B, Herman B (1998) Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy. Biophys J 74:2702–2713. doi:10.1016/S0006-3495(98)77976-7

    Article  CAS  PubMed  Google Scholar 

  5. Jares-Erijman EA, Jovin TM (2003) FRET imaging. Nat Biotechnol 21:1387–1395. doi:10.1038/nbt896

    Article  CAS  PubMed  Google Scholar 

  6. Chen Y, Periasamy A (2006) Intensity range based quantitative FRET data analysis to localize protein molecules in live cell nuclei. J Fluoresc 16:95–104. doi:10.1007/s10895-005-0024-1

    Article  CAS  PubMed  Google Scholar 

  7. Becker W, Bergmann A, Hink MA, König K, Benndorf K, Biskup C (2004) Fluorescence lifetime imaging by time-correlated single-photon counting. Microsc Res Tech 63:58–66. doi:10.1002/jemt.10421

    Article  CAS  PubMed  Google Scholar 

  8. Hoppe A, Christensen K, Swanson JA (2002) Fluorescence resonance energy transfer-based stoichiometry in living cells. Biophys J 83:3652–3664. doi:10.1016/S0006-3495(02)75365-4

    Article  CAS  PubMed  Google Scholar 

  9. Pelet S, Previte MJR, So PTC (2006) Comparing the quantification of Förster resonance energy transfer measurement accuracies based on intensity, spectral, and lifetime imaging. J Biomed Opt 11:034017. doi:10.1117/1.2203664

    Article  Google Scholar 

  10. Kenworthy AK (2002) Imaging protein-protein interactions using fluorescence resonance energy transfer microscopy. Methods 24:289–296. doi:10.1006/meth.2001.1189

    Article  Google Scholar 

  11. Gu Y, Di WL, Kelsell DP, Zicha D (2004) Quantitative fluorescence resonance energy transfer (FRET) measurement with acceptor photobleaching and spectral unmixing. J Micro 15:162–173. doi:10.1111/j.0022-2720.2004.01365.x

    Article  Google Scholar 

  12. Hoffmann B, Zimmer T, Klöcker N, Kelbauskas L, König K, Benndorf K, Biskup C (2008) Prolonged irradiation of enhanced cyan fluorescent protein or Cerulean can invalidate Förster resonance energy transfer measurements. J Biomed Opt 13:031205. doi:10.1117/1.2937829

    Article  PubMed  Google Scholar 

  13. Berney C, Danuser G (2003) FRET or no FRET: a quantitative study. Biophys J 84:3992–4010. doi:10.1016/S0006-3495(03)75126-1

    Article  CAS  PubMed  Google Scholar 

  14. Gordon GW, Berry G, Liang XH, Levine B, Herman B (1998) Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy. Biophys J 74:2702–2713. doi:10.1016/S0006-3495(98)77976-7

    Article  CAS  PubMed  Google Scholar 

  15. Xia ZP, Liu YC (2001) Reliable and global measurement of fluorescence energy transfer using fluorescence microscopes. Biophys J 81:2395–2402. doi:10.1016/S0006-3495(01)75886-9

    Article  CAS  PubMed  Google Scholar 

  16. Kerr JF, Winterford CM, Harmon BV (1994) Apoptosis: its significance in cancer and cancer therapy. Cancer 73:2013–2026. doi:10.1002/1097-0142(19940415)73:8<2013::AID-CNCR2820730802>3.0.CO;2-J

    Article  CAS  PubMed  Google Scholar 

  17. Green DR (1998) Apoptotic pathways: the roads to ruin. Cell 94:695–698. doi:10.1016/S0092-8674(00)81728-6

    Article  CAS  PubMed  Google Scholar 

  18. Kiwamu T, Takeharu N, Atsushi M, Masayuki M (2003) Spatio-temporal activation of caspase revealed by indicator that is insensitive to environmental effects. J Cell Biol 160:235–243. doi:10.1083/jcb.200207111

    Article  Google Scholar 

  19. Wu YX, Xing D, Chen WR (2006) Single cell FRET imaging for determination of pathway of tumor cell apoptosis induced by photofrin-PDT. Cell Cycle 5:729–734

    CAS  PubMed  Google Scholar 

  20. Wu YX, Xing D, Luo SM, Tang YH, Chen Q (2006) Detection of caspase-3 activation in single cells by fluorescence resonance energy transfer during photodynamic therapy induced apoptosis. Cancer Lett 235:239–247. doi:10.1016/j.canlet.2005.04.036

    Article  CAS  PubMed  Google Scholar 

  21. Wu YY, Xing D, Chen WR, Wang XC (2007) Bid is not required for Bax translocation during UV-induced apoptosis. Cell Signal 19:2468–2478. doi:10.1016/j.cellsig.2007.07.024

    Article  CAS  PubMed  Google Scholar 

  22. Liu L, Xing D, Chen WR, Chen TS, Pei YH, Gao XJ (2008) Calpain-mediated pathway dominates cisplatin-induced apoptosis in human lung adenocarcinoma cells as determined by real-time single cell analysis. Int J Cancer 122:2210–2222. doi:10.1002/ijc.23378

    Article  CAS  PubMed  Google Scholar 

  23. Salako MA, Carter MJ, Kass GEN (2006) Coxsackievirus protein 2BC blocks host cell apoptosis by inhibiting caspase-3. J Biol Chem 281:16296–16304. doi:10.1074/jbc.M510662200

    Article  CAS  PubMed  Google Scholar 

  24. Rehm M, Düßmann H, Jänicke RU, Tavaré JM, Kögel D, Prehn JH (2002) Single cell fluorescence resonance energy transfer analysis demonstrates that caspase activation during apoptosis is a rapid process: role of caspase-3. J. Biol. Chem. 277:24506–24514. doi:10.1074/jbc.M110789200

    Article  CAS  PubMed  Google Scholar 

  25. Andersson M, Sjöstrand J, Petersen A, Honarvar AKS, Karlsson JO (2000) Caspase and proteasome activity during staurosporine-induced apoptosis in lens epithelial cells. Invest Ophthalmol Vis Sci 41:2623–2632

    CAS  PubMed  Google Scholar 

  26. Xue LY, Chiu SM, Oleinick NL (2003) Staurosporine-induced death of MCF-7 human breast cancer cells: distinction between caspase-3-dependent steps of apoptosis and the critical lethal lesions. Exp Cell Res 283:135–145. doi:10.1016/S0014-4827(02)00032-0

    Article  CAS  PubMed  Google Scholar 

  27. Pepperkok R, Squire A, Geley S, Bastiaens PI (1999) Simultaneous detection of multiple green fluorescent proteins in live cells by fluorescence lifetime imaging microscopy. Curr Biol 9:269–272. doi:10.1016/S0960-9822(99)80117-1

    Article  CAS  PubMed  Google Scholar 

  28. Pan WL, Qu JL, Chen TS, Sun L (2009) FLIM and emission spectral analysis of caspase-3 activation inside single living cell during anticancer drug-induced cell death. Eur Biophys J. doi:10.1007/s00249-008-0390-0

    PubMed  Google Scholar 

  29. Zeng SQ, Lv XH, Zhan C, Chen WR, Xiong WH, Jacuqes SL, Luo QM (2006) Simultaneous compensation for spatial and temporal dispersion of acousto-optical deflectors for two-dimensional scanning with a single prism. Opt Lett 31:1091–1093. doi:10.1364/OL.31.001091

    Article  PubMed  Google Scholar 

  30. Zeng SQ, Li DR, Luo QM (2007) Pulse broadening of the femtosecond pulses in a Gaussian beam passing an angular disperser. Opt Lett 32:1180–1182. doi:10.1364/OL.32.001180

    Article  PubMed  Google Scholar 

  31. Tsien RY (1998) The green fluorescent protein. Annu Rev Biochem 67:509–544. doi:10.1146/annurev.biochem.67.1.509

    Article  CAS  PubMed  Google Scholar 

  32. Wei WS (2005) Fluorescent proteins as tools to aid protein production. Microb Cell Fact 4:12. doi:10.1186/1475-2859-4-12

    Google Scholar 

  33. Partikian A, Ölveczky B, Swaminathan R, Li YX, Verkman AS (1998) Rapid diffusion of green fluorescent protein in the mitochondrial matrix. J Cell Biol 140:821–829. doi:10.1083/jcb.140.4.821

    Article  CAS  PubMed  Google Scholar 

  34. Elangovan M, Wallrabe H, Chen Y, Day RN, Barroso M, Eriasam A (2003) Characterization of one- and two-photon excitation fluorescence resonance energy transfer microscopy. Methods 291:58–73. doi:10.1016/S1046-2023(02)00283-9

    Article  Google Scholar 

  35. Hoppe A, Christensen K, Swanson JA (2002) Fluorescence resonance energy transfer-based stoichiometry in living cells. Biophys J 83:3652–3664. doi:10.1016/S0006-3495(02)75365-4

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank Prof. M. Miura for providing us with the SCAT3 plasmid. This study was supported by National Natural Science Foundation of China (Grant No. 30670507 and 60627003), the Natural Science Foundation of Guangdong Province (F051001), “Shuguang Scholor” (Grant No. 07SG05) of Education Commision and “Leading Academic Development Project” (Grant No. B109) of the Science & Technology Commission of Shanghai Municipality.

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Authors and Affiliations

  1. MOE Key laboratory of Laser Life Science & Institute of Laser Life Science, South China Normal University, Guangzhou, 510631, China

    Longxiang Wang & Tongsheng Chen

  2. Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, Institute of Optoelectronics, Shenzhen University, Shenzhen, 518060, China

    Junle Qu

  3. Institutes of Biomedical Sciences, Fudan University, Shanghai, 200032, China

    Xunbin Wei

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  1. Longxiang Wang
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  2. Tongsheng Chen
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  3. Junle Qu
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Correspondence to Tongsheng Chen or Xunbin Wei.

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Wang, L., Chen, T., Qu, J. et al. Photobleaching-Based Quantitative Analysis of Fluorescence Resonance Energy Transfer inside Single Living Cell. J Fluoresc 20, 27–35 (2010). https://doi.org/10.1007/s10895-009-0518-3

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  • DOI: https://doi.org/10.1007/s10895-009-0518-3

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