Hostname: page-component-848d4c4894-ndmmz Total loading time: 0 Render date: 2024-06-02T21:37:43.364Z Has data issue: false hasContentIssue false
  • English
  • Français

Partial Acceptor Photobleaching-Based Quantitative FRET Method Completely Overcoming Emission Spectral Crosstalks

Published online by Cambridge University Press:  02 October 2012

Huali Li ,
Huaina Yu  and
Tongsheng Chen
Huali Li
Affiliation:
MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, China
Huaina Yu
Affiliation:
MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, China
Tongsheng Chen*
Affiliation:
MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, China
*
*Corresponding author. E-mail: chentsh@scnu.edu.cn
Get access

Abstract

Based on the quantitative fluorescence resonance energy transfer (FRET) method named PbFRET we reported recently, we herein developed a partial acceptor photobleaching-based quantitative FRET algorithm named B-PbFRET method. B-PbFRET overcomes not only the acceptor excitation crosstalk and donor emission spectral crosstalk but also the acceptor emission spectral crosstalk that harasses previous methods including fluorescence lifetime (FLIM), fluorescence recovery of donor after acceptor photobleaching, and acceptor sensitized emission (SE)-based methods. B-PbFRET method is implemented by simultaneously measuring the fluorescence intensity of both donor and acceptor channels at donor excitation before and after partial acceptor photobleaching, and it can directly measure the FRET efficiency (E) without any verified references. Based on the theoretical analysis of B-PbFRET, we also developed a more straightforward correction method named C-PbFRET to obtain the absolute E from the value measured by PbFRET for a given donor-acceptor pair. We validated both B-PbFRET and C-PbFRET methods by measuring the E of two linked constructs, 18AA and SCAT3 proteins, in single living cells, and our data demonstrated that both B-PbFRET and C-PbFRET methods can directly measure the absolute E of the linked constructs inside living cells under different degrees of acceptor emission spectral crosstalk.

Keywords

FRETpartial acceptor photobleachingdonor-acceptor pairacceptor emission spectral crosstalkfluorescent proteinmicroscopy
Type
Techniques and Equipment Development
Information
Microscopy and Microanalysis , Volume 18 , Issue 5 , October 2012 , pp. 1021 - 1029
Copyright
Copyright © Microscopy Society of America 2012

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Bastiaens, P.I. & Squire, A. (1999). Fluorescence lifetime imaging microscopy: Spatial resolution of biochemical processes in the cell. Trends Cell Biol 9(2), 48–52.Google Scholar
Chan, F.K., Siegel, R.M., Zacharias, D., Swofford, R., Holmes, K.L., Tsien, R.Y. & Lenardo, M.J. (2001). Fluorescence resonance energy transfer analysis of cell surface receptor interactions and signaling using spectral variants of the green fluorescent protein. Cytometry 44(4), 361–368.Google Scholar
Chen, H.M., Puhl, H.L. 3rd, Koushik, S.V., Vogel, S.S. & Ikeda, S.R. (2006). Measurement of FRET efficiency and ratio of donor to acceptor concentration in living cells. Biophys J 91(5), L39–L41.Google Scholar
Chen, Y.E., Mauldin, J.P., Day, R.N. & Periasamy, A. (2007). Characterization of spectral FRET imaging microscopy for monitoring nuclear protein interactions. J Microsc 228 (Pt 2), 139–152.Google Scholar
Day, R.N., Periasamy, A. & Schaufele, F. (2001). Fluorescence resonance energy transfer microscopy of localized protein interactions in the living cell nucleus. Methods 25(1), 4–18.Google Scholar
Düssmann, H., Rehm, M., Concannon, C.G., Anguissola, S., Würstle, M., Kacmar, S., Völler, P., Huber, H.J. & Prehn, J.H. (2010). Single-cell quantification of Bax activation and mathematical modelling suggest pore formation on minimal mitochondrial Bax accumulation. Cell Death Differ 17(2), 278–290.Google Scholar
Elder, A.D., Domin, A., Kaminski Schierle, G.S., Lindon, C., Pines, J., Esposito, A. & Kaminskiet, C.F. (2009). A quantitative protocol for dynamic measurements of protein interactions by Förster resonance energy transfer-sensitized fluorescence emission. J R Soc Interface 6(S1), S59–81.Google Scholar
Erickson, M., Alseikhan, B., Peterson, B. & Yue, D. (2001). Preassociation of calmodulin with voltage-gated Ca2+ channels revealed by FRET in single living cells. Neuron 31(6), 973–985.Google Scholar
Gordon, G.W., Berry, G.., Liang, X.H., Levine, B. & Herman, B. (1998). Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy. Biophys J 74(5), 2702–2713.Google Scholar
Grailhe, R., Merola, F., Ridard, J., Couvignou, S., Poupon, C.L., Changeux, J.P. & Laguitton-Pasquier, H. (2006). Monitoring protein interactions in the living cell through the fluorescence decays of the cyan fluorescent protein. Chemphyschem 7(7), 1442–1454.CrossRefGoogle ScholarPubMed
Gu, Y., Di, W.L., Kelsell, D.P. & Zicha, D. (2004). Quantitative fluorescence resonance energy transfer (FRET) measurement with acceptor photobleaching and spectral unmixing. J Microsc 215(Pt 2), 162–173.Google Scholar
Hoppe, A., Christensen, K. & Swanson, J.A. (2002). Fluorescence resonance energy transfer-based stoichiometry in living cells. Biophys J 83(6), 3652–3664.Google Scholar
Jalink, K. & Van Rheenen, J. (2009). FilterFRET: Quantitative imaging of sensitized emission. In FRET and FLIM Techniques, Gadella, T.W.J. (Ed.), pp. 289–350. Amsterdam: Elsevier.CrossRefGoogle Scholar
Jares-Erijman, E.A. & Jovin, T.M. (2003). FRET imaging. Nat Biotechnol 21(11), 1387–1395.Google Scholar
Kenworthy, A.K. (2001). Imaging protein–protein interactions using fluorescence resonance energy transfer microscopy. Methods 24(3), 289–296.Google Scholar
Kenworthy, A.K., Petranova, N. & Edidin, M. (2000). High-resolution FRET microscopy of cholera toxin B-subunit and GPI-anchored proteins in cell plasma membranes. Mol Biol Cell 11 (5), 1645–1655.Google Scholar
Kölner, M. & Wolfrum, J. (1992). How many photons are necessary for fluorescence lifetime measurements? Chem Phys Lett 200(1-2), 199–204.CrossRefGoogle Scholar
Kusumi, A., Tsuji, A., Murata, M., Sako, Y., Yoshizawa, A.C., Kagiwada, S., Hayakawa, T. & Ohnishi, S. (1991). Development of a streak-camera-based time-resolved microscope fluorimeter and its application to studies of membrane fusion in single cells. Biochemistry 30(26), 6517–6527.Google Scholar
Lakowicz, J.R. (2006). Principles of Fluorescence Spectroscopy. New York: Springer.Google Scholar
Levy, S., Wilms, C.D., Brumer, E., Kahn, J., Pnueli, L., Arava, Y., Eilers, J. & Gitler, D. (2011). SpRET: Highly sensitive and reliable spectral measurement of absolute FRET efficiency. Microsc Microanal 17(2), 176–190.Google Scholar
Millington, M., Grindlay, G.,J., Altenbach, K., Neely, R.K., Kolch, W., Bencina, M., Read, N.D., Jones, A.C., Dryden, D.T. & Magennis, S.W. (2007). High-precision FLIM–FRET in fixed and living cells reveals heterogeneity in a simple CFP–YFP fusion protein. Biophys Chem 127(3), 155–164.Google Scholar
Nagai, T., Ibata, K., Park, E.S., Kubota, M., Mikoshiba, K. & Miyawaki, A. (2002). A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat Biotechnol 20(1), 87–90.Google Scholar
Neher, R.A. & Neher, E. (2004). Applying spectral fingerprinting to the analysis of FRET images. Microsc Res Tech 64 (2), 185–195.Google Scholar
Pan, W.L., Qu, J.L., Chen, T.S, Sun, L. & Qi, J. (2009). FLIM and emission spectral analysis of caspase-3 activation inside single living cell during anticancer drug-induced cell death. Eur Biophys J 38(4), 447–456.Google Scholar
Pelet, S., Previte, M.J. & So, P.T. (2006). Comparing the quantification of Forster resonance energy transfer measurement accuracies based on intensity, spectral, and lifetime imaging. J Biomed Opt 11(3), 34017.Google Scholar
Sarkar, P., Koushik, S.V., Vogel, S.S., Gryczynski, I. & Gryczyn-ski, Z. (2009). Photophysical properties of cerulean and venus fluorescent proteins. J Biomed Opt 14(3), 034047.CrossRefGoogle ScholarPubMed
Takemoto, K., Nagai, T., Miyawaki, A. & Miura, M. (2003). Spatio-temporal activation of caspase revealed by indicator that is insensitive to environmental effects. J Cell Biol 160(2), 235–243.Google Scholar
Thaler, C., Koushik, S.V., Blank, P.S. & Vogel, S.S. (2005). Quantitative multiphoton spectral imaging and its use for measuring resonance energy transfer. Biophys J 89(4), 2736–2749.Google Scholar
Tramier, M., Zahid, M., Mevel, J.C., Masse, M.J. & Coppey-Moisan, M. (2006). Sensitivity of CFP/YFP and GFP/mCherry pairs to donor photobleaching on FRET determination by fluorescence lifetime imaging microscopy in living cells. Microsc Res Tech 69(11), 933–939.Google Scholar
Tsien, R.Y. (1998). The green fluorescent protein. Annu Rev Biochem 67, 509–544.Google Scholar
Wang, L.X., Chen, T.S., Qu, J.L. & Wei, X.B. (2009). Quantitative analysis of caspase-3 activation by fitting fluorescence emission spectra in living cells. Micron 40(8), 811–820.Google Scholar
Wang, L.X., Chen, T.S., Qu, J.L. & Wei, X.B. (2010). Photobleaching-based quantitative analysis of fluorescence resonance energy transfer inside single living cell. J Fluoresc 20(1), 27–35.Google Scholar
Wei, W.S. (2005). Fluorescent proteins as tools to aid protein production. Microb Cell Fact 4, 12.Google Scholar
Wouters, F.S., Bastiaens, P.I., Wirtz, K.W. & Jovin, T.M. (1998). FRET microscopy demonstrates molecular association of non-specific lipid transfer protein (nsL-TP) with fatty acid oxidation enzymes in peroxisomes. EMBO J 17(24), 7179–7189.Google Scholar
Wu, Y.X., Xing, D. & Chen, W.R. (2006a). Single cell FRET imaging for determination of pathway of tumor cell apoptosis induced by photofrin-PDT. Cell Cycle 5(7), 729–734.Google Scholar
Wu, Y.X., Xing, D., Luo, S.M., Tang, Y.H. & Chen, Q. (2006b). Detection of caspase-3 activation in single cells by fluorescence resonance energy transfer during photodynamic therapy induced apoptosis. Cancer Lett 235(2), 239–247.Google Scholar
Zacharias, D.A., Violin, J.D., Newton, A.C. & Tsien, R.Y. (2002). Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science 296(5569), 913–916.Google Scholar
Zal, T. & Gascoigne, N.R. (2004). Photobleaching-corrected FRET efficiency imaging of live cells. Biophys J 86 (6), 3923–3939.Google Scholar
Zhang, Y.J., Xing, D. & Liu, L. (2009). PUMA promotes Bax translocation by both directly activating Bax and antagonizing Bcl-XL during UV-induced apoptosis. Mol Biol Cell 20(13), 3077–3087.Google Scholar
Zimmermann, T. (2005). Spectral imaging and linear unmixing in light microscopy. Adv Biochem Eng Biotechnol 95, 245–265.Google ScholarPubMed