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

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 (CH1) to selectively detect the fluorescence from donor, and a FRET channel (CH2) 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.

[1]  T. Jovin,et al.  FRET imaging , 2003, Nature Biotechnology.

[2]  Wei Wen Su,et al.  Fluorescent proteins as tools to aid protein production , 2005, Microbial cell factories.

[3]  Tongsheng Chen,et al.  FLIM and emission spectral analysis of caspase-3 activation inside single living cell during anticancer drug-induced cell death , 2009, European Biophysics Journal.

[4]  Y. Liu,et al.  Reliable and global measurement of fluorescence resonance energy transfer using fluorescence microscopes. , 2001, Biophysical journal.

[5]  R. Tsien,et al.  green fluorescent protein , 2020, Catalysis from A to Z.

[6]  Markus Rehm,et al.  Single-cell Fluorescence Resonance Energy Transfer Analysis Demonstrates That Caspase Activation during Apoptosis Is a Rapid Process , 2002, The Journal of Biological Chemistry.

[7]  J. Swanson,et al.  Fluorescence resonance energy transfer-based stoichiometry in living cells. , 2002, Biophysical journal.

[8]  Ammasi Periasamy,et al.  Intensity Range Based Quantitative FRET Data Analysis to Localize Protein Molecules in Live Cell Nuclei , 2006, Journal of Fluorescence.

[9]  Gaudenz Danuser,et al.  FRET or no FRET: a quantitative comparison. , 2003, Biophysical journal.

[10]  C. Winterford,et al.  Apoptosis. Its significance in cancer and cancer Therapy , 1994, Cancer.

[11]  D. Xing,et al.  Calpain‐mediated pathway dominates cisplatin‐induced apoptosis in human lung adenocarcinoma cells as determined by real‐time single cell analysis , 2008, International journal of cancer.

[12]  N. Oleinick,et al.  Staurosporine-induced death of MCF-7 human breast cancer cells: a distinction between caspase-3-dependent steps of apoptosis and the critical lethal lesions. , 2003, Experimental cell research.

[13]  Takeharu Nagai,et al.  Spatio-temporal activation of caspase revealed by indicator that is insensitive to environmental effects , 2003, The Journal of cell biology.

[14]  Da Xing,et al.  Bid is not required for Bax translocation during UV-induced apoptosis. , 2007, Cellular signalling.

[15]  Horst Wallrabe,et al.  Characterization of one- and two-photon excitation fluorescence resonance energy transfer microscopy. , 2003, Methods.

[16]  G. Kass,et al.  Coxsackievirus Protein 2BC Blocks Host Cell Apoptosis by Inhibiting Caspase-3* , 2006, Journal of Biological Chemistry.

[17]  B. Herman,et al.  Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy. , 1998, Biophysical journal.

[18]  D. Green.  Apoptotic Pathways The Roads to Ruin , 1998, Cell.

[19]  K. König,et al.  Fluorescence lifetime imaging by time‐correlated single‐photon counting , 2004, Microscopy research and technique.

[20]  Th. Förster.  Zwischenmolekulare Energiewanderung und Fluoreszenz , 1948 .

[21]  J. Lakowicz.  Principles of fluorescence spectroscopy , 1983 .

[22]  Rainer Pepperkok,et al.  Simultaneous detection of multiple green fluorescent proteins in live cells by fluorescence lifetime imaging microscopy , 1999, Current Biology.

[23]  Shaoqun Zeng,et al.  Simultaneous compensation for spatial and temporal dispersion of acousto-optical deflectors for two-dimensional scanning with a single prism. , 2006, Optics letters.

[24]  L. Stryer,et al.  Energy transfer: a spectroscopic ruler. , 1967, Proceedings of the National Academy of Sciences of the United States of America.

[25]  Bence Ölveczky,et al.  Rapid Diffusion of Green Fluorescent Protein in the Mitochondrial Matrix , 1998, The Journal of cell biology.

[26]  Karsten König,et al.  Prolonged irradiation of enhanced cyan fluorescent protein or Cerulean can invalidate Forster resonance energy transfer measurements. , 2008, Journal of biomedical optics.

[27]  D Zicha,et al.  Quantitative fluorescence resonance energy transfer (FRET) measurement with acceptor photobleaching and spectral unmixing , 2004, Journal of microscopy.

[28]  A. Kenworthy,et al.  Imaging protein-protein interactions using fluorescence resonance energy transfer microscopy. , 2001, Methods.

[29]  Da Xing,et al.  Single Cell FRET Imaging for Determination of Pathway of Tumor Cell Apoptosis Induced by Photofrin-PDT , 2006, Cell cycle.

[30]  J. Sjöstrand,et al.  Caspase and proteasome activity during staurosporin-induced apoptosis in lens epithelial cells. , 2000, Investigative ophthalmology & visual science.

[31]  Q. Luo,et al.  Pulse broadening of the femtosecond pulses in a Gaussian beam passing an angular disperser. , 2007, Optics letters.

[32]  P. So,et al.  Comparing the quantification of Forster resonance energy transfer measurement accuracies based on intensity, spectral, and lifetime imaging. , 2006, Journal of Biomedical Optics.

[33]  Da Xing,et al.  Detection of caspase-3 activation in single cells by fluorescence resonance energy transfer during photodynamic therapy induced apoptosis. , 2006, Cancer letters.