Abstract

Simultaneous linear unmixing of excitation-emission spectra (ExEm-unmixing)-based fluorescence resonance energy transfer (FRET) two-hybrid assay method, named as ExEm-FRET two-hybrid assay, was developed for evaluating the stoichiometric ratio of macromolecular complexes in living cells. Linear unmixing of the excitation-emission spectra (SDA) of cells obtains the weight factors of donor (WD), acceptor (WA) and acceptor sensitization (WS), yielding ED and EA (donor- and acceptor-centric FRET efficiency) images. ExEm-FRET two-hybrid assay employs pixel-to-pixel titration curves of ED/EA versus the free acceptor (Ca)/donor (Cd) concentration deduced from the three weight factors to obtain EA,max and ED,max (the maximal EA and ED), thus yielding the stoichiometric ratio (EA,max/ED,max) of donor-tagged protein to acceptor-tagged protein.

© 2019 Optical Society of America under the terms of the OSA Open Access Publishing Agreement

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  1. E. S. Butz, M. Ben-Johny, M. Shen, P. S. Yang, L. Sang, M. Biel, D. T. Yue, and C. Wahl-Schott, “Quantifying macromolecular interactions in living cells using FRET two-hybrid assays,” Nat. Protoc. 11(12), 2470–2498 (2016).
    [Crossref] [PubMed]
  2. M. Y. Du, F. F. Yang, Z. H. Mai, W. F. Qu, F. R. Lin, L. C. Wei, and T. S. Chen, “FRET two-hybrid assay linearly fitting FRET efficiency to concentration ratio between acceptor and donor,” Appl. Phys. Lett. 112(15), 153702 (2018).
    [Crossref]
  3. M. G. Erickson, B. A. Alseikhan, B. Z. Peterson, and D. T. Yue, “Preassociation of calmodulin with voltage-gated Ca(2+) channels revealed by FRET in single living cells,” Neuron 31(6), 973–985 (2001).
    [Crossref] [PubMed]
  4. T. Zal and N. R. Gascoigne, “Photobleaching-corrected FRET efficiency imaging of live cells,” Biophys. J. 86(6), 3923–3939 (2004).
    [Crossref] [PubMed]
  5. A. D. Hoppe, B. L. Scott, T. P. Welliver, S. W. Straight, and J. A. Swanson, “N-way FRET microscopy of multiple protein-protein interactions in live cells,” PLoS One 8(6), e64760 (2013).
    [Crossref] [PubMed]
  6. F. Lin, C. Zhang, M. Du, L. Wang, Z. Mai, and T. Chen, “Superior robustness of ExEm-spFRET to IIem-spFRET method in live-cell FRET measurement,” J. Microsc. 272(2), 145–150 (2018).
    [Crossref] [PubMed]
  7. M. Du, L. Zhang, S. Xie, and T. Chen, “Wide-field microscopic FRET imaging using simultaneous spectral unmixing of excitation and emission spectra,” Opt. Express 24(14), 16037–16051 (2016).
    [Crossref] [PubMed]
  8. Y. Sun, H. Wallrabe, C. F. Booker, R. N. Day, and A. Periasamy, “Three-color spectral FRET microscopy localizes three interacting proteins in living cells,” Biophys. J. 99(4), 1274–1283 (2010).
    [Crossref] [PubMed]
  9. J. Wlodarczyk, A. Woehler, F. Kobe, E. Ponimaskin, A. Zeug, and E. Neher, “Analysis of FRET signals in the presence of free donors and acceptors,” Biophys. J. 94(3), 986–1000 (2008).
    [Crossref] [PubMed]
  10. C. Zhang, F. Lin, M. Du, W. Qu, Z. Mai, J. Qu, and T. Chen, “Simultaneous measurement of quantum yield ratio and absorption ratio between acceptor and donor by linearly unmixing excitation-emission spectra,” J. Microsc. 270(3), 335–342 (2018).
    [Crossref] [PubMed]
  11. W. Su, M. Du, F. Lin, C. Zhang, and T. Chen, “Quantitative FRET measurement based on spectral unmixing of donor, acceptor and spontaneous excitation-emission spectra,” J. Biophotonics 12(4), e201800314 (2019).
    [Crossref] [PubMed]
  12. G. Heras-Martinez, J. Andrieu, B. Larijani, and J. Requejo-Isidro, “Quantifying intracellular equilibrium dissociation constants using single-channel time-resolved FRET,” J. Biophotonics 11(1), e201600272 (2018).
    [Crossref]
  13. C. Zhang, Y. Liu, H. Sun, F. Lin, Y. Ma, W. Qu, and T. Chen, “Automated E-FRET microscope for dynamical live-cell FRET imaging,” J. Microsc. 274(1), 45–54 (2019).
    [Crossref] [PubMed]
  14. F. R. Lin, M. Y. Du, F. F. Yang, L. C. Wei, and T. S. Chen, “Improved spectrometer-microscope for quantitative FRET measurement based on simultaneous spectral unmixing of excitation and emission spectra,” J. Biomed. Opt. 23(1), 1–10 (2018).
    [PubMed]
  15. A. J. Valentijn, A. D. Metcalfe, J. Kott, C. H. Streuli, and A. P. Gilmore, “Spatial and temporal changes in Bax subcellular localization during anoikis,” J. Cell Biol. 162(4), 599–612 (2003).
    [PubMed]
  16. A. Aranovich, Q. Liu, T. Collins, F. Geng, S. Dixit, B. Leber, and D. W. Andrews, “Differences in the mechanisms of proapoptotic BH3 proteins binding to Bcl-XL and Bcl-2 quantified in live MCF-7 cells,” Mol. Cell 45(6), 754–763 (2012).
    [Crossref] [PubMed]
  17. C. King, S. Sarabipour, P. Byrne, D. J. Leahy, and K. Hristova, “The FRET signatures of noninteracting proteins in membranes: simulations and experiments,” Biophys. J. 106(6), 1309–1317 (2014).
    [Crossref] [PubMed]
  18. F. Edlich, S. Banerjee, M. Suzuki, M. M. Cleland, D. Arnoult, C. Wang, A. Neutzner, N. Tjandra, and R. J. Youle, “Bcl-x(L) retrotranslocates Bax from the mitochondria into the cytosol,” Cell 145(1), 104–116 (2011).
    [Crossref] [PubMed]
  19. H. Düssmann, M. Rehm, C. G. Concannon, S. Anguissola, M. Würstle, S. Kacmar, P. Völler, H. J. Huber, and J. H. Prehn, “Single-cell quantification of Bax activation and mathematical modelling suggest pore formation on minimal mitochondrial Bax accumulation,” Cell Death Differ. 17(2), 278–290 (2010).
    [Crossref] [PubMed]
  20. A. Kelekar, B. S. Chang, J. E. Harlan, S. W. Fesik, and C. B. Thompson, “Bad is a BH3 domain-containing protein that forms an inactivating dimer with Bcl-XL,” Mol. Cell. Biol. 17(12), 7040–7046 (1997).
    [Crossref] [PubMed]
  21. J. Zha, H. Harada, K. Osipov, J. Jockel, G. Waksman, and S. J. Korsmeyer, “BH3 domain of BAD is required for heterodimerization with BCL-XL and pro-apoptotic activity,” J. Biol. Chem. 272(39), 24101–24104 (1997).
    [Crossref] [PubMed]
  22. E. Yang, J. Zha, J. Jockel, L. H. Boise, C. B. Thompson, and S. J. Korsmeyer, “Bad, a heterodimeric partner for Bcl-XL and Bcl-2, displaces Bax and promotes cell death,” Cell 80(2), 285–291 (1995).
    [Crossref] [PubMed]
  23. A. M. Petros, D. G. Nettesheim, Y. Wang, E. T. Olejniczak, R. P. Meadows, J. Mack, K. Swift, E. D. Matayoshi, H. Zhang, C. B. Thompson, and S. W. Fesik, “Rationale for Bcl-xL/Bad peptide complex formation from structure, mutagenesis, and biophysical studies,” Protein Sci. 9(12), 2528–2534 (2000).
    [Crossref] [PubMed]

2019 (2)

W. Su, M. Du, F. Lin, C. Zhang, and T. Chen, “Quantitative FRET measurement based on spectral unmixing of donor, acceptor and spontaneous excitation-emission spectra,” J. Biophotonics 12(4), e201800314 (2019).
[Crossref] [PubMed]

C. Zhang, Y. Liu, H. Sun, F. Lin, Y. Ma, W. Qu, and T. Chen, “Automated E-FRET microscope for dynamical live-cell FRET imaging,” J. Microsc. 274(1), 45–54 (2019).
[Crossref] [PubMed]

2018 (5)

F. R. Lin, M. Y. Du, F. F. Yang, L. C. Wei, and T. S. Chen, “Improved spectrometer-microscope for quantitative FRET measurement based on simultaneous spectral unmixing of excitation and emission spectra,” J. Biomed. Opt. 23(1), 1–10 (2018).
[PubMed]

C. Zhang, F. Lin, M. Du, W. Qu, Z. Mai, J. Qu, and T. Chen, “Simultaneous measurement of quantum yield ratio and absorption ratio between acceptor and donor by linearly unmixing excitation-emission spectra,” J. Microsc. 270(3), 335–342 (2018).
[Crossref] [PubMed]

G. Heras-Martinez, J. Andrieu, B. Larijani, and J. Requejo-Isidro, “Quantifying intracellular equilibrium dissociation constants using single-channel time-resolved FRET,” J. Biophotonics 11(1), e201600272 (2018).
[Crossref]

M. Y. Du, F. F. Yang, Z. H. Mai, W. F. Qu, F. R. Lin, L. C. Wei, and T. S. Chen, “FRET two-hybrid assay linearly fitting FRET efficiency to concentration ratio between acceptor and donor,” Appl. Phys. Lett. 112(15), 153702 (2018).
[Crossref]

F. Lin, C. Zhang, M. Du, L. Wang, Z. Mai, and T. Chen, “Superior robustness of ExEm-spFRET to IIem-spFRET method in live-cell FRET measurement,” J. Microsc. 272(2), 145–150 (2018).
[Crossref] [PubMed]

2016 (2)

M. Du, L. Zhang, S. Xie, and T. Chen, “Wide-field microscopic FRET imaging using simultaneous spectral unmixing of excitation and emission spectra,” Opt. Express 24(14), 16037–16051 (2016).
[Crossref] [PubMed]

E. S. Butz, M. Ben-Johny, M. Shen, P. S. Yang, L. Sang, M. Biel, D. T. Yue, and C. Wahl-Schott, “Quantifying macromolecular interactions in living cells using FRET two-hybrid assays,” Nat. Protoc. 11(12), 2470–2498 (2016).
[Crossref] [PubMed]

2014 (1)

C. King, S. Sarabipour, P. Byrne, D. J. Leahy, and K. Hristova, “The FRET signatures of noninteracting proteins in membranes: simulations and experiments,” Biophys. J. 106(6), 1309–1317 (2014).
[Crossref] [PubMed]

2013 (1)

A. D. Hoppe, B. L. Scott, T. P. Welliver, S. W. Straight, and J. A. Swanson, “N-way FRET microscopy of multiple protein-protein interactions in live cells,” PLoS One 8(6), e64760 (2013).
[Crossref] [PubMed]

2012 (1)

A. Aranovich, Q. Liu, T. Collins, F. Geng, S. Dixit, B. Leber, and D. W. Andrews, “Differences in the mechanisms of proapoptotic BH3 proteins binding to Bcl-XL and Bcl-2 quantified in live MCF-7 cells,” Mol. Cell 45(6), 754–763 (2012).
[Crossref] [PubMed]

2011 (1)

F. Edlich, S. Banerjee, M. Suzuki, M. M. Cleland, D. Arnoult, C. Wang, A. Neutzner, N. Tjandra, and R. J. Youle, “Bcl-x(L) retrotranslocates Bax from the mitochondria into the cytosol,” Cell 145(1), 104–116 (2011).
[Crossref] [PubMed]

2010 (2)

H. Düssmann, M. Rehm, C. G. Concannon, S. Anguissola, M. Würstle, S. Kacmar, P. Völler, H. J. Huber, and J. H. Prehn, “Single-cell quantification of Bax activation and mathematical modelling suggest pore formation on minimal mitochondrial Bax accumulation,” Cell Death Differ. 17(2), 278–290 (2010).
[Crossref] [PubMed]

Y. Sun, H. Wallrabe, C. F. Booker, R. N. Day, and A. Periasamy, “Three-color spectral FRET microscopy localizes three interacting proteins in living cells,” Biophys. J. 99(4), 1274–1283 (2010).
[Crossref] [PubMed]

2008 (1)

J. Wlodarczyk, A. Woehler, F. Kobe, E. Ponimaskin, A. Zeug, and E. Neher, “Analysis of FRET signals in the presence of free donors and acceptors,” Biophys. J. 94(3), 986–1000 (2008).
[Crossref] [PubMed]

2004 (1)

T. Zal and N. R. Gascoigne, “Photobleaching-corrected FRET efficiency imaging of live cells,” Biophys. J. 86(6), 3923–3939 (2004).
[Crossref] [PubMed]

2003 (1)

A. J. Valentijn, A. D. Metcalfe, J. Kott, C. H. Streuli, and A. P. Gilmore, “Spatial and temporal changes in Bax subcellular localization during anoikis,” J. Cell Biol. 162(4), 599–612 (2003).
[PubMed]

2001 (1)

M. G. Erickson, B. A. Alseikhan, B. Z. Peterson, and D. T. Yue, “Preassociation of calmodulin with voltage-gated Ca(2+) channels revealed by FRET in single living cells,” Neuron 31(6), 973–985 (2001).
[Crossref] [PubMed]

2000 (1)

A. M. Petros, D. G. Nettesheim, Y. Wang, E. T. Olejniczak, R. P. Meadows, J. Mack, K. Swift, E. D. Matayoshi, H. Zhang, C. B. Thompson, and S. W. Fesik, “Rationale for Bcl-xL/Bad peptide complex formation from structure, mutagenesis, and biophysical studies,” Protein Sci. 9(12), 2528–2534 (2000).
[Crossref] [PubMed]

1997 (2)

A. Kelekar, B. S. Chang, J. E. Harlan, S. W. Fesik, and C. B. Thompson, “Bad is a BH3 domain-containing protein that forms an inactivating dimer with Bcl-XL,” Mol. Cell. Biol. 17(12), 7040–7046 (1997).
[Crossref] [PubMed]

J. Zha, H. Harada, K. Osipov, J. Jockel, G. Waksman, and S. J. Korsmeyer, “BH3 domain of BAD is required for heterodimerization with BCL-XL and pro-apoptotic activity,” J. Biol. Chem. 272(39), 24101–24104 (1997).
[Crossref] [PubMed]

1995 (1)

E. Yang, J. Zha, J. Jockel, L. H. Boise, C. B. Thompson, and S. J. Korsmeyer, “Bad, a heterodimeric partner for Bcl-XL and Bcl-2, displaces Bax and promotes cell death,” Cell 80(2), 285–291 (1995).
[Crossref] [PubMed]

Alseikhan, B. A.

M. G. Erickson, B. A. Alseikhan, B. Z. Peterson, and D. T. Yue, “Preassociation of calmodulin with voltage-gated Ca(2+) channels revealed by FRET in single living cells,” Neuron 31(6), 973–985 (2001).
[Crossref] [PubMed]

Andrews, D. W.

A. Aranovich, Q. Liu, T. Collins, F. Geng, S. Dixit, B. Leber, and D. W. Andrews, “Differences in the mechanisms of proapoptotic BH3 proteins binding to Bcl-XL and Bcl-2 quantified in live MCF-7 cells,” Mol. Cell 45(6), 754–763 (2012).
[Crossref] [PubMed]

Andrieu, J.

G. Heras-Martinez, J. Andrieu, B. Larijani, and J. Requejo-Isidro, “Quantifying intracellular equilibrium dissociation constants using single-channel time-resolved FRET,” J. Biophotonics 11(1), e201600272 (2018).
[Crossref]

Anguissola, S.

H. Düssmann, M. Rehm, C. G. Concannon, S. Anguissola, M. Würstle, S. Kacmar, P. Völler, H. J. Huber, and J. H. Prehn, “Single-cell quantification of Bax activation and mathematical modelling suggest pore formation on minimal mitochondrial Bax accumulation,” Cell Death Differ. 17(2), 278–290 (2010).
[Crossref] [PubMed]

Aranovich, A.

A. Aranovich, Q. Liu, T. Collins, F. Geng, S. Dixit, B. Leber, and D. W. Andrews, “Differences in the mechanisms of proapoptotic BH3 proteins binding to Bcl-XL and Bcl-2 quantified in live MCF-7 cells,” Mol. Cell 45(6), 754–763 (2012).
[Crossref] [PubMed]

Arnoult, D.

F. Edlich, S. Banerjee, M. Suzuki, M. M. Cleland, D. Arnoult, C. Wang, A. Neutzner, N. Tjandra, and R. J. Youle, “Bcl-x(L) retrotranslocates Bax from the mitochondria into the cytosol,” Cell 145(1), 104–116 (2011).
[Crossref] [PubMed]

Banerjee, S.

F. Edlich, S. Banerjee, M. Suzuki, M. M. Cleland, D. Arnoult, C. Wang, A. Neutzner, N. Tjandra, and R. J. Youle, “Bcl-x(L) retrotranslocates Bax from the mitochondria into the cytosol,” Cell 145(1), 104–116 (2011).
[Crossref] [PubMed]

Ben-Johny, M.

E. S. Butz, M. Ben-Johny, M. Shen, P. S. Yang, L. Sang, M. Biel, D. T. Yue, and C. Wahl-Schott, “Quantifying macromolecular interactions in living cells using FRET two-hybrid assays,” Nat. Protoc. 11(12), 2470–2498 (2016).
[Crossref] [PubMed]

Biel, M.

E. S. Butz, M. Ben-Johny, M. Shen, P. S. Yang, L. Sang, M. Biel, D. T. Yue, and C. Wahl-Schott, “Quantifying macromolecular interactions in living cells using FRET two-hybrid assays,” Nat. Protoc. 11(12), 2470–2498 (2016).
[Crossref] [PubMed]

Boise, L. H.

E. Yang, J. Zha, J. Jockel, L. H. Boise, C. B. Thompson, and S. J. Korsmeyer, “Bad, a heterodimeric partner for Bcl-XL and Bcl-2, displaces Bax and promotes cell death,” Cell 80(2), 285–291 (1995).
[Crossref] [PubMed]

Booker, C. F.

Y. Sun, H. Wallrabe, C. F. Booker, R. N. Day, and A. Periasamy, “Three-color spectral FRET microscopy localizes three interacting proteins in living cells,” Biophys. J. 99(4), 1274–1283 (2010).
[Crossref] [PubMed]

Butz, E. S.

E. S. Butz, M. Ben-Johny, M. Shen, P. S. Yang, L. Sang, M. Biel, D. T. Yue, and C. Wahl-Schott, “Quantifying macromolecular interactions in living cells using FRET two-hybrid assays,” Nat. Protoc. 11(12), 2470–2498 (2016).
[Crossref] [PubMed]

Byrne, P.

C. King, S. Sarabipour, P. Byrne, D. J. Leahy, and K. Hristova, “The FRET signatures of noninteracting proteins in membranes: simulations and experiments,” Biophys. J. 106(6), 1309–1317 (2014).
[Crossref] [PubMed]

Chang, B. S.

A. Kelekar, B. S. Chang, J. E. Harlan, S. W. Fesik, and C. B. Thompson, “Bad is a BH3 domain-containing protein that forms an inactivating dimer with Bcl-XL,” Mol. Cell. Biol. 17(12), 7040–7046 (1997).
[Crossref] [PubMed]

Chen, T.

W. Su, M. Du, F. Lin, C. Zhang, and T. Chen, “Quantitative FRET measurement based on spectral unmixing of donor, acceptor and spontaneous excitation-emission spectra,” J. Biophotonics 12(4), e201800314 (2019).
[Crossref] [PubMed]

C. Zhang, Y. Liu, H. Sun, F. Lin, Y. Ma, W. Qu, and T. Chen, “Automated E-FRET microscope for dynamical live-cell FRET imaging,” J. Microsc. 274(1), 45–54 (2019).
[Crossref] [PubMed]

C. Zhang, F. Lin, M. Du, W. Qu, Z. Mai, J. Qu, and T. Chen, “Simultaneous measurement of quantum yield ratio and absorption ratio between acceptor and donor by linearly unmixing excitation-emission spectra,” J. Microsc. 270(3), 335–342 (2018).
[Crossref] [PubMed]

F. Lin, C. Zhang, M. Du, L. Wang, Z. Mai, and T. Chen, “Superior robustness of ExEm-spFRET to IIem-spFRET method in live-cell FRET measurement,” J. Microsc. 272(2), 145–150 (2018).
[Crossref] [PubMed]

M. Du, L. Zhang, S. Xie, and T. Chen, “Wide-field microscopic FRET imaging using simultaneous spectral unmixing of excitation and emission spectra,” Opt. Express 24(14), 16037–16051 (2016).
[Crossref] [PubMed]

Chen, T. S.

M. Y. Du, F. F. Yang, Z. H. Mai, W. F. Qu, F. R. Lin, L. C. Wei, and T. S. Chen, “FRET two-hybrid assay linearly fitting FRET efficiency to concentration ratio between acceptor and donor,” Appl. Phys. Lett. 112(15), 153702 (2018).
[Crossref]

F. R. Lin, M. Y. Du, F. F. Yang, L. C. Wei, and T. S. Chen, “Improved spectrometer-microscope for quantitative FRET measurement based on simultaneous spectral unmixing of excitation and emission spectra,” J. Biomed. Opt. 23(1), 1–10 (2018).
[PubMed]

Cleland, M. M.

F. Edlich, S. Banerjee, M. Suzuki, M. M. Cleland, D. Arnoult, C. Wang, A. Neutzner, N. Tjandra, and R. J. Youle, “Bcl-x(L) retrotranslocates Bax from the mitochondria into the cytosol,” Cell 145(1), 104–116 (2011).
[Crossref] [PubMed]

Collins, T.

A. Aranovich, Q. Liu, T. Collins, F. Geng, S. Dixit, B. Leber, and D. W. Andrews, “Differences in the mechanisms of proapoptotic BH3 proteins binding to Bcl-XL and Bcl-2 quantified in live MCF-7 cells,” Mol. Cell 45(6), 754–763 (2012).
[Crossref] [PubMed]

Concannon, C. G.

H. Düssmann, M. Rehm, C. G. Concannon, S. Anguissola, M. Würstle, S. Kacmar, P. Völler, H. J. Huber, and J. H. Prehn, “Single-cell quantification of Bax activation and mathematical modelling suggest pore formation on minimal mitochondrial Bax accumulation,” Cell Death Differ. 17(2), 278–290 (2010).
[Crossref] [PubMed]

Day, R. N.

Y. Sun, H. Wallrabe, C. F. Booker, R. N. Day, and A. Periasamy, “Three-color spectral FRET microscopy localizes three interacting proteins in living cells,” Biophys. J. 99(4), 1274–1283 (2010).
[Crossref] [PubMed]

Dixit, S.

A. Aranovich, Q. Liu, T. Collins, F. Geng, S. Dixit, B. Leber, and D. W. Andrews, “Differences in the mechanisms of proapoptotic BH3 proteins binding to Bcl-XL and Bcl-2 quantified in live MCF-7 cells,” Mol. Cell 45(6), 754–763 (2012).
[Crossref] [PubMed]

Du, M.

W. Su, M. Du, F. Lin, C. Zhang, and T. Chen, “Quantitative FRET measurement based on spectral unmixing of donor, acceptor and spontaneous excitation-emission spectra,” J. Biophotonics 12(4), e201800314 (2019).
[Crossref] [PubMed]

C. Zhang, F. Lin, M. Du, W. Qu, Z. Mai, J. Qu, and T. Chen, “Simultaneous measurement of quantum yield ratio and absorption ratio between acceptor and donor by linearly unmixing excitation-emission spectra,” J. Microsc. 270(3), 335–342 (2018).
[Crossref] [PubMed]

F. Lin, C. Zhang, M. Du, L. Wang, Z. Mai, and T. Chen, “Superior robustness of ExEm-spFRET to IIem-spFRET method in live-cell FRET measurement,” J. Microsc. 272(2), 145–150 (2018).
[Crossref] [PubMed]

M. Du, L. Zhang, S. Xie, and T. Chen, “Wide-field microscopic FRET imaging using simultaneous spectral unmixing of excitation and emission spectra,” Opt. Express 24(14), 16037–16051 (2016).
[Crossref] [PubMed]

Du, M. Y.

M. Y. Du, F. F. Yang, Z. H. Mai, W. F. Qu, F. R. Lin, L. C. Wei, and T. S. Chen, “FRET two-hybrid assay linearly fitting FRET efficiency to concentration ratio between acceptor and donor,” Appl. Phys. Lett. 112(15), 153702 (2018).
[Crossref]

F. R. Lin, M. Y. Du, F. F. Yang, L. C. Wei, and T. S. Chen, “Improved spectrometer-microscope for quantitative FRET measurement based on simultaneous spectral unmixing of excitation and emission spectra,” J. Biomed. Opt. 23(1), 1–10 (2018).
[PubMed]

Düssmann, H.

H. Düssmann, M. Rehm, C. G. Concannon, S. Anguissola, M. Würstle, S. Kacmar, P. Völler, H. J. Huber, and J. H. Prehn, “Single-cell quantification of Bax activation and mathematical modelling suggest pore formation on minimal mitochondrial Bax accumulation,” Cell Death Differ. 17(2), 278–290 (2010).
[Crossref] [PubMed]

Edlich, F.

F. Edlich, S. Banerjee, M. Suzuki, M. M. Cleland, D. Arnoult, C. Wang, A. Neutzner, N. Tjandra, and R. J. Youle, “Bcl-x(L) retrotranslocates Bax from the mitochondria into the cytosol,” Cell 145(1), 104–116 (2011).
[Crossref] [PubMed]

Erickson, M. G.

M. G. Erickson, B. A. Alseikhan, B. Z. Peterson, and D. T. Yue, “Preassociation of calmodulin with voltage-gated Ca(2+) channels revealed by FRET in single living cells,” Neuron 31(6), 973–985 (2001).
[Crossref] [PubMed]

Fesik, S. W.

A. M. Petros, D. G. Nettesheim, Y. Wang, E. T. Olejniczak, R. P. Meadows, J. Mack, K. Swift, E. D. Matayoshi, H. Zhang, C. B. Thompson, and S. W. Fesik, “Rationale for Bcl-xL/Bad peptide complex formation from structure, mutagenesis, and biophysical studies,” Protein Sci. 9(12), 2528–2534 (2000).
[Crossref] [PubMed]

A. Kelekar, B. S. Chang, J. E. Harlan, S. W. Fesik, and C. B. Thompson, “Bad is a BH3 domain-containing protein that forms an inactivating dimer with Bcl-XL,” Mol. Cell. Biol. 17(12), 7040–7046 (1997).
[Crossref] [PubMed]

Gascoigne, N. R.

T. Zal and N. R. Gascoigne, “Photobleaching-corrected FRET efficiency imaging of live cells,” Biophys. J. 86(6), 3923–3939 (2004).
[Crossref] [PubMed]

Geng, F.

A. Aranovich, Q. Liu, T. Collins, F. Geng, S. Dixit, B. Leber, and D. W. Andrews, “Differences in the mechanisms of proapoptotic BH3 proteins binding to Bcl-XL and Bcl-2 quantified in live MCF-7 cells,” Mol. Cell 45(6), 754–763 (2012).
[Crossref] [PubMed]

Gilmore, A. P.

A. J. Valentijn, A. D. Metcalfe, J. Kott, C. H. Streuli, and A. P. Gilmore, “Spatial and temporal changes in Bax subcellular localization during anoikis,” J. Cell Biol. 162(4), 599–612 (2003).
[PubMed]

Harada, H.

J. Zha, H. Harada, K. Osipov, J. Jockel, G. Waksman, and S. J. Korsmeyer, “BH3 domain of BAD is required for heterodimerization with BCL-XL and pro-apoptotic activity,” J. Biol. Chem. 272(39), 24101–24104 (1997).
[Crossref] [PubMed]

Harlan, J. E.

A. Kelekar, B. S. Chang, J. E. Harlan, S. W. Fesik, and C. B. Thompson, “Bad is a BH3 domain-containing protein that forms an inactivating dimer with Bcl-XL,” Mol. Cell. Biol. 17(12), 7040–7046 (1997).
[Crossref] [PubMed]

Heras-Martinez, G.

G. Heras-Martinez, J. Andrieu, B. Larijani, and J. Requejo-Isidro, “Quantifying intracellular equilibrium dissociation constants using single-channel time-resolved FRET,” J. Biophotonics 11(1), e201600272 (2018).
[Crossref]

Hoppe, A. D.

A. D. Hoppe, B. L. Scott, T. P. Welliver, S. W. Straight, and J. A. Swanson, “N-way FRET microscopy of multiple protein-protein interactions in live cells,” PLoS One 8(6), e64760 (2013).
[Crossref] [PubMed]

Hristova, K.

C. King, S. Sarabipour, P. Byrne, D. J. Leahy, and K. Hristova, “The FRET signatures of noninteracting proteins in membranes: simulations and experiments,” Biophys. J. 106(6), 1309–1317 (2014).
[Crossref] [PubMed]

Huber, H. J.

H. Düssmann, M. Rehm, C. G. Concannon, S. Anguissola, M. Würstle, S. Kacmar, P. Völler, H. J. Huber, and J. H. Prehn, “Single-cell quantification of Bax activation and mathematical modelling suggest pore formation on minimal mitochondrial Bax accumulation,” Cell Death Differ. 17(2), 278–290 (2010).
[Crossref] [PubMed]

Jockel, J.

J. Zha, H. Harada, K. Osipov, J. Jockel, G. Waksman, and S. J. Korsmeyer, “BH3 domain of BAD is required for heterodimerization with BCL-XL and pro-apoptotic activity,” J. Biol. Chem. 272(39), 24101–24104 (1997).
[Crossref] [PubMed]

E. Yang, J. Zha, J. Jockel, L. H. Boise, C. B. Thompson, and S. J. Korsmeyer, “Bad, a heterodimeric partner for Bcl-XL and Bcl-2, displaces Bax and promotes cell death,” Cell 80(2), 285–291 (1995).
[Crossref] [PubMed]

Kacmar, S.

H. Düssmann, M. Rehm, C. G. Concannon, S. Anguissola, M. Würstle, S. Kacmar, P. Völler, H. J. Huber, and J. H. Prehn, “Single-cell quantification of Bax activation and mathematical modelling suggest pore formation on minimal mitochondrial Bax accumulation,” Cell Death Differ. 17(2), 278–290 (2010).
[Crossref] [PubMed]

Kelekar, A.

A. Kelekar, B. S. Chang, J. E. Harlan, S. W. Fesik, and C. B. Thompson, “Bad is a BH3 domain-containing protein that forms an inactivating dimer with Bcl-XL,” Mol. Cell. Biol. 17(12), 7040–7046 (1997).
[Crossref] [PubMed]

King, C.

C. King, S. Sarabipour, P. Byrne, D. J. Leahy, and K. Hristova, “The FRET signatures of noninteracting proteins in membranes: simulations and experiments,” Biophys. J. 106(6), 1309–1317 (2014).
[Crossref] [PubMed]

Kobe, F.

J. Wlodarczyk, A. Woehler, F. Kobe, E. Ponimaskin, A. Zeug, and E. Neher, “Analysis of FRET signals in the presence of free donors and acceptors,” Biophys. J. 94(3), 986–1000 (2008).
[Crossref] [PubMed]

Korsmeyer, S. J.

J. Zha, H. Harada, K. Osipov, J. Jockel, G. Waksman, and S. J. Korsmeyer, “BH3 domain of BAD is required for heterodimerization with BCL-XL and pro-apoptotic activity,” J. Biol. Chem. 272(39), 24101–24104 (1997).
[Crossref] [PubMed]

E. Yang, J. Zha, J. Jockel, L. H. Boise, C. B. Thompson, and S. J. Korsmeyer, “Bad, a heterodimeric partner for Bcl-XL and Bcl-2, displaces Bax and promotes cell death,” Cell 80(2), 285–291 (1995).
[Crossref] [PubMed]

Kott, J.

A. J. Valentijn, A. D. Metcalfe, J. Kott, C. H. Streuli, and A. P. Gilmore, “Spatial and temporal changes in Bax subcellular localization during anoikis,” J. Cell Biol. 162(4), 599–612 (2003).
[PubMed]

Larijani, B.

G. Heras-Martinez, J. Andrieu, B. Larijani, and J. Requejo-Isidro, “Quantifying intracellular equilibrium dissociation constants using single-channel time-resolved FRET,” J. Biophotonics 11(1), e201600272 (2018).
[Crossref]

Leahy, D. J.

C. King, S. Sarabipour, P. Byrne, D. J. Leahy, and K. Hristova, “The FRET signatures of noninteracting proteins in membranes: simulations and experiments,” Biophys. J. 106(6), 1309–1317 (2014).
[Crossref] [PubMed]

Leber, B.

A. Aranovich, Q. Liu, T. Collins, F. Geng, S. Dixit, B. Leber, and D. W. Andrews, “Differences in the mechanisms of proapoptotic BH3 proteins binding to Bcl-XL and Bcl-2 quantified in live MCF-7 cells,” Mol. Cell 45(6), 754–763 (2012).
[Crossref] [PubMed]

Lin, F.

W. Su, M. Du, F. Lin, C. Zhang, and T. Chen, “Quantitative FRET measurement based on spectral unmixing of donor, acceptor and spontaneous excitation-emission spectra,” J. Biophotonics 12(4), e201800314 (2019).
[Crossref] [PubMed]

C. Zhang, Y. Liu, H. Sun, F. Lin, Y. Ma, W. Qu, and T. Chen, “Automated E-FRET microscope for dynamical live-cell FRET imaging,” J. Microsc. 274(1), 45–54 (2019).
[Crossref] [PubMed]

C. Zhang, F. Lin, M. Du, W. Qu, Z. Mai, J. Qu, and T. Chen, “Simultaneous measurement of quantum yield ratio and absorption ratio between acceptor and donor by linearly unmixing excitation-emission spectra,” J. Microsc. 270(3), 335–342 (2018).
[Crossref] [PubMed]

F. Lin, C. Zhang, M. Du, L. Wang, Z. Mai, and T. Chen, “Superior robustness of ExEm-spFRET to IIem-spFRET method in live-cell FRET measurement,” J. Microsc. 272(2), 145–150 (2018).
[Crossref] [PubMed]

Lin, F. R.

M. Y. Du, F. F. Yang, Z. H. Mai, W. F. Qu, F. R. Lin, L. C. Wei, and T. S. Chen, “FRET two-hybrid assay linearly fitting FRET efficiency to concentration ratio between acceptor and donor,” Appl. Phys. Lett. 112(15), 153702 (2018).
[Crossref]

F. R. Lin, M. Y. Du, F. F. Yang, L. C. Wei, and T. S. Chen, “Improved spectrometer-microscope for quantitative FRET measurement based on simultaneous spectral unmixing of excitation and emission spectra,” J. Biomed. Opt. 23(1), 1–10 (2018).
[PubMed]

Liu, Q.

A. Aranovich, Q. Liu, T. Collins, F. Geng, S. Dixit, B. Leber, and D. W. Andrews, “Differences in the mechanisms of proapoptotic BH3 proteins binding to Bcl-XL and Bcl-2 quantified in live MCF-7 cells,” Mol. Cell 45(6), 754–763 (2012).
[Crossref] [PubMed]

Liu, Y.

C. Zhang, Y. Liu, H. Sun, F. Lin, Y. Ma, W. Qu, and T. Chen, “Automated E-FRET microscope for dynamical live-cell FRET imaging,” J. Microsc. 274(1), 45–54 (2019).
[Crossref] [PubMed]

Ma, Y.

C. Zhang, Y. Liu, H. Sun, F. Lin, Y. Ma, W. Qu, and T. Chen, “Automated E-FRET microscope for dynamical live-cell FRET imaging,” J. Microsc. 274(1), 45–54 (2019).
[Crossref] [PubMed]

Mack, J.

A. M. Petros, D. G. Nettesheim, Y. Wang, E. T. Olejniczak, R. P. Meadows, J. Mack, K. Swift, E. D. Matayoshi, H. Zhang, C. B. Thompson, and S. W. Fesik, “Rationale for Bcl-xL/Bad peptide complex formation from structure, mutagenesis, and biophysical studies,” Protein Sci. 9(12), 2528–2534 (2000).
[Crossref] [PubMed]

Mai, Z.

C. Zhang, F. Lin, M. Du, W. Qu, Z. Mai, J. Qu, and T. Chen, “Simultaneous measurement of quantum yield ratio and absorption ratio between acceptor and donor by linearly unmixing excitation-emission spectra,” J. Microsc. 270(3), 335–342 (2018).
[Crossref] [PubMed]

F. Lin, C. Zhang, M. Du, L. Wang, Z. Mai, and T. Chen, “Superior robustness of ExEm-spFRET to IIem-spFRET method in live-cell FRET measurement,” J. Microsc. 272(2), 145–150 (2018).
[Crossref] [PubMed]

Mai, Z. H.

M. Y. Du, F. F. Yang, Z. H. Mai, W. F. Qu, F. R. Lin, L. C. Wei, and T. S. Chen, “FRET two-hybrid assay linearly fitting FRET efficiency to concentration ratio between acceptor and donor,” Appl. Phys. Lett. 112(15), 153702 (2018).
[Crossref]

Matayoshi, E. D.

A. M. Petros, D. G. Nettesheim, Y. Wang, E. T. Olejniczak, R. P. Meadows, J. Mack, K. Swift, E. D. Matayoshi, H. Zhang, C. B. Thompson, and S. W. Fesik, “Rationale for Bcl-xL/Bad peptide complex formation from structure, mutagenesis, and biophysical studies,” Protein Sci. 9(12), 2528–2534 (2000).
[Crossref] [PubMed]

Meadows, R. P.

A. M. Petros, D. G. Nettesheim, Y. Wang, E. T. Olejniczak, R. P. Meadows, J. Mack, K. Swift, E. D. Matayoshi, H. Zhang, C. B. Thompson, and S. W. Fesik, “Rationale for Bcl-xL/Bad peptide complex formation from structure, mutagenesis, and biophysical studies,” Protein Sci. 9(12), 2528–2534 (2000).
[Crossref] [PubMed]

Metcalfe, A. D.

A. J. Valentijn, A. D. Metcalfe, J. Kott, C. H. Streuli, and A. P. Gilmore, “Spatial and temporal changes in Bax subcellular localization during anoikis,” J. Cell Biol. 162(4), 599–612 (2003).
[PubMed]

Neher, E.

J. Wlodarczyk, A. Woehler, F. Kobe, E. Ponimaskin, A. Zeug, and E. Neher, “Analysis of FRET signals in the presence of free donors and acceptors,” Biophys. J. 94(3), 986–1000 (2008).
[Crossref] [PubMed]

Nettesheim, D. G.

A. M. Petros, D. G. Nettesheim, Y. Wang, E. T. Olejniczak, R. P. Meadows, J. Mack, K. Swift, E. D. Matayoshi, H. Zhang, C. B. Thompson, and S. W. Fesik, “Rationale for Bcl-xL/Bad peptide complex formation from structure, mutagenesis, and biophysical studies,” Protein Sci. 9(12), 2528–2534 (2000).
[Crossref] [PubMed]

Neutzner, A.

F. Edlich, S. Banerjee, M. Suzuki, M. M. Cleland, D. Arnoult, C. Wang, A. Neutzner, N. Tjandra, and R. J. Youle, “Bcl-x(L) retrotranslocates Bax from the mitochondria into the cytosol,” Cell 145(1), 104–116 (2011).
[Crossref] [PubMed]

Olejniczak, E. T.

A. M. Petros, D. G. Nettesheim, Y. Wang, E. T. Olejniczak, R. P. Meadows, J. Mack, K. Swift, E. D. Matayoshi, H. Zhang, C. B. Thompson, and S. W. Fesik, “Rationale for Bcl-xL/Bad peptide complex formation from structure, mutagenesis, and biophysical studies,” Protein Sci. 9(12), 2528–2534 (2000).
[Crossref] [PubMed]

Osipov, K.

J. Zha, H. Harada, K. Osipov, J. Jockel, G. Waksman, and S. J. Korsmeyer, “BH3 domain of BAD is required for heterodimerization with BCL-XL and pro-apoptotic activity,” J. Biol. Chem. 272(39), 24101–24104 (1997).
[Crossref] [PubMed]

Periasamy, A.

Y. Sun, H. Wallrabe, C. F. Booker, R. N. Day, and A. Periasamy, “Three-color spectral FRET microscopy localizes three interacting proteins in living cells,” Biophys. J. 99(4), 1274–1283 (2010).
[Crossref] [PubMed]

Peterson, B. Z.

M. G. Erickson, B. A. Alseikhan, B. Z. Peterson, and D. T. Yue, “Preassociation of calmodulin with voltage-gated Ca(2+) channels revealed by FRET in single living cells,” Neuron 31(6), 973–985 (2001).
[Crossref] [PubMed]

Petros, A. M.

A. M. Petros, D. G. Nettesheim, Y. Wang, E. T. Olejniczak, R. P. Meadows, J. Mack, K. Swift, E. D. Matayoshi, H. Zhang, C. B. Thompson, and S. W. Fesik, “Rationale for Bcl-xL/Bad peptide complex formation from structure, mutagenesis, and biophysical studies,” Protein Sci. 9(12), 2528–2534 (2000).
[Crossref] [PubMed]

Ponimaskin, E.

J. Wlodarczyk, A. Woehler, F. Kobe, E. Ponimaskin, A. Zeug, and E. Neher, “Analysis of FRET signals in the presence of free donors and acceptors,” Biophys. J. 94(3), 986–1000 (2008).
[Crossref] [PubMed]

Prehn, J. H.

H. Düssmann, M. Rehm, C. G. Concannon, S. Anguissola, M. Würstle, S. Kacmar, P. Völler, H. J. Huber, and J. H. Prehn, “Single-cell quantification of Bax activation and mathematical modelling suggest pore formation on minimal mitochondrial Bax accumulation,” Cell Death Differ. 17(2), 278–290 (2010).
[Crossref] [PubMed]

Qu, J.

C. Zhang, F. Lin, M. Du, W. Qu, Z. Mai, J. Qu, and T. Chen, “Simultaneous measurement of quantum yield ratio and absorption ratio between acceptor and donor by linearly unmixing excitation-emission spectra,” J. Microsc. 270(3), 335–342 (2018).
[Crossref] [PubMed]

Qu, W.

C. Zhang, Y. Liu, H. Sun, F. Lin, Y. Ma, W. Qu, and T. Chen, “Automated E-FRET microscope for dynamical live-cell FRET imaging,” J. Microsc. 274(1), 45–54 (2019).
[Crossref] [PubMed]

C. Zhang, F. Lin, M. Du, W. Qu, Z. Mai, J. Qu, and T. Chen, “Simultaneous measurement of quantum yield ratio and absorption ratio between acceptor and donor by linearly unmixing excitation-emission spectra,” J. Microsc. 270(3), 335–342 (2018).
[Crossref] [PubMed]

Qu, W. F.

M. Y. Du, F. F. Yang, Z. H. Mai, W. F. Qu, F. R. Lin, L. C. Wei, and T. S. Chen, “FRET two-hybrid assay linearly fitting FRET efficiency to concentration ratio between acceptor and donor,” Appl. Phys. Lett. 112(15), 153702 (2018).
[Crossref]

Rehm, M.

H. Düssmann, M. Rehm, C. G. Concannon, S. Anguissola, M. Würstle, S. Kacmar, P. Völler, H. J. Huber, and J. H. Prehn, “Single-cell quantification of Bax activation and mathematical modelling suggest pore formation on minimal mitochondrial Bax accumulation,” Cell Death Differ. 17(2), 278–290 (2010).
[Crossref] [PubMed]

Requejo-Isidro, J.

G. Heras-Martinez, J. Andrieu, B. Larijani, and J. Requejo-Isidro, “Quantifying intracellular equilibrium dissociation constants using single-channel time-resolved FRET,” J. Biophotonics 11(1), e201600272 (2018).
[Crossref]

Sang, L.

E. S. Butz, M. Ben-Johny, M. Shen, P. S. Yang, L. Sang, M. Biel, D. T. Yue, and C. Wahl-Schott, “Quantifying macromolecular interactions in living cells using FRET two-hybrid assays,” Nat. Protoc. 11(12), 2470–2498 (2016).
[Crossref] [PubMed]

Sarabipour, S.

C. King, S. Sarabipour, P. Byrne, D. J. Leahy, and K. Hristova, “The FRET signatures of noninteracting proteins in membranes: simulations and experiments,” Biophys. J. 106(6), 1309–1317 (2014).
[Crossref] [PubMed]

Scott, B. L.

A. D. Hoppe, B. L. Scott, T. P. Welliver, S. W. Straight, and J. A. Swanson, “N-way FRET microscopy of multiple protein-protein interactions in live cells,” PLoS One 8(6), e64760 (2013).
[Crossref] [PubMed]

Shen, M.

E. S. Butz, M. Ben-Johny, M. Shen, P. S. Yang, L. Sang, M. Biel, D. T. Yue, and C. Wahl-Schott, “Quantifying macromolecular interactions in living cells using FRET two-hybrid assays,” Nat. Protoc. 11(12), 2470–2498 (2016).
[Crossref] [PubMed]

Straight, S. W.

A. D. Hoppe, B. L. Scott, T. P. Welliver, S. W. Straight, and J. A. Swanson, “N-way FRET microscopy of multiple protein-protein interactions in live cells,” PLoS One 8(6), e64760 (2013).
[Crossref] [PubMed]

Streuli, C. H.

A. J. Valentijn, A. D. Metcalfe, J. Kott, C. H. Streuli, and A. P. Gilmore, “Spatial and temporal changes in Bax subcellular localization during anoikis,” J. Cell Biol. 162(4), 599–612 (2003).
[PubMed]

Su, W.

W. Su, M. Du, F. Lin, C. Zhang, and T. Chen, “Quantitative FRET measurement based on spectral unmixing of donor, acceptor and spontaneous excitation-emission spectra,” J. Biophotonics 12(4), e201800314 (2019).
[Crossref] [PubMed]

Sun, H.

C. Zhang, Y. Liu, H. Sun, F. Lin, Y. Ma, W. Qu, and T. Chen, “Automated E-FRET microscope for dynamical live-cell FRET imaging,” J. Microsc. 274(1), 45–54 (2019).
[Crossref] [PubMed]

Sun, Y.

Y. Sun, H. Wallrabe, C. F. Booker, R. N. Day, and A. Periasamy, “Three-color spectral FRET microscopy localizes three interacting proteins in living cells,” Biophys. J. 99(4), 1274–1283 (2010).
[Crossref] [PubMed]

Suzuki, M.

F. Edlich, S. Banerjee, M. Suzuki, M. M. Cleland, D. Arnoult, C. Wang, A. Neutzner, N. Tjandra, and R. J. Youle, “Bcl-x(L) retrotranslocates Bax from the mitochondria into the cytosol,” Cell 145(1), 104–116 (2011).
[Crossref] [PubMed]

Swanson, J. A.

A. D. Hoppe, B. L. Scott, T. P. Welliver, S. W. Straight, and J. A. Swanson, “N-way FRET microscopy of multiple protein-protein interactions in live cells,” PLoS One 8(6), e64760 (2013).
[Crossref] [PubMed]

Swift, K.

A. M. Petros, D. G. Nettesheim, Y. Wang, E. T. Olejniczak, R. P. Meadows, J. Mack, K. Swift, E. D. Matayoshi, H. Zhang, C. B. Thompson, and S. W. Fesik, “Rationale for Bcl-xL/Bad peptide complex formation from structure, mutagenesis, and biophysical studies,” Protein Sci. 9(12), 2528–2534 (2000).
[Crossref] [PubMed]

Thompson, C. B.

A. M. Petros, D. G. Nettesheim, Y. Wang, E. T. Olejniczak, R. P. Meadows, J. Mack, K. Swift, E. D. Matayoshi, H. Zhang, C. B. Thompson, and S. W. Fesik, “Rationale for Bcl-xL/Bad peptide complex formation from structure, mutagenesis, and biophysical studies,” Protein Sci. 9(12), 2528–2534 (2000).
[Crossref] [PubMed]

A. Kelekar, B. S. Chang, J. E. Harlan, S. W. Fesik, and C. B. Thompson, “Bad is a BH3 domain-containing protein that forms an inactivating dimer with Bcl-XL,” Mol. Cell. Biol. 17(12), 7040–7046 (1997).
[Crossref] [PubMed]

E. Yang, J. Zha, J. Jockel, L. H. Boise, C. B. Thompson, and S. J. Korsmeyer, “Bad, a heterodimeric partner for Bcl-XL and Bcl-2, displaces Bax and promotes cell death,” Cell 80(2), 285–291 (1995).
[Crossref] [PubMed]

Tjandra, N.

F. Edlich, S. Banerjee, M. Suzuki, M. M. Cleland, D. Arnoult, C. Wang, A. Neutzner, N. Tjandra, and R. J. Youle, “Bcl-x(L) retrotranslocates Bax from the mitochondria into the cytosol,” Cell 145(1), 104–116 (2011).
[Crossref] [PubMed]

Valentijn, A. J.

A. J. Valentijn, A. D. Metcalfe, J. Kott, C. H. Streuli, and A. P. Gilmore, “Spatial and temporal changes in Bax subcellular localization during anoikis,” J. Cell Biol. 162(4), 599–612 (2003).
[PubMed]

Völler, P.

H. Düssmann, M. Rehm, C. G. Concannon, S. Anguissola, M. Würstle, S. Kacmar, P. Völler, H. J. Huber, and J. H. Prehn, “Single-cell quantification of Bax activation and mathematical modelling suggest pore formation on minimal mitochondrial Bax accumulation,” Cell Death Differ. 17(2), 278–290 (2010).
[Crossref] [PubMed]

Wahl-Schott, C.

E. S. Butz, M. Ben-Johny, M. Shen, P. S. Yang, L. Sang, M. Biel, D. T. Yue, and C. Wahl-Schott, “Quantifying macromolecular interactions in living cells using FRET two-hybrid assays,” Nat. Protoc. 11(12), 2470–2498 (2016).
[Crossref] [PubMed]

Waksman, G.

J. Zha, H. Harada, K. Osipov, J. Jockel, G. Waksman, and S. J. Korsmeyer, “BH3 domain of BAD is required for heterodimerization with BCL-XL and pro-apoptotic activity,” J. Biol. Chem. 272(39), 24101–24104 (1997).
[Crossref] [PubMed]

Wallrabe, H.

Y. Sun, H. Wallrabe, C. F. Booker, R. N. Day, and A. Periasamy, “Three-color spectral FRET microscopy localizes three interacting proteins in living cells,” Biophys. J. 99(4), 1274–1283 (2010).
[Crossref] [PubMed]

Wang, C.

F. Edlich, S. Banerjee, M. Suzuki, M. M. Cleland, D. Arnoult, C. Wang, A. Neutzner, N. Tjandra, and R. J. Youle, “Bcl-x(L) retrotranslocates Bax from the mitochondria into the cytosol,” Cell 145(1), 104–116 (2011).
[Crossref] [PubMed]

Wang, L.

F. Lin, C. Zhang, M. Du, L. Wang, Z. Mai, and T. Chen, “Superior robustness of ExEm-spFRET to IIem-spFRET method in live-cell FRET measurement,” J. Microsc. 272(2), 145–150 (2018).
[Crossref] [PubMed]

Wang, Y.

A. M. Petros, D. G. Nettesheim, Y. Wang, E. T. Olejniczak, R. P. Meadows, J. Mack, K. Swift, E. D. Matayoshi, H. Zhang, C. B. Thompson, and S. W. Fesik, “Rationale for Bcl-xL/Bad peptide complex formation from structure, mutagenesis, and biophysical studies,” Protein Sci. 9(12), 2528–2534 (2000).
[Crossref] [PubMed]

Wei, L. C.

F. R. Lin, M. Y. Du, F. F. Yang, L. C. Wei, and T. S. Chen, “Improved spectrometer-microscope for quantitative FRET measurement based on simultaneous spectral unmixing of excitation and emission spectra,” J. Biomed. Opt. 23(1), 1–10 (2018).
[PubMed]

M. Y. Du, F. F. Yang, Z. H. Mai, W. F. Qu, F. R. Lin, L. C. Wei, and T. S. Chen, “FRET two-hybrid assay linearly fitting FRET efficiency to concentration ratio between acceptor and donor,” Appl. Phys. Lett. 112(15), 153702 (2018).
[Crossref]

Welliver, T. P.

A. D. Hoppe, B. L. Scott, T. P. Welliver, S. W. Straight, and J. A. Swanson, “N-way FRET microscopy of multiple protein-protein interactions in live cells,” PLoS One 8(6), e64760 (2013).
[Crossref] [PubMed]

Wlodarczyk, J.

J. Wlodarczyk, A. Woehler, F. Kobe, E. Ponimaskin, A. Zeug, and E. Neher, “Analysis of FRET signals in the presence of free donors and acceptors,” Biophys. J. 94(3), 986–1000 (2008).
[Crossref] [PubMed]

Woehler, A.

J. Wlodarczyk, A. Woehler, F. Kobe, E. Ponimaskin, A. Zeug, and E. Neher, “Analysis of FRET signals in the presence of free donors and acceptors,” Biophys. J. 94(3), 986–1000 (2008).
[Crossref] [PubMed]

Würstle, M.

H. Düssmann, M. Rehm, C. G. Concannon, S. Anguissola, M. Würstle, S. Kacmar, P. Völler, H. J. Huber, and J. H. Prehn, “Single-cell quantification of Bax activation and mathematical modelling suggest pore formation on minimal mitochondrial Bax accumulation,” Cell Death Differ. 17(2), 278–290 (2010).
[Crossref] [PubMed]

Xie, S.

Yang, E.

E. Yang, J. Zha, J. Jockel, L. H. Boise, C. B. Thompson, and S. J. Korsmeyer, “Bad, a heterodimeric partner for Bcl-XL and Bcl-2, displaces Bax and promotes cell death,” Cell 80(2), 285–291 (1995).
[Crossref] [PubMed]

Yang, F. F.

F. R. Lin, M. Y. Du, F. F. Yang, L. C. Wei, and T. S. Chen, “Improved spectrometer-microscope for quantitative FRET measurement based on simultaneous spectral unmixing of excitation and emission spectra,” J. Biomed. Opt. 23(1), 1–10 (2018).
[PubMed]

M. Y. Du, F. F. Yang, Z. H. Mai, W. F. Qu, F. R. Lin, L. C. Wei, and T. S. Chen, “FRET two-hybrid assay linearly fitting FRET efficiency to concentration ratio between acceptor and donor,” Appl. Phys. Lett. 112(15), 153702 (2018).
[Crossref]

Yang, P. S.

E. S. Butz, M. Ben-Johny, M. Shen, P. S. Yang, L. Sang, M. Biel, D. T. Yue, and C. Wahl-Schott, “Quantifying macromolecular interactions in living cells using FRET two-hybrid assays,” Nat. Protoc. 11(12), 2470–2498 (2016).
[Crossref] [PubMed]

Youle, R. J.

F. Edlich, S. Banerjee, M. Suzuki, M. M. Cleland, D. Arnoult, C. Wang, A. Neutzner, N. Tjandra, and R. J. Youle, “Bcl-x(L) retrotranslocates Bax from the mitochondria into the cytosol,” Cell 145(1), 104–116 (2011).
[Crossref] [PubMed]

Yue, D. T.

E. S. Butz, M. Ben-Johny, M. Shen, P. S. Yang, L. Sang, M. Biel, D. T. Yue, and C. Wahl-Schott, “Quantifying macromolecular interactions in living cells using FRET two-hybrid assays,” Nat. Protoc. 11(12), 2470–2498 (2016).
[Crossref] [PubMed]

M. G. Erickson, B. A. Alseikhan, B. Z. Peterson, and D. T. Yue, “Preassociation of calmodulin with voltage-gated Ca(2+) channels revealed by FRET in single living cells,” Neuron 31(6), 973–985 (2001).
[Crossref] [PubMed]

Zal, T.

T. Zal and N. R. Gascoigne, “Photobleaching-corrected FRET efficiency imaging of live cells,” Biophys. J. 86(6), 3923–3939 (2004).
[Crossref] [PubMed]

Zeug, A.

J. Wlodarczyk, A. Woehler, F. Kobe, E. Ponimaskin, A. Zeug, and E. Neher, “Analysis of FRET signals in the presence of free donors and acceptors,” Biophys. J. 94(3), 986–1000 (2008).
[Crossref] [PubMed]

Zha, J.

J. Zha, H. Harada, K. Osipov, J. Jockel, G. Waksman, and S. J. Korsmeyer, “BH3 domain of BAD is required for heterodimerization with BCL-XL and pro-apoptotic activity,” J. Biol. Chem. 272(39), 24101–24104 (1997).
[Crossref] [PubMed]

E. Yang, J. Zha, J. Jockel, L. H. Boise, C. B. Thompson, and S. J. Korsmeyer, “Bad, a heterodimeric partner for Bcl-XL and Bcl-2, displaces Bax and promotes cell death,” Cell 80(2), 285–291 (1995).
[Crossref] [PubMed]

Zhang, C.

W. Su, M. Du, F. Lin, C. Zhang, and T. Chen, “Quantitative FRET measurement based on spectral unmixing of donor, acceptor and spontaneous excitation-emission spectra,” J. Biophotonics 12(4), e201800314 (2019).
[Crossref] [PubMed]

C. Zhang, Y. Liu, H. Sun, F. Lin, Y. Ma, W. Qu, and T. Chen, “Automated E-FRET microscope for dynamical live-cell FRET imaging,” J. Microsc. 274(1), 45–54 (2019).
[Crossref] [PubMed]

C. Zhang, F. Lin, M. Du, W. Qu, Z. Mai, J. Qu, and T. Chen, “Simultaneous measurement of quantum yield ratio and absorption ratio between acceptor and donor by linearly unmixing excitation-emission spectra,” J. Microsc. 270(3), 335–342 (2018).
[Crossref] [PubMed]

F. Lin, C. Zhang, M. Du, L. Wang, Z. Mai, and T. Chen, “Superior robustness of ExEm-spFRET to IIem-spFRET method in live-cell FRET measurement,” J. Microsc. 272(2), 145–150 (2018).
[Crossref] [PubMed]

Zhang, H.

A. M. Petros, D. G. Nettesheim, Y. Wang, E. T. Olejniczak, R. P. Meadows, J. Mack, K. Swift, E. D. Matayoshi, H. Zhang, C. B. Thompson, and S. W. Fesik, “Rationale for Bcl-xL/Bad peptide complex formation from structure, mutagenesis, and biophysical studies,” Protein Sci. 9(12), 2528–2534 (2000).
[Crossref] [PubMed]

Zhang, L.

Appl. Phys. Lett. (1)

M. Y. Du, F. F. Yang, Z. H. Mai, W. F. Qu, F. R. Lin, L. C. Wei, and T. S. Chen, “FRET two-hybrid assay linearly fitting FRET efficiency to concentration ratio between acceptor and donor,” Appl. Phys. Lett. 112(15), 153702 (2018).
[Crossref]

Biophys. J. (4)

T. Zal and N. R. Gascoigne, “Photobleaching-corrected FRET efficiency imaging of live cells,” Biophys. J. 86(6), 3923–3939 (2004).
[Crossref] [PubMed]

Y. Sun, H. Wallrabe, C. F. Booker, R. N. Day, and A. Periasamy, “Three-color spectral FRET microscopy localizes three interacting proteins in living cells,” Biophys. J. 99(4), 1274–1283 (2010).
[Crossref] [PubMed]

J. Wlodarczyk, A. Woehler, F. Kobe, E. Ponimaskin, A. Zeug, and E. Neher, “Analysis of FRET signals in the presence of free donors and acceptors,” Biophys. J. 94(3), 986–1000 (2008).
[Crossref] [PubMed]

C. King, S. Sarabipour, P. Byrne, D. J. Leahy, and K. Hristova, “The FRET signatures of noninteracting proteins in membranes: simulations and experiments,” Biophys. J. 106(6), 1309–1317 (2014).
[Crossref] [PubMed]

Cell (2)

F. Edlich, S. Banerjee, M. Suzuki, M. M. Cleland, D. Arnoult, C. Wang, A. Neutzner, N. Tjandra, and R. J. Youle, “Bcl-x(L) retrotranslocates Bax from the mitochondria into the cytosol,” Cell 145(1), 104–116 (2011).
[Crossref] [PubMed]

E. Yang, J. Zha, J. Jockel, L. H. Boise, C. B. Thompson, and S. J. Korsmeyer, “Bad, a heterodimeric partner for Bcl-XL and Bcl-2, displaces Bax and promotes cell death,” Cell 80(2), 285–291 (1995).
[Crossref] [PubMed]

Cell Death Differ. (1)

H. Düssmann, M. Rehm, C. G. Concannon, S. Anguissola, M. Würstle, S. Kacmar, P. Völler, H. J. Huber, and J. H. Prehn, “Single-cell quantification of Bax activation and mathematical modelling suggest pore formation on minimal mitochondrial Bax accumulation,” Cell Death Differ. 17(2), 278–290 (2010).
[Crossref] [PubMed]

J. Biol. Chem. (1)

J. Zha, H. Harada, K. Osipov, J. Jockel, G. Waksman, and S. J. Korsmeyer, “BH3 domain of BAD is required for heterodimerization with BCL-XL and pro-apoptotic activity,” J. Biol. Chem. 272(39), 24101–24104 (1997).
[Crossref] [PubMed]

J. Biomed. Opt. (1)

F. R. Lin, M. Y. Du, F. F. Yang, L. C. Wei, and T. S. Chen, “Improved spectrometer-microscope for quantitative FRET measurement based on simultaneous spectral unmixing of excitation and emission spectra,” J. Biomed. Opt. 23(1), 1–10 (2018).
[PubMed]

J. Biophotonics (2)

W. Su, M. Du, F. Lin, C. Zhang, and T. Chen, “Quantitative FRET measurement based on spectral unmixing of donor, acceptor and spontaneous excitation-emission spectra,” J. Biophotonics 12(4), e201800314 (2019).
[Crossref] [PubMed]

G. Heras-Martinez, J. Andrieu, B. Larijani, and J. Requejo-Isidro, “Quantifying intracellular equilibrium dissociation constants using single-channel time-resolved FRET,” J. Biophotonics 11(1), e201600272 (2018).
[Crossref]

J. Cell Biol. (1)

A. J. Valentijn, A. D. Metcalfe, J. Kott, C. H. Streuli, and A. P. Gilmore, “Spatial and temporal changes in Bax subcellular localization during anoikis,” J. Cell Biol. 162(4), 599–612 (2003).
[PubMed]

J. Microsc. (3)

C. Zhang, Y. Liu, H. Sun, F. Lin, Y. Ma, W. Qu, and T. Chen, “Automated E-FRET microscope for dynamical live-cell FRET imaging,” J. Microsc. 274(1), 45–54 (2019).
[Crossref] [PubMed]

C. Zhang, F. Lin, M. Du, W. Qu, Z. Mai, J. Qu, and T. Chen, “Simultaneous measurement of quantum yield ratio and absorption ratio between acceptor and donor by linearly unmixing excitation-emission spectra,” J. Microsc. 270(3), 335–342 (2018).
[Crossref] [PubMed]

F. Lin, C. Zhang, M. Du, L. Wang, Z. Mai, and T. Chen, “Superior robustness of ExEm-spFRET to IIem-spFRET method in live-cell FRET measurement,” J. Microsc. 272(2), 145–150 (2018).
[Crossref] [PubMed]

Mol. Cell (1)

A. Aranovich, Q. Liu, T. Collins, F. Geng, S. Dixit, B. Leber, and D. W. Andrews, “Differences in the mechanisms of proapoptotic BH3 proteins binding to Bcl-XL and Bcl-2 quantified in live MCF-7 cells,” Mol. Cell 45(6), 754–763 (2012).
[Crossref] [PubMed]

Mol. Cell. Biol. (1)

A. Kelekar, B. S. Chang, J. E. Harlan, S. W. Fesik, and C. B. Thompson, “Bad is a BH3 domain-containing protein that forms an inactivating dimer with Bcl-XL,” Mol. Cell. Biol. 17(12), 7040–7046 (1997).
[Crossref] [PubMed]

Nat. Protoc. (1)

E. S. Butz, M. Ben-Johny, M. Shen, P. S. Yang, L. Sang, M. Biel, D. T. Yue, and C. Wahl-Schott, “Quantifying macromolecular interactions in living cells using FRET two-hybrid assays,” Nat. Protoc. 11(12), 2470–2498 (2016).
[Crossref] [PubMed]

Neuron (1)

M. G. Erickson, B. A. Alseikhan, B. Z. Peterson, and D. T. Yue, “Preassociation of calmodulin with voltage-gated Ca(2+) channels revealed by FRET in single living cells,” Neuron 31(6), 973–985 (2001).
[Crossref] [PubMed]

Opt. Express (1)

PLoS One (1)

A. D. Hoppe, B. L. Scott, T. P. Welliver, S. W. Straight, and J. A. Swanson, “N-way FRET microscopy of multiple protein-protein interactions in live cells,” PLoS One 8(6), e64760 (2013).
[Crossref] [PubMed]

Protein Sci. (1)

A. M. Petros, D. G. Nettesheim, Y. Wang, E. T. Olejniczak, R. P. Meadows, J. Mack, K. Swift, E. D. Matayoshi, H. Zhang, C. B. Thompson, and S. W. Fesik, “Rationale for Bcl-xL/Bad peptide complex formation from structure, mutagenesis, and biophysical studies,” Protein Sci. 9(12), 2528–2534 (2000).
[Crossref] [PubMed]

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Figures (7)

Fig. 1
Fig. 1 Illustration of live-cell FRET binding curves. (a) Acceptor-centric FRET efficiency (EA) values measured from single pixels were plotted versus the concentration of free donor (Cd) to form EA-Cd binding curve. The EA values of the pixels with excess donor molecules are extremely close to the value of EA,max (the maximal EA, assuming each acceptor molecule binds to donor). (b) Donor-centric FRET efficiency (ED) values measured from single pixels were plotted versus the concentration of free acceptor (Ca) to form ED-Ca binding curve. The ED values of the pixels with excess acceptor molecules are extremely close to the value of ED,max (the maximal ED, assuming each donor molecule binds to acceptor).
Fig. 2
Fig. 2 Illustration of ExEm-FRET experiments. (a) Diagram of the wide-field automated FRET microscope. A 435/20 nm band-pass excitation filter and a 455 nm dichroic mirror are placed in Cube 435; A 470/20 nm band-pass excitation filter and a 490 nm dichroic mirror are placed in Cube 470. The emission filter wheel contains 470/20 nm, 490/20 nm, 510/20 nm, 530/20 nm, and 550/20 nm band-pass emission filters. (b) An SDA contains five images with 435 nm excitation and three images with 470 nm excitation).
Fig. 3
Fig. 3 Excitation-emission spectral signatures of CFP (SD), YFP (SA) and CFP-YFP sensitisation (SS). (a) Left: emission-spectral images of CFP and YFP with 435 nm excitation and 470 nm excitation, respectively; right: average emission spectra of the pixels of the cells separately expressing CFP and YFP, respectively. (b) Top: excitation spectral images of cells expressing CFP and YFP with 435 nm excitation and 470 nm excitation, respectively; bottom: average excitation spectra of the pixels of the cells separately expressing CFP and YFP, respectively. (c) Pseudo-colour images of SD, SA and SS.
Fig. 4
Fig. 4 Measuring collisional FRET in living Hela cells. (a) A representative excitation-emission spectral image (SDA) of cells co-expressing untagged CFP and untagged YFP. Scale bar: 40 μm. (b) Top: pixel-to-pixel images of WA, WS and WD corresponding to (a); Bottom: the pixel-to-pixel images of CtD, EA, CtA and ED. (c) Pixel-to-pixel EA- CtD plot with a slope of 3.4 × 10−6. (d) Pixel-to-pixel ED- CtA plot with a slope of 3.4 × 10−6.
Fig. 5
Fig. 5 Stoichiometric ratio of Bad binding to Bcl-XL in healthy cells. (a) An SDA of living cells co-expressing Bad-YFP and CFP-Bcl-XL and the corresponding WA, WD and WS images. Scale bar: 40 μm. (b) The corresponding pixel-to-pixel images of EA, CtD ,CtA and ED. (c) Left: pixel-to-pixel EA-Cd plot with an EA,max value of 0.364; Right: pixel-to-pixel ED-Ca plot with a ED,max value of 0.274.
Fig. 6
Fig. 6 Stoichiometric ratio of Bad binding to Bcl-XL in apoptotic cells treated with 1 μM STS for 7 h. (a) A SDA of apoptotic cells co-expressing Bad-YFP and CFP-Bcl-XL and the corresponding WA, WD and WS images. Scale bar: 40 μm. (b) The corresponding pixel-to-pixel images of EA, CtD, CtA and ED. (c) Left: pixel-to-pixel EA-Cd plot with an EA,max value of 0.377; Right: pixel-to-pixel ED-Ca plot with a ED,max value of 0.202.
Fig. 7
Fig. 7 Stoichiometric ratio of Bad binding to Bcl-XL in the cell indicated by a red box. (a) An SDA of the cell indicated by the red box. (b) Left: pixel-to-pixel EA-Cd plot of the cell indicated by red box in (a) with an EA,max value of 0.381; Right: pixel-to-pixel ED-Ca plot with a ED,max value of 0.307.

Equations (21)

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S DA =( C d K D Q D + C da K D (1E) Q D ) S D + C da K D E Q A S S + C A t K A Q A S A = W D S D + W S S S + W A S A ,
C A t = 1 K A Q A W A ,
C D t = C d + C da = 1 K D Q D ( W D + Q D Q A W S ),
E A =E C da C A t = K A K D W S W A ,
E D =E C da C D t = W S W S + Q A Q D W D .
n D D+ n A A K d D n D A n A ,
K d = C d C a C DA ,
C A t = C a + C DA ,
C D t = C d + n D C DA .
C d 2 +( K d + n D C A t C D t ) C d K d C D t =0.
C d = b+ b 2 +4c 2 ,
b= 1 K D Q D ( W D + Q D Q A W S ) n D 1 K A Q A W A K d ,
c= K d 1 K D Q D ( W D + Q D Q A W S ).
C a = C A t C D t C d n D = 1 K A Q A W A 1 n D [ 1 K D Q D ( W D + Q D Q A W S ) C d ].
n D n A = E A,max E D,max .
E A,spurious =Slop e spuriousFRET C D t =Slop e spuriousFRET 1 K D Q D ( W D + Q D Q A W S ),
E D,spurious =Slop e spuriousFRET C A t =Slop e spuriousFRET 1 K A Q A W A .
E A,corrected = E A Slop e spuriousFRET C D t ,
E D,corrected = E D Slop e spuriousFRET C A t .
E A,pred = E A,max C DA C A t = E A,max C d C d + K d ,
E D,pred = E D,max n D C DA C D t = E D,max C a C a + K d 1 n D .

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