Abstract

Simultaneous spectral unmixing of excitation and emission spectra (ExEm unmixing) has the inherent ability to resolve donor emission, fluorescence resonance energy transfer (FRET)-sensitized acceptor emission and directly excited acceptor emission. We here develop an ExEm unmixing-based quantitative FRET measurement method (EES-FRET) independent of excitation intensity and detector parameter setting. The ratio factor (rK), predetermined using a donor-acceptor tandem construct, of total acceptor absorption to total donor absorption in excitation wavelengths used is introduced for determining the concentration ratio of acceptor to donor. We implemented EES-FRET method on a wide-field microscope to image living cells expressing tandem FRET constructs with different donor-acceptor stoichiometry.

© 2016 Optical Society of America

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References

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  3. S. Sarabipour and K. Hristova, “Mechanism of FGF receptor dimerization and activation,” Nat. Commun. 7, 10262 (2016).
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  4. M. Waldeck-Weiermair, R. Malli, W. Parichatikanond, B. Gottschalk, C. T. Madreiter-Sokolowski, C. Klec, R. Rost, and W. F. Graier, “Rearrangement of MICU1 multimers for activation of MCU is solely controlled by cytosolic Ca(2.),” Sci. Rep. 5, 15602 (2015).
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  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).
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  8. M. A. Rizzo, G. Springer, K. Segawa, W. R. Zipfel, and D. W. Piston, “Optimization of pairings and detection conditions for measurement of FRET between cyan and yellow fluorescent proteins,” Microsc. Microanal. 12(3), 238–254 (2006).
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  13. S. Levy, C. D. Wilms, E. Brumer, J. Kahn, L. Pnueli, Y. Arava, J. Eilers, and D. Gitler, “SpRET: highly sensitive and reliable spectral measurement of absolute FRET efficiency,” Microsc. Microanal. 17(2), 176–190 (2011).
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  14. C. Dinant, M. E. van Royen, W. Vermeulen, and A. B. Houtsmuller, “Fluorescence resonance energy transfer of GFP and YFP by spectral imaging and quantitative acceptor photobleaching,” J. Microsc. 231(1), 97–104 (2008).
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  17. J. Yuan, L. Peng, B. E. Bouma, and G. J. Tearney, “Quantitative FRET measurement by high-speed fluorescence excitation and emission spectrometer,” Opt. Express 18(18), 18839–18851 (2010).
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    [Crossref] [PubMed]
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    [Crossref] [PubMed]
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    [Crossref] [PubMed]
  23. L. Zhang, G. Qin, L. Chai, J. Zhang, F. Yang, H. Yang, S. Xie, and T. Chen, “Spectral wide-field microscopic fluorescence resonance energy transfer imaging in live cells,” J. Biomed. Opt. 20(8), 086011 (2015).
    [Crossref] [PubMed]
  24. L. Zhang, H. Yu, J. Zhang, and T. Chen, “Binomial distribution-based quantitative measurement of multiple-acceptors fluorescence resonance energy transfer by partially photobleaching acceptor,” Appl. Phys. Lett. 104(24), 243706 (2014).
    [Crossref]
  25. T. Zal and N. R. J. Gascoigne, “Photobleaching-corrected FRET efficiency imaging of live cells,” Biophys. J. 86(6), 3923–3939 (2004).
    [Crossref] [PubMed]
  26. H. Chen, H. L. Puhl, S. V. Koushik, S. S. Vogel, and S. R. Ikeda, “Measurement of FRET efficiency and ratio of donor to acceptor concentration in living cells,” Biophys. J. 91(5), L39–L41 (2006).
    [Crossref] [PubMed]
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    [Crossref] [PubMed]
  28. J. R. Lakowicz, Principles of Fluorescence Spectroscopy (Springer, 2006), Chap. 2.
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    [Crossref] [PubMed]
  30. M. A. Rizzo, G. H. Springer, B. Granada, and D. W. Piston, “An improved cyan fluorescent protein variant useful for FRET,” Nat. Biotechnol. 22(4), 445–449 (2004).
    [Crossref] [PubMed]
  31. T. Nagai, K. Ibata, E. S. Park, M. Kubota, K. Mikoshiba, and A. Miyawaki, “A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications,” Nat. Biotechnol. 20(1), 87–90 (2002).
    [Crossref] [PubMed]

2016 (1)

S. Sarabipour and K. Hristova, “Mechanism of FGF receptor dimerization and activation,” Nat. Commun. 7, 10262 (2016).
[Crossref] [PubMed]

2015 (4)

M. Waldeck-Weiermair, R. Malli, W. Parichatikanond, B. Gottschalk, C. T. Madreiter-Sokolowski, C. Klec, R. Rost, and W. F. Graier, “Rearrangement of MICU1 multimers for activation of MCU is solely controlled by cytosolic Ca(2.),” Sci. Rep. 5, 15602 (2015).
[Crossref] [PubMed]

J. Zhang, H. Li, L. Chai, L. Zhang, J. Qu, and T. Chen, “Quantitative FRET measurement using emission-spectral unmixing with independent excitation crosstalk correction,” J. Microsc. 257(2), 104–116 (2015).
[Crossref] [PubMed]

L. Chai, J. Zhang, L. Zhang, and T. Chen, “Miniature fiber optic spectrometer-based quantitative fluorescence resonance energy transfer measurement in single living cells,” J. Biomed. Opt. 20(3), 037008 (2015).
[Crossref] [PubMed]

L. Zhang, G. Qin, L. Chai, J. Zhang, F. Yang, H. Yang, S. Xie, and T. Chen, “Spectral wide-field microscopic fluorescence resonance energy transfer imaging in live cells,” J. Biomed. Opt. 20(8), 086011 (2015).
[Crossref] [PubMed]

2014 (1)

L. Zhang, H. Yu, J. Zhang, and T. Chen, “Binomial distribution-based quantitative measurement of multiple-acceptors fluorescence resonance energy transfer by partially photobleaching acceptor,” Appl. Phys. Lett. 104(24), 243706 (2014).
[Crossref]

2013 (3)

S. Mustafa, J. Hannagan, P. Rigby, K. Pfleger, and B. Corry, “Quantitative Förster resonance energy transfer efficiency measurements using simultaneous spectral unmixing of excitation and emission spectra,” J. Biomed. Opt. 18(2), 026024 (2013).
[Crossref] [PubMed]

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]

A. Woehler, “Simultaneous quantitative live cell imaging of multiple FRET-based biosensors,” PLoS One 8(4), e61096 (2013).
[Crossref] [PubMed]

2011 (1)

S. Levy, C. D. Wilms, E. Brumer, J. Kahn, L. Pnueli, Y. Arava, J. Eilers, and D. Gitler, “SpRET: highly sensitive and reliable spectral measurement of absolute FRET efficiency,” Microsc. Microanal. 17(2), 176–190 (2011).
[Crossref] [PubMed]

2010 (4)

J. Yuan, L. Peng, B. E. Bouma, and G. J. Tearney, “Quantitative FRET measurement by high-speed fluorescence excitation and emission spectrometer,” Opt. Express 18(18), 18839–18851 (2010).
[Crossref] [PubMed]

H. Düssmann, M. Rehm, C. G. Concannon, S. Anguissola, M. Würstle, S. Kacmar, P. Völler, H. J. Huber, and J. H. M. 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]

H. Yu, T. Chen, and J. Qu, “Improving FRET efficiency measurement in confocal microscopy imaging,” Chin. Opt. Lett. 8(10), 947–949 (2010).
[Crossref]

L. Wang, T. Chen, J. Qu, and X. Wei, “Photobleaching-based quantitative analysis of fluorescence resonance energy transfer inside single living cell,” J. Fluoresc. 20(1), 27–35 (2010).
[Crossref] [PubMed]

2009 (3)

S. V. Koushik, P. S. Blank, and S. S. Vogel, “Anomalous surplus energy transfer observed with multiple FRET acceptors,” PLoS One 4(11), e8031 (2009).
[Crossref] [PubMed]

Y. Zhang, D. Xing, and L. Liu, “PUMA promotes Bax translocation by both directly interacting with Bax and by competitive binding to Bcl-X L during UV-induced apoptosis,” Mol. Biol. Cell 20(13), 3077–3087 (2009).
[Crossref] [PubMed]

D. Megías, R. Marrero, B. Martínez Del Peso, M. Á. García, J. J. Bravo-Cordero, A. García-Grande, A. Santos, and M. C. Montoya, “Novel lambda FRET spectral confocal microscopy imaging method,” Microsc. Res. Tech. 72(1), 1–11 (2009).
[Crossref] [PubMed]

2008 (2)

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. Dinant, M. E. van Royen, W. Vermeulen, and A. B. Houtsmuller, “Fluorescence resonance energy transfer of GFP and YFP by spectral imaging and quantitative acceptor photobleaching,” J. Microsc. 231(1), 97–104 (2008).
[Crossref] [PubMed]

2007 (1)

Y. Chen, J. P. Mauldin, R. N. Day, and A. Periasamy, “Characterization of spectral FRET imaging microscopy for monitoring nuclear protein interactions,” J. Microsc. 228(2), 139–152 (2007).
[Crossref] [PubMed]

2006 (2)

H. Chen, H. L. Puhl, S. V. Koushik, S. S. Vogel, and S. R. Ikeda, “Measurement of FRET efficiency and ratio of donor to acceptor concentration in living cells,” Biophys. J. 91(5), L39–L41 (2006).
[Crossref] [PubMed]

M. A. Rizzo, G. Springer, K. Segawa, W. R. Zipfel, and D. W. Piston, “Optimization of pairings and detection conditions for measurement of FRET between cyan and yellow fluorescent proteins,” Microsc. Microanal. 12(3), 238–254 (2006).
[Crossref] [PubMed]

2005 (1)

C. Thaler, S. V. Koushik, P. S. Blank, and S. S. Vogel, “Quantitative multiphoton spectral imaging and its use for measuring resonance energy transfer,” Biophys. J. 89(4), 2736–2749 (2005).
[Crossref] [PubMed]

2004 (4)

Y. Gu, W. L. Di, D. P. Kelsell, and D. Zicha, “Quantitative fluorescence resonance energy transfer (FRET) measurement with acceptor photobleaching and spectral unmixing,” J. Microsc. 215(2), 162–173 (2004).
[Crossref] [PubMed]

E. Galperin, V. V. Verkhusha, and A. Sorkin, “Three-chromophore FRET microscopy to analyze multiprotein interactions in living cells,” Nat. Methods 1(3), 209–217 (2004).
[Crossref] [PubMed]

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

M. A. Rizzo, G. H. Springer, B. Granada, and D. W. Piston, “An improved cyan fluorescent protein variant useful for FRET,” Nat. Biotechnol. 22(4), 445–449 (2004).
[Crossref] [PubMed]

2003 (1)

M. Elangovan, H. Wallrabe, Y. Chen, R. N. Day, M. Barroso, and A. Periasamy, “Characterization of one- and two-photon excitation fluorescence resonance energy transfer microscopy,” Methods 29(1), 58–73 (2003).
[Crossref] [PubMed]

2002 (1)

T. Nagai, K. Ibata, E. S. Park, M. Kubota, K. Mikoshiba, and A. Miyawaki, “A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications,” Nat. Biotechnol. 20(1), 87–90 (2002).
[Crossref] [PubMed]

1992 (1)

R. M. Clegg, “Fluorescence resonance energy transfer and nucleic acids,” Methods Enzymol. 211, 353–388 (1992).
[Crossref] [PubMed]

Anguissola, S.

H. Düssmann, M. Rehm, C. G. Concannon, S. Anguissola, M. Würstle, S. Kacmar, P. Völler, H. J. Huber, and J. H. M. 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]

Arava, Y.

S. Levy, C. D. Wilms, E. Brumer, J. Kahn, L. Pnueli, Y. Arava, J. Eilers, and D. Gitler, “SpRET: highly sensitive and reliable spectral measurement of absolute FRET efficiency,” Microsc. Microanal. 17(2), 176–190 (2011).
[Crossref] [PubMed]

Barroso, M.

M. Elangovan, H. Wallrabe, Y. Chen, R. N. Day, M. Barroso, and A. Periasamy, “Characterization of one- and two-photon excitation fluorescence resonance energy transfer microscopy,” Methods 29(1), 58–73 (2003).
[Crossref] [PubMed]

Blank, P. S.

S. V. Koushik, P. S. Blank, and S. S. Vogel, “Anomalous surplus energy transfer observed with multiple FRET acceptors,” PLoS One 4(11), e8031 (2009).
[Crossref] [PubMed]

C. Thaler, S. V. Koushik, P. S. Blank, and S. S. Vogel, “Quantitative multiphoton spectral imaging and its use for measuring resonance energy transfer,” Biophys. J. 89(4), 2736–2749 (2005).
[Crossref] [PubMed]

Bouma, B. E.

Bravo-Cordero, J. J.

D. Megías, R. Marrero, B. Martínez Del Peso, M. Á. García, J. J. Bravo-Cordero, A. García-Grande, A. Santos, and M. C. Montoya, “Novel lambda FRET spectral confocal microscopy imaging method,” Microsc. Res. Tech. 72(1), 1–11 (2009).
[Crossref] [PubMed]

Brumer, E.

S. Levy, C. D. Wilms, E. Brumer, J. Kahn, L. Pnueli, Y. Arava, J. Eilers, and D. Gitler, “SpRET: highly sensitive and reliable spectral measurement of absolute FRET efficiency,” Microsc. Microanal. 17(2), 176–190 (2011).
[Crossref] [PubMed]

Chai, L.

J. Zhang, H. Li, L. Chai, L. Zhang, J. Qu, and T. Chen, “Quantitative FRET measurement using emission-spectral unmixing with independent excitation crosstalk correction,” J. Microsc. 257(2), 104–116 (2015).
[Crossref] [PubMed]

L. Chai, J. Zhang, L. Zhang, and T. Chen, “Miniature fiber optic spectrometer-based quantitative fluorescence resonance energy transfer measurement in single living cells,” J. Biomed. Opt. 20(3), 037008 (2015).
[Crossref] [PubMed]

L. Zhang, G. Qin, L. Chai, J. Zhang, F. Yang, H. Yang, S. Xie, and T. Chen, “Spectral wide-field microscopic fluorescence resonance energy transfer imaging in live cells,” J. Biomed. Opt. 20(8), 086011 (2015).
[Crossref] [PubMed]

Chen, H.

H. Chen, H. L. Puhl, S. V. Koushik, S. S. Vogel, and S. R. Ikeda, “Measurement of FRET efficiency and ratio of donor to acceptor concentration in living cells,” Biophys. J. 91(5), L39–L41 (2006).
[Crossref] [PubMed]

Chen, T.

L. Zhang, G. Qin, L. Chai, J. Zhang, F. Yang, H. Yang, S. Xie, and T. Chen, “Spectral wide-field microscopic fluorescence resonance energy transfer imaging in live cells,” J. Biomed. Opt. 20(8), 086011 (2015).
[Crossref] [PubMed]

L. Chai, J. Zhang, L. Zhang, and T. Chen, “Miniature fiber optic spectrometer-based quantitative fluorescence resonance energy transfer measurement in single living cells,” J. Biomed. Opt. 20(3), 037008 (2015).
[Crossref] [PubMed]

J. Zhang, H. Li, L. Chai, L. Zhang, J. Qu, and T. Chen, “Quantitative FRET measurement using emission-spectral unmixing with independent excitation crosstalk correction,” J. Microsc. 257(2), 104–116 (2015).
[Crossref] [PubMed]

L. Zhang, H. Yu, J. Zhang, and T. Chen, “Binomial distribution-based quantitative measurement of multiple-acceptors fluorescence resonance energy transfer by partially photobleaching acceptor,” Appl. Phys. Lett. 104(24), 243706 (2014).
[Crossref]

L. Wang, T. Chen, J. Qu, and X. Wei, “Photobleaching-based quantitative analysis of fluorescence resonance energy transfer inside single living cell,” J. Fluoresc. 20(1), 27–35 (2010).
[Crossref] [PubMed]

H. Yu, T. Chen, and J. Qu, “Improving FRET efficiency measurement in confocal microscopy imaging,” Chin. Opt. Lett. 8(10), 947–949 (2010).
[Crossref]

Chen, Y.

Y. Chen, J. P. Mauldin, R. N. Day, and A. Periasamy, “Characterization of spectral FRET imaging microscopy for monitoring nuclear protein interactions,” J. Microsc. 228(2), 139–152 (2007).
[Crossref] [PubMed]

M. Elangovan, H. Wallrabe, Y. Chen, R. N. Day, M. Barroso, and A. Periasamy, “Characterization of one- and two-photon excitation fluorescence resonance energy transfer microscopy,” Methods 29(1), 58–73 (2003).
[Crossref] [PubMed]

Clegg, R. M.

R. M. Clegg, “Fluorescence resonance energy transfer and nucleic acids,” Methods Enzymol. 211, 353–388 (1992).
[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. M. 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]

Corry, B.

S. Mustafa, J. Hannagan, P. Rigby, K. Pfleger, and B. Corry, “Quantitative Förster resonance energy transfer efficiency measurements using simultaneous spectral unmixing of excitation and emission spectra,” J. Biomed. Opt. 18(2), 026024 (2013).
[Crossref] [PubMed]

Day, R. N.

Y. Chen, J. P. Mauldin, R. N. Day, and A. Periasamy, “Characterization of spectral FRET imaging microscopy for monitoring nuclear protein interactions,” J. Microsc. 228(2), 139–152 (2007).
[Crossref] [PubMed]

M. Elangovan, H. Wallrabe, Y. Chen, R. N. Day, M. Barroso, and A. Periasamy, “Characterization of one- and two-photon excitation fluorescence resonance energy transfer microscopy,” Methods 29(1), 58–73 (2003).
[Crossref] [PubMed]

Di, W. L.

Y. Gu, W. L. Di, D. P. Kelsell, and D. Zicha, “Quantitative fluorescence resonance energy transfer (FRET) measurement with acceptor photobleaching and spectral unmixing,” J. Microsc. 215(2), 162–173 (2004).
[Crossref] [PubMed]

Dinant, C.

C. Dinant, M. E. van Royen, W. Vermeulen, and A. B. Houtsmuller, “Fluorescence resonance energy transfer of GFP and YFP by spectral imaging and quantitative acceptor photobleaching,” J. Microsc. 231(1), 97–104 (2008).
[Crossref] [PubMed]

Düssmann, H.

H. Düssmann, M. Rehm, C. G. Concannon, S. Anguissola, M. Würstle, S. Kacmar, P. Völler, H. J. Huber, and J. H. M. 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]

Eilers, J.

S. Levy, C. D. Wilms, E. Brumer, J. Kahn, L. Pnueli, Y. Arava, J. Eilers, and D. Gitler, “SpRET: highly sensitive and reliable spectral measurement of absolute FRET efficiency,” Microsc. Microanal. 17(2), 176–190 (2011).
[Crossref] [PubMed]

Elangovan, M.

M. Elangovan, H. Wallrabe, Y. Chen, R. N. Day, M. Barroso, and A. Periasamy, “Characterization of one- and two-photon excitation fluorescence resonance energy transfer microscopy,” Methods 29(1), 58–73 (2003).
[Crossref] [PubMed]

Galperin, E.

E. Galperin, V. V. Verkhusha, and A. Sorkin, “Three-chromophore FRET microscopy to analyze multiprotein interactions in living cells,” Nat. Methods 1(3), 209–217 (2004).
[Crossref] [PubMed]

García, M. Á.

D. Megías, R. Marrero, B. Martínez Del Peso, M. Á. García, J. J. Bravo-Cordero, A. García-Grande, A. Santos, and M. C. Montoya, “Novel lambda FRET spectral confocal microscopy imaging method,” Microsc. Res. Tech. 72(1), 1–11 (2009).
[Crossref] [PubMed]

García-Grande, A.

D. Megías, R. Marrero, B. Martínez Del Peso, M. Á. García, J. J. Bravo-Cordero, A. García-Grande, A. Santos, and M. C. Montoya, “Novel lambda FRET spectral confocal microscopy imaging method,” Microsc. Res. Tech. 72(1), 1–11 (2009).
[Crossref] [PubMed]

Gascoigne, N. R. J.

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

Gitler, D.

S. Levy, C. D. Wilms, E. Brumer, J. Kahn, L. Pnueli, Y. Arava, J. Eilers, and D. Gitler, “SpRET: highly sensitive and reliable spectral measurement of absolute FRET efficiency,” Microsc. Microanal. 17(2), 176–190 (2011).
[Crossref] [PubMed]

Gottschalk, B.

M. Waldeck-Weiermair, R. Malli, W. Parichatikanond, B. Gottschalk, C. T. Madreiter-Sokolowski, C. Klec, R. Rost, and W. F. Graier, “Rearrangement of MICU1 multimers for activation of MCU is solely controlled by cytosolic Ca(2.),” Sci. Rep. 5, 15602 (2015).
[Crossref] [PubMed]

Graier, W. F.

M. Waldeck-Weiermair, R. Malli, W. Parichatikanond, B. Gottschalk, C. T. Madreiter-Sokolowski, C. Klec, R. Rost, and W. F. Graier, “Rearrangement of MICU1 multimers for activation of MCU is solely controlled by cytosolic Ca(2.),” Sci. Rep. 5, 15602 (2015).
[Crossref] [PubMed]

Granada, B.

M. A. Rizzo, G. H. Springer, B. Granada, and D. W. Piston, “An improved cyan fluorescent protein variant useful for FRET,” Nat. Biotechnol. 22(4), 445–449 (2004).
[Crossref] [PubMed]

Gu, Y.

Y. Gu, W. L. Di, D. P. Kelsell, and D. Zicha, “Quantitative fluorescence resonance energy transfer (FRET) measurement with acceptor photobleaching and spectral unmixing,” J. Microsc. 215(2), 162–173 (2004).
[Crossref] [PubMed]

Hannagan, J.

S. Mustafa, J. Hannagan, P. Rigby, K. Pfleger, and B. Corry, “Quantitative Förster resonance energy transfer efficiency measurements using simultaneous spectral unmixing of excitation and emission spectra,” J. Biomed. Opt. 18(2), 026024 (2013).
[Crossref] [PubMed]

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]

Houtsmuller, A. B.

C. Dinant, M. E. van Royen, W. Vermeulen, and A. B. Houtsmuller, “Fluorescence resonance energy transfer of GFP and YFP by spectral imaging and quantitative acceptor photobleaching,” J. Microsc. 231(1), 97–104 (2008).
[Crossref] [PubMed]

Hristova, K.

S. Sarabipour and K. Hristova, “Mechanism of FGF receptor dimerization and activation,” Nat. Commun. 7, 10262 (2016).
[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. M. 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]

Ibata, K.

T. Nagai, K. Ibata, E. S. Park, M. Kubota, K. Mikoshiba, and A. Miyawaki, “A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications,” Nat. Biotechnol. 20(1), 87–90 (2002).
[Crossref] [PubMed]

Ikeda, S. R.

H. Chen, H. L. Puhl, S. V. Koushik, S. S. Vogel, and S. R. Ikeda, “Measurement of FRET efficiency and ratio of donor to acceptor concentration in living cells,” Biophys. J. 91(5), L39–L41 (2006).
[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. M. 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]

Kahn, J.

S. Levy, C. D. Wilms, E. Brumer, J. Kahn, L. Pnueli, Y. Arava, J. Eilers, and D. Gitler, “SpRET: highly sensitive and reliable spectral measurement of absolute FRET efficiency,” Microsc. Microanal. 17(2), 176–190 (2011).
[Crossref] [PubMed]

Kelsell, D. P.

Y. Gu, W. L. Di, D. P. Kelsell, and D. Zicha, “Quantitative fluorescence resonance energy transfer (FRET) measurement with acceptor photobleaching and spectral unmixing,” J. Microsc. 215(2), 162–173 (2004).
[Crossref] [PubMed]

Klec, C.

M. Waldeck-Weiermair, R. Malli, W. Parichatikanond, B. Gottschalk, C. T. Madreiter-Sokolowski, C. Klec, R. Rost, and W. F. Graier, “Rearrangement of MICU1 multimers for activation of MCU is solely controlled by cytosolic Ca(2.),” Sci. Rep. 5, 15602 (2015).
[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]

Koushik, S. V.

S. V. Koushik, P. S. Blank, and S. S. Vogel, “Anomalous surplus energy transfer observed with multiple FRET acceptors,” PLoS One 4(11), e8031 (2009).
[Crossref] [PubMed]

H. Chen, H. L. Puhl, S. V. Koushik, S. S. Vogel, and S. R. Ikeda, “Measurement of FRET efficiency and ratio of donor to acceptor concentration in living cells,” Biophys. J. 91(5), L39–L41 (2006).
[Crossref] [PubMed]

C. Thaler, S. V. Koushik, P. S. Blank, and S. S. Vogel, “Quantitative multiphoton spectral imaging and its use for measuring resonance energy transfer,” Biophys. J. 89(4), 2736–2749 (2005).
[Crossref] [PubMed]

Kubota, M.

T. Nagai, K. Ibata, E. S. Park, M. Kubota, K. Mikoshiba, and A. Miyawaki, “A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications,” Nat. Biotechnol. 20(1), 87–90 (2002).
[Crossref] [PubMed]

Levy, S.

S. Levy, C. D. Wilms, E. Brumer, J. Kahn, L. Pnueli, Y. Arava, J. Eilers, and D. Gitler, “SpRET: highly sensitive and reliable spectral measurement of absolute FRET efficiency,” Microsc. Microanal. 17(2), 176–190 (2011).
[Crossref] [PubMed]

Li, H.

J. Zhang, H. Li, L. Chai, L. Zhang, J. Qu, and T. Chen, “Quantitative FRET measurement using emission-spectral unmixing with independent excitation crosstalk correction,” J. Microsc. 257(2), 104–116 (2015).
[Crossref] [PubMed]

Liu, L.

Y. Zhang, D. Xing, and L. Liu, “PUMA promotes Bax translocation by both directly interacting with Bax and by competitive binding to Bcl-X L during UV-induced apoptosis,” Mol. Biol. Cell 20(13), 3077–3087 (2009).
[Crossref] [PubMed]

Madreiter-Sokolowski, C. T.

M. Waldeck-Weiermair, R. Malli, W. Parichatikanond, B. Gottschalk, C. T. Madreiter-Sokolowski, C. Klec, R. Rost, and W. F. Graier, “Rearrangement of MICU1 multimers for activation of MCU is solely controlled by cytosolic Ca(2.),” Sci. Rep. 5, 15602 (2015).
[Crossref] [PubMed]

Malli, R.

M. Waldeck-Weiermair, R. Malli, W. Parichatikanond, B. Gottschalk, C. T. Madreiter-Sokolowski, C. Klec, R. Rost, and W. F. Graier, “Rearrangement of MICU1 multimers for activation of MCU is solely controlled by cytosolic Ca(2.),” Sci. Rep. 5, 15602 (2015).
[Crossref] [PubMed]

Marrero, R.

D. Megías, R. Marrero, B. Martínez Del Peso, M. Á. García, J. J. Bravo-Cordero, A. García-Grande, A. Santos, and M. C. Montoya, “Novel lambda FRET spectral confocal microscopy imaging method,” Microsc. Res. Tech. 72(1), 1–11 (2009).
[Crossref] [PubMed]

Martínez Del Peso, B.

D. Megías, R. Marrero, B. Martínez Del Peso, M. Á. García, J. J. Bravo-Cordero, A. García-Grande, A. Santos, and M. C. Montoya, “Novel lambda FRET spectral confocal microscopy imaging method,” Microsc. Res. Tech. 72(1), 1–11 (2009).
[Crossref] [PubMed]

Mauldin, J. P.

Y. Chen, J. P. Mauldin, R. N. Day, and A. Periasamy, “Characterization of spectral FRET imaging microscopy for monitoring nuclear protein interactions,” J. Microsc. 228(2), 139–152 (2007).
[Crossref] [PubMed]

Megías, D.

D. Megías, R. Marrero, B. Martínez Del Peso, M. Á. García, J. J. Bravo-Cordero, A. García-Grande, A. Santos, and M. C. Montoya, “Novel lambda FRET spectral confocal microscopy imaging method,” Microsc. Res. Tech. 72(1), 1–11 (2009).
[Crossref] [PubMed]

Mikoshiba, K.

T. Nagai, K. Ibata, E. S. Park, M. Kubota, K. Mikoshiba, and A. Miyawaki, “A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications,” Nat. Biotechnol. 20(1), 87–90 (2002).
[Crossref] [PubMed]

Miyawaki, A.

T. Nagai, K. Ibata, E. S. Park, M. Kubota, K. Mikoshiba, and A. Miyawaki, “A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications,” Nat. Biotechnol. 20(1), 87–90 (2002).
[Crossref] [PubMed]

Montoya, M. C.

D. Megías, R. Marrero, B. Martínez Del Peso, M. Á. García, J. J. Bravo-Cordero, A. García-Grande, A. Santos, and M. C. Montoya, “Novel lambda FRET spectral confocal microscopy imaging method,” Microsc. Res. Tech. 72(1), 1–11 (2009).
[Crossref] [PubMed]

Mustafa, S.

S. Mustafa, J. Hannagan, P. Rigby, K. Pfleger, and B. Corry, “Quantitative Förster resonance energy transfer efficiency measurements using simultaneous spectral unmixing of excitation and emission spectra,” J. Biomed. Opt. 18(2), 026024 (2013).
[Crossref] [PubMed]

Nagai, T.

T. Nagai, K. Ibata, E. S. Park, M. Kubota, K. Mikoshiba, and A. Miyawaki, “A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications,” Nat. Biotechnol. 20(1), 87–90 (2002).
[Crossref] [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]

Parichatikanond, W.

M. Waldeck-Weiermair, R. Malli, W. Parichatikanond, B. Gottschalk, C. T. Madreiter-Sokolowski, C. Klec, R. Rost, and W. F. Graier, “Rearrangement of MICU1 multimers for activation of MCU is solely controlled by cytosolic Ca(2.),” Sci. Rep. 5, 15602 (2015).
[Crossref] [PubMed]

Park, E. S.

T. Nagai, K. Ibata, E. S. Park, M. Kubota, K. Mikoshiba, and A. Miyawaki, “A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications,” Nat. Biotechnol. 20(1), 87–90 (2002).
[Crossref] [PubMed]

Peng, L.

Periasamy, A.

Y. Chen, J. P. Mauldin, R. N. Day, and A. Periasamy, “Characterization of spectral FRET imaging microscopy for monitoring nuclear protein interactions,” J. Microsc. 228(2), 139–152 (2007).
[Crossref] [PubMed]

M. Elangovan, H. Wallrabe, Y. Chen, R. N. Day, M. Barroso, and A. Periasamy, “Characterization of one- and two-photon excitation fluorescence resonance energy transfer microscopy,” Methods 29(1), 58–73 (2003).
[Crossref] [PubMed]

Pfleger, K.

S. Mustafa, J. Hannagan, P. Rigby, K. Pfleger, and B. Corry, “Quantitative Förster resonance energy transfer efficiency measurements using simultaneous spectral unmixing of excitation and emission spectra,” J. Biomed. Opt. 18(2), 026024 (2013).
[Crossref] [PubMed]

Piston, D. W.

M. A. Rizzo, G. Springer, K. Segawa, W. R. Zipfel, and D. W. Piston, “Optimization of pairings and detection conditions for measurement of FRET between cyan and yellow fluorescent proteins,” Microsc. Microanal. 12(3), 238–254 (2006).
[Crossref] [PubMed]

M. A. Rizzo, G. H. Springer, B. Granada, and D. W. Piston, “An improved cyan fluorescent protein variant useful for FRET,” Nat. Biotechnol. 22(4), 445–449 (2004).
[Crossref] [PubMed]

Pnueli, L.

S. Levy, C. D. Wilms, E. Brumer, J. Kahn, L. Pnueli, Y. Arava, J. Eilers, and D. Gitler, “SpRET: highly sensitive and reliable spectral measurement of absolute FRET efficiency,” Microsc. Microanal. 17(2), 176–190 (2011).
[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. M.

H. Düssmann, M. Rehm, C. G. Concannon, S. Anguissola, M. Würstle, S. Kacmar, P. Völler, H. J. Huber, and J. H. M. 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]

Puhl, H. L.

H. Chen, H. L. Puhl, S. V. Koushik, S. S. Vogel, and S. R. Ikeda, “Measurement of FRET efficiency and ratio of donor to acceptor concentration in living cells,” Biophys. J. 91(5), L39–L41 (2006).
[Crossref] [PubMed]

Qin, G.

L. Zhang, G. Qin, L. Chai, J. Zhang, F. Yang, H. Yang, S. Xie, and T. Chen, “Spectral wide-field microscopic fluorescence resonance energy transfer imaging in live cells,” J. Biomed. Opt. 20(8), 086011 (2015).
[Crossref] [PubMed]

Qu, J.

J. Zhang, H. Li, L. Chai, L. Zhang, J. Qu, and T. Chen, “Quantitative FRET measurement using emission-spectral unmixing with independent excitation crosstalk correction,” J. Microsc. 257(2), 104–116 (2015).
[Crossref] [PubMed]

H. Yu, T. Chen, and J. Qu, “Improving FRET efficiency measurement in confocal microscopy imaging,” Chin. Opt. Lett. 8(10), 947–949 (2010).
[Crossref]

L. Wang, T. Chen, J. Qu, and X. Wei, “Photobleaching-based quantitative analysis of fluorescence resonance energy transfer inside single living cell,” J. Fluoresc. 20(1), 27–35 (2010).
[Crossref] [PubMed]

Rehm, M.

H. Düssmann, M. Rehm, C. G. Concannon, S. Anguissola, M. Würstle, S. Kacmar, P. Völler, H. J. Huber, and J. H. M. 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]

Rigby, P.

S. Mustafa, J. Hannagan, P. Rigby, K. Pfleger, and B. Corry, “Quantitative Förster resonance energy transfer efficiency measurements using simultaneous spectral unmixing of excitation and emission spectra,” J. Biomed. Opt. 18(2), 026024 (2013).
[Crossref] [PubMed]

Rizzo, M. A.

M. A. Rizzo, G. Springer, K. Segawa, W. R. Zipfel, and D. W. Piston, “Optimization of pairings and detection conditions for measurement of FRET between cyan and yellow fluorescent proteins,” Microsc. Microanal. 12(3), 238–254 (2006).
[Crossref] [PubMed]

M. A. Rizzo, G. H. Springer, B. Granada, and D. W. Piston, “An improved cyan fluorescent protein variant useful for FRET,” Nat. Biotechnol. 22(4), 445–449 (2004).
[Crossref] [PubMed]

Rost, R.

M. Waldeck-Weiermair, R. Malli, W. Parichatikanond, B. Gottschalk, C. T. Madreiter-Sokolowski, C. Klec, R. Rost, and W. F. Graier, “Rearrangement of MICU1 multimers for activation of MCU is solely controlled by cytosolic Ca(2.),” Sci. Rep. 5, 15602 (2015).
[Crossref] [PubMed]

Santos, A.

D. Megías, R. Marrero, B. Martínez Del Peso, M. Á. García, J. J. Bravo-Cordero, A. García-Grande, A. Santos, and M. C. Montoya, “Novel lambda FRET spectral confocal microscopy imaging method,” Microsc. Res. Tech. 72(1), 1–11 (2009).
[Crossref] [PubMed]

Sarabipour, S.

S. Sarabipour and K. Hristova, “Mechanism of FGF receptor dimerization and activation,” Nat. Commun. 7, 10262 (2016).
[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]

Segawa, K.

M. A. Rizzo, G. Springer, K. Segawa, W. R. Zipfel, and D. W. Piston, “Optimization of pairings and detection conditions for measurement of FRET between cyan and yellow fluorescent proteins,” Microsc. Microanal. 12(3), 238–254 (2006).
[Crossref] [PubMed]

Sorkin, A.

E. Galperin, V. V. Verkhusha, and A. Sorkin, “Three-chromophore FRET microscopy to analyze multiprotein interactions in living cells,” Nat. Methods 1(3), 209–217 (2004).
[Crossref] [PubMed]

Springer, G.

M. A. Rizzo, G. Springer, K. Segawa, W. R. Zipfel, and D. W. Piston, “Optimization of pairings and detection conditions for measurement of FRET between cyan and yellow fluorescent proteins,” Microsc. Microanal. 12(3), 238–254 (2006).
[Crossref] [PubMed]

Springer, G. H.

M. A. Rizzo, G. H. Springer, B. Granada, and D. W. Piston, “An improved cyan fluorescent protein variant useful for FRET,” Nat. Biotechnol. 22(4), 445–449 (2004).
[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]

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]

Tearney, G. J.

Thaler, C.

C. Thaler, S. V. Koushik, P. S. Blank, and S. S. Vogel, “Quantitative multiphoton spectral imaging and its use for measuring resonance energy transfer,” Biophys. J. 89(4), 2736–2749 (2005).
[Crossref] [PubMed]

van Royen, M. E.

C. Dinant, M. E. van Royen, W. Vermeulen, and A. B. Houtsmuller, “Fluorescence resonance energy transfer of GFP and YFP by spectral imaging and quantitative acceptor photobleaching,” J. Microsc. 231(1), 97–104 (2008).
[Crossref] [PubMed]

Verkhusha, V. V.

E. Galperin, V. V. Verkhusha, and A. Sorkin, “Three-chromophore FRET microscopy to analyze multiprotein interactions in living cells,” Nat. Methods 1(3), 209–217 (2004).
[Crossref] [PubMed]

Vermeulen, W.

C. Dinant, M. E. van Royen, W. Vermeulen, and A. B. Houtsmuller, “Fluorescence resonance energy transfer of GFP and YFP by spectral imaging and quantitative acceptor photobleaching,” J. Microsc. 231(1), 97–104 (2008).
[Crossref] [PubMed]

Vogel, S. S.

S. V. Koushik, P. S. Blank, and S. S. Vogel, “Anomalous surplus energy transfer observed with multiple FRET acceptors,” PLoS One 4(11), e8031 (2009).
[Crossref] [PubMed]

H. Chen, H. L. Puhl, S. V. Koushik, S. S. Vogel, and S. R. Ikeda, “Measurement of FRET efficiency and ratio of donor to acceptor concentration in living cells,” Biophys. J. 91(5), L39–L41 (2006).
[Crossref] [PubMed]

C. Thaler, S. V. Koushik, P. S. Blank, and S. S. Vogel, “Quantitative multiphoton spectral imaging and its use for measuring resonance energy transfer,” Biophys. J. 89(4), 2736–2749 (2005).
[Crossref] [PubMed]

Völler, P.

H. Düssmann, M. Rehm, C. G. Concannon, S. Anguissola, M. Würstle, S. Kacmar, P. Völler, H. J. Huber, and J. H. M. 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]

Waldeck-Weiermair, M.

M. Waldeck-Weiermair, R. Malli, W. Parichatikanond, B. Gottschalk, C. T. Madreiter-Sokolowski, C. Klec, R. Rost, and W. F. Graier, “Rearrangement of MICU1 multimers for activation of MCU is solely controlled by cytosolic Ca(2.),” Sci. Rep. 5, 15602 (2015).
[Crossref] [PubMed]

Wallrabe, H.

M. Elangovan, H. Wallrabe, Y. Chen, R. N. Day, M. Barroso, and A. Periasamy, “Characterization of one- and two-photon excitation fluorescence resonance energy transfer microscopy,” Methods 29(1), 58–73 (2003).
[Crossref] [PubMed]

Wang, L.

L. Wang, T. Chen, J. Qu, and X. Wei, “Photobleaching-based quantitative analysis of fluorescence resonance energy transfer inside single living cell,” J. Fluoresc. 20(1), 27–35 (2010).
[Crossref] [PubMed]

Wei, X.

L. Wang, T. Chen, J. Qu, and X. Wei, “Photobleaching-based quantitative analysis of fluorescence resonance energy transfer inside single living cell,” J. Fluoresc. 20(1), 27–35 (2010).
[Crossref] [PubMed]

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]

Wilms, C. D.

S. Levy, C. D. Wilms, E. Brumer, J. Kahn, L. Pnueli, Y. Arava, J. Eilers, and D. Gitler, “SpRET: highly sensitive and reliable spectral measurement of absolute FRET efficiency,” Microsc. Microanal. 17(2), 176–190 (2011).
[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.

A. Woehler, “Simultaneous quantitative live cell imaging of multiple FRET-based biosensors,” PLoS One 8(4), e61096 (2013).
[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]

Würstle, M.

H. Düssmann, M. Rehm, C. G. Concannon, S. Anguissola, M. Würstle, S. Kacmar, P. Völler, H. J. Huber, and J. H. M. 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.

L. Zhang, G. Qin, L. Chai, J. Zhang, F. Yang, H. Yang, S. Xie, and T. Chen, “Spectral wide-field microscopic fluorescence resonance energy transfer imaging in live cells,” J. Biomed. Opt. 20(8), 086011 (2015).
[Crossref] [PubMed]

Xing, D.

Y. Zhang, D. Xing, and L. Liu, “PUMA promotes Bax translocation by both directly interacting with Bax and by competitive binding to Bcl-X L during UV-induced apoptosis,” Mol. Biol. Cell 20(13), 3077–3087 (2009).
[Crossref] [PubMed]

Yang, F.

L. Zhang, G. Qin, L. Chai, J. Zhang, F. Yang, H. Yang, S. Xie, and T. Chen, “Spectral wide-field microscopic fluorescence resonance energy transfer imaging in live cells,” J. Biomed. Opt. 20(8), 086011 (2015).
[Crossref] [PubMed]

Yang, H.

L. Zhang, G. Qin, L. Chai, J. Zhang, F. Yang, H. Yang, S. Xie, and T. Chen, “Spectral wide-field microscopic fluorescence resonance energy transfer imaging in live cells,” J. Biomed. Opt. 20(8), 086011 (2015).
[Crossref] [PubMed]

Yu, H.

L. Zhang, H. Yu, J. Zhang, and T. Chen, “Binomial distribution-based quantitative measurement of multiple-acceptors fluorescence resonance energy transfer by partially photobleaching acceptor,” Appl. Phys. Lett. 104(24), 243706 (2014).
[Crossref]

H. Yu, T. Chen, and J. Qu, “Improving FRET efficiency measurement in confocal microscopy imaging,” Chin. Opt. Lett. 8(10), 947–949 (2010).
[Crossref]

Yuan, J.

Zal, T.

T. Zal and N. R. J. 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]

Zhang, J.

J. Zhang, H. Li, L. Chai, L. Zhang, J. Qu, and T. Chen, “Quantitative FRET measurement using emission-spectral unmixing with independent excitation crosstalk correction,” J. Microsc. 257(2), 104–116 (2015).
[Crossref] [PubMed]

L. Chai, J. Zhang, L. Zhang, and T. Chen, “Miniature fiber optic spectrometer-based quantitative fluorescence resonance energy transfer measurement in single living cells,” J. Biomed. Opt. 20(3), 037008 (2015).
[Crossref] [PubMed]

L. Zhang, G. Qin, L. Chai, J. Zhang, F. Yang, H. Yang, S. Xie, and T. Chen, “Spectral wide-field microscopic fluorescence resonance energy transfer imaging in live cells,” J. Biomed. Opt. 20(8), 086011 (2015).
[Crossref] [PubMed]

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Figures (7)

Fig. 1
Fig. 1 Scheme of quantitative EES-FRET measurement. Tandem: a tandem construct with known acceptor-donor concentration ratio.
Fig. 2
Fig. 2 Illustration on the spectral wide-field microscope. 405: ET405/20x bandpass excitation filter (405 nm excitation); 436: BP436/20 bandpass excitation filter (436 nm excitation); D455: E455Ipv2 dichroic mirror (455 nm dichroic mirror, D455); 470: a cube contains a BP470/40 bandpass excitation filter (470 nm excitation) and a FT495 dichroic mirror (495 nm dichroic mirror, D495); 510: a cube contains a BP510/17 bandpass excitation filter (510 nm excitation) and a DFT520 dichroic mirror (520 nm dichroic mirror, D520); DD: a cube contains filters for donor excitation and donor emission; AA: a cube contains filters for acceptor excitation and acceptor emission; DA: a cube contains filters for donor excitation and acceptor emission; LCTF: liquid crystal tunable filter (Varispec LCTF, VIS-10-20-STD, CRI, Cambridge); CCD1: CCD camera for quantitative EES-FRET measurement; CCD2: CCD camera for three-cube-based quantitative FRET measurement; a: for CCD1 detection; b: for CCD2 detection.
Fig. 3
Fig. 3 Excitation intensity spectra measured by recording the emission intensity at 630 nm of 7 g/l rhodamine B in ethylene glycol with 405, 436, 470 and 510 nm excitation, respectively. (a) Excitation intensity spectra (normalized to the maximum excitation intensity) measured under three attenuation degrees (Transmission: T = 0.25%, 0.5% and 1%). (b) Normalized excitation intensity spectra measured from 16 December to 20 December under T = 0.25%.
Fig. 4
Fig. 4 Emission-spectral responses of the spectral wide-field microscope. (a) Standard emission spectra of a precalibrated light source (LS-1-CAL). Open circles: intensity spectrum. Shaded circles: photon spectrum. (b) Emission-spectral responses (obtained by comparing the ratio between the measured and standard emission spectrum of the LS-1-CAL) of our system with different dichroic mirrors: D455, D495 and D520. (c) 13 emission-spectral responses of our system with D455 measured from 12 December to 28 December.
Fig. 5
Fig. 5 Three excitation-emission spectral signatures. (a) Excitation spectra of Cerulean (Sex D) and Venus (Sex A) (Left), and emission spectra of Cerulean (Sem D) and Venus (Sem A) (Right). These spectra were measured by recording the excitation and emission spectra of living HepG2 cells separately expressing donor (Cerulean) and acceptor (Venus). (b) Fitted pseudo-color images of three excitation-emission spectral signatures (SD, SA, SS) from the excitation and emission spectra (a) using Eq. (2).
Fig. 6
Fig. 6 Determination of rK factor using a donor-acceptor tandem sample (living HepG2 cells expressing CTV tandem construct with RC = 1). (a) A representative excitation-emission spectral image-stack of living cells expressing CTV construct. (b and c) Pixel-to-pixel rK image (b) and histograms (c) corresponding to (a).
Fig. 7
Fig. 7 Microscopic EES-FRET imaging of living cells expressing constructs with different donor-acceptor stoichiometry. (a) Representative spectral image-stacks (SDA) for living HepG2 cells expressing C32V, CVC and VCV, respectively. (b) The measured and fitted excitation-emission spectra of the brightest pixel in (a). (c and d) Pixel-to-pixel E images (c) and histograms (d) corresponding to (a). (e and f) Pixel-to-pixel RC images (e) and histograms (f) corresponding to (a).

Tables (1)

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Table 1 Compilation of Symbols Used in the Article.

Equations (10)

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S DA = C d K D Q D S 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 .
S D = S D ex S D em S A = S A ex S A em . S S = S D ex S A em
S DA = K D Q D ( C D t E C da ) 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 ,
W D = K D Q D ( C D t E C da ) W S =E K D Q A C da W A = K A Q A C A t
C D t = W D K D Q D +E C da ,
E C da = W S K D Q A ,
C A t = W A K A Q A .
E app =E C da C D t = W S W D r Q + W S ,
R C = C A t C D t = W A r K r Q W D + r K W S ,
r K = W A R C ( W D r Q + W S ) .

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