Abstract

Acceptor-sensitized quantitative Förster resonance energy transfer (FRET) measurement (E-FRET) is mainly impeded by donor emission crosstalk and acceptor direct excitation crosstalk. In this paper, we develop a novel E-FRET approach (Lux-E-FRET) based on linear unmixing (Lux) of the fluorescence intensity ratio between two detection channels with each excitation of two different wavelengths. The two detection channels need not to selectively collect the emission of donor or acceptor, and the excitation wavelengths need not to selectively excite donor or acceptor. For a tandem FRET sensor, Lux-E-FRET only needs single excitation wavelength. We performed Lux-E-FRET measurements on our dual-channel wide-field fluorescence microscope for FRET constructs in living cells, and obtained consistent FRET efficiencies with those measured by other methods. Collectively, Lux-E-FRET completely overcomes all spectral crosstalks and thus is applicable to the donor-acceptor pair with larger spectral overlapping.

© 2017 Optical Society of America

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References

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    [PubMed]

2017 (1)

2016 (4)

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).
[PubMed]

R. N. Day, W. Tao, and K. W. Dunn, “A simple approach for measuring FRET in fluorescent biosensors using two-photon microscopy,” Nat. Protoc. 11(11), 2066–2080 (2016).
[PubMed]

M. B Johny, D. N Yue, and D. T Yue,“Detecting stoichiometry of macromolecular complexes in live cells using FRET,” Nat. Commun. 7, 13709 (2016).

J. Zhang, L. Zhang, L. Chai, F. Yang, M. Du, and T. Chen, “Reliable measurement of the FRET sensitized-quenching transition factor for FRET quantification in living cells,” Micron 88, 7–15 (2016).
[PubMed]

2015 (3)

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), 86011 (2015).
[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).
[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).
[PubMed]

2013 (2)

J. A. Broussard, B. Rappaz, D. J. Webb, and C. M. Brown, “Fluorescence resonance energy transfer microscopy as demonstrated by measuring the activation of the serine/threonine kinase Akt,” Nat. Protoc. 8(2), 265–281 (2013).
[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).
[PubMed]

2012 (2)

K. Aoki, N. Komatsu, E. Hirata, Y. Kamioka, and M. Matsuda, “Stable expression of FRET biosensors: a new light in cancer research,” Cancer Sci. 103(4), 614–619 (2012).
[PubMed]

S. Okumoto, A. Jones, and W. B. Frommer, “Quantitative imaging with fluorescent biosensors,” Annu. Rev. Plant Biol. 63, 663–706 (2012).
[PubMed]

2011 (2)

R. H. Newman, M. D. Fosbrink, and J. Zhang, “Genetically encodable fluorescent biosensors for tracking signaling dynamics in living cells,” Chem. Rev. 111(5), 3614–3666 (2011).
[PubMed]

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).
[PubMed]

2009 (1)

A. D. Elder, A. Domin, G. S. Kaminski Schierle, C. Lindon, J. Pines, A. Esponsito, and C. F. Kaminski, “A quantitative protocol for dynamic measurements of protein interactions by Forster resonance energy transfer-sensitized fluorescence emission,” J. R. Soc. Interface 6(S1), S59–S81 (2009).

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).
[PubMed]

2006 (2)

I. T. Li, E. Pham, and K. Truong, “Protein biosensors based on the principle of fluorescence resonance energy transfer for monitoring cellular dynamics,” Biotechnol. Lett. 28(24), 1971–1982 (2006).
[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).
[PubMed]

2004 (1)

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

2002 (3)

T. Zimmermann, J. Rietdorf, A. Girod, V. Georget, and R. Pepperkok, “Spectral imaging and linear un-mixing enables improved FRET efficiency with a novel GFP2-YFP FRET pair,” FEBS Lett. 531(2), 245–249 (2002).
[PubMed]

S. B. Fowler, R. B. Best, J. L. Toca Herrera, T. J. Rutherford, A. Steward, E. Paci, M. Karplus, and J. Clarke, “Mechanical unfolding of a titin Ig domain: structure of unfolding intermediate revealed by combining AFM, molecular dynamics simulations, NMR and protein engineering,” J. Mol. Biol. 322(4), 841–849 (2002).
[PubMed]

D. J. Brockwell, G. S. Beddard, J. Clarkson, R. C. Zinober, A. W. Blake, J. Trinick, P. D. Olmsted, D. A. Smith, and S. E. Radford, “The effect of core destabilization on the mechanical resistance of I27,” Biophys. J. 83(1), 458–472 (2002).
[PubMed]

1999 (1)

M. Carrion-Vazquez, A. F. Oberhauser, S. B. Fowler, P. E. Marszalek, S. E. Broedel, J. Clarke, and J. M. Fernandez, “Mechanical and chemical unfolding of a single protein: a comparison,” Proc. Natl. Acad. Sci. U.S.A. 96(7), 3694–3699 (1999).
[PubMed]

1998 (1)

G. W. Gordon, G. Berry, X. H. Liang, B. Levine, and B. Herman, “Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy,” Biophys. J. 74(5), 2702–2713 (1998).
[PubMed]

1948 (1)

T. H. Förster, “Zwischenmolekulare energiewanderung und fluoreszenz,” Ann Phys-Berlin. 437(1‐2), 55–75 (1948).

Aoki, K.

K. Aoki, N. Komatsu, E. Hirata, Y. Kamioka, and M. Matsuda, “Stable expression of FRET biosensors: a new light in cancer research,” Cancer Sci. 103(4), 614–619 (2012).
[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).
[PubMed]

Beddard, G. S.

D. J. Brockwell, G. S. Beddard, J. Clarkson, R. C. Zinober, A. W. Blake, J. Trinick, P. D. Olmsted, D. A. Smith, and S. E. Radford, “The effect of core destabilization on the mechanical resistance of I27,” Biophys. J. 83(1), 458–472 (2002).
[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).
[PubMed]

Berry, G.

G. W. Gordon, G. Berry, X. H. Liang, B. Levine, and B. Herman, “Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy,” Biophys. J. 74(5), 2702–2713 (1998).
[PubMed]

Best, R. B.

S. B. Fowler, R. B. Best, J. L. Toca Herrera, T. J. Rutherford, A. Steward, E. Paci, M. Karplus, and J. Clarke, “Mechanical unfolding of a titin Ig domain: structure of unfolding intermediate revealed by combining AFM, molecular dynamics simulations, NMR and protein engineering,” J. Mol. Biol. 322(4), 841–849 (2002).
[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).
[PubMed]

Blake, A. W.

D. J. Brockwell, G. S. Beddard, J. Clarkson, R. C. Zinober, A. W. Blake, J. Trinick, P. D. Olmsted, D. A. Smith, and S. E. Radford, “The effect of core destabilization on the mechanical resistance of I27,” Biophys. J. 83(1), 458–472 (2002).
[PubMed]

Brockwell, D. J.

D. J. Brockwell, G. S. Beddard, J. Clarkson, R. C. Zinober, A. W. Blake, J. Trinick, P. D. Olmsted, D. A. Smith, and S. E. Radford, “The effect of core destabilization on the mechanical resistance of I27,” Biophys. J. 83(1), 458–472 (2002).
[PubMed]

Broedel, S. E.

M. Carrion-Vazquez, A. F. Oberhauser, S. B. Fowler, P. E. Marszalek, S. E. Broedel, J. Clarke, and J. M. Fernandez, “Mechanical and chemical unfolding of a single protein: a comparison,” Proc. Natl. Acad. Sci. U.S.A. 96(7), 3694–3699 (1999).
[PubMed]

Broussard, J. A.

J. A. Broussard, B. Rappaz, D. J. Webb, and C. M. Brown, “Fluorescence resonance energy transfer microscopy as demonstrated by measuring the activation of the serine/threonine kinase Akt,” Nat. Protoc. 8(2), 265–281 (2013).
[PubMed]

Brown, C. M.

J. A. Broussard, B. Rappaz, D. J. Webb, and C. M. Brown, “Fluorescence resonance energy transfer microscopy as demonstrated by measuring the activation of the serine/threonine kinase Akt,” Nat. Protoc. 8(2), 265–281 (2013).
[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).
[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).
[PubMed]

Carrion-Vazquez, M.

M. Carrion-Vazquez, A. F. Oberhauser, S. B. Fowler, P. E. Marszalek, S. E. Broedel, J. Clarke, and J. M. Fernandez, “Mechanical and chemical unfolding of a single protein: a comparison,” Proc. Natl. Acad. Sci. U.S.A. 96(7), 3694–3699 (1999).
[PubMed]

Chai, L.

J. Zhang, L. Zhang, L. Chai, F. Yang, M. Du, and T. Chen, “Reliable measurement of the FRET sensitized-quenching transition factor for FRET quantification in living cells,” Micron 88, 7–15 (2016).
[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), 86011 (2015).
[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).
[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).
[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).
[PubMed]

Chen, T.

J. Zhang, L. Zhang, L. Chai, F. Yang, M. Du, and T. Chen, “Reliable measurement of the FRET sensitized-quenching transition factor for FRET quantification in living cells,” Micron 88, 7–15 (2016).
[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), 86011 (2015).
[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).
[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).
[PubMed]

Clarke, J.

S. B. Fowler, R. B. Best, J. L. Toca Herrera, T. J. Rutherford, A. Steward, E. Paci, M. Karplus, and J. Clarke, “Mechanical unfolding of a titin Ig domain: structure of unfolding intermediate revealed by combining AFM, molecular dynamics simulations, NMR and protein engineering,” J. Mol. Biol. 322(4), 841–849 (2002).
[PubMed]

M. Carrion-Vazquez, A. F. Oberhauser, S. B. Fowler, P. E. Marszalek, S. E. Broedel, J. Clarke, and J. M. Fernandez, “Mechanical and chemical unfolding of a single protein: a comparison,” Proc. Natl. Acad. Sci. U.S.A. 96(7), 3694–3699 (1999).
[PubMed]

Clarkson, J.

D. J. Brockwell, G. S. Beddard, J. Clarkson, R. C. Zinober, A. W. Blake, J. Trinick, P. D. Olmsted, D. A. Smith, and S. E. Radford, “The effect of core destabilization on the mechanical resistance of I27,” Biophys. J. 83(1), 458–472 (2002).
[PubMed]

Day, R. N.

R. N. Day, W. Tao, and K. W. Dunn, “A simple approach for measuring FRET in fluorescent biosensors using two-photon microscopy,” Nat. Protoc. 11(11), 2066–2080 (2016).
[PubMed]

Domin, A.

A. D. Elder, A. Domin, G. S. Kaminski Schierle, C. Lindon, J. Pines, A. Esponsito, and C. F. Kaminski, “A quantitative protocol for dynamic measurements of protein interactions by Forster resonance energy transfer-sensitized fluorescence emission,” J. R. Soc. Interface 6(S1), S59–S81 (2009).

Du, M.

J. Zhang, L. Zhang, L. Chai, F. Yang, M. Du, and T. Chen, “Reliable measurement of the FRET sensitized-quenching transition factor for FRET quantification in living cells,” Micron 88, 7–15 (2016).
[PubMed]

Dunn, K. W.

R. N. Day, W. Tao, and K. W. Dunn, “A simple approach for measuring FRET in fluorescent biosensors using two-photon microscopy,” Nat. Protoc. 11(11), 2066–2080 (2016).
[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).
[PubMed]

Elder, A. D.

A. D. Elder, A. Domin, G. S. Kaminski Schierle, C. Lindon, J. Pines, A. Esponsito, and C. F. Kaminski, “A quantitative protocol for dynamic measurements of protein interactions by Forster resonance energy transfer-sensitized fluorescence emission,” J. R. Soc. Interface 6(S1), S59–S81 (2009).

Esponsito, A.

A. D. Elder, A. Domin, G. S. Kaminski Schierle, C. Lindon, J. Pines, A. Esponsito, and C. F. Kaminski, “A quantitative protocol for dynamic measurements of protein interactions by Forster resonance energy transfer-sensitized fluorescence emission,” J. R. Soc. Interface 6(S1), S59–S81 (2009).

Feeks, J. A.

Fernandez, J. M.

M. Carrion-Vazquez, A. F. Oberhauser, S. B. Fowler, P. E. Marszalek, S. E. Broedel, J. Clarke, and J. M. Fernandez, “Mechanical and chemical unfolding of a single protein: a comparison,” Proc. Natl. Acad. Sci. U.S.A. 96(7), 3694–3699 (1999).
[PubMed]

Förster, T. H.

T. H. Förster, “Zwischenmolekulare energiewanderung und fluoreszenz,” Ann Phys-Berlin. 437(1‐2), 55–75 (1948).

Fosbrink, M. D.

R. H. Newman, M. D. Fosbrink, and J. Zhang, “Genetically encodable fluorescent biosensors for tracking signaling dynamics in living cells,” Chem. Rev. 111(5), 3614–3666 (2011).
[PubMed]

Fowler, S. B.

S. B. Fowler, R. B. Best, J. L. Toca Herrera, T. J. Rutherford, A. Steward, E. Paci, M. Karplus, and J. Clarke, “Mechanical unfolding of a titin Ig domain: structure of unfolding intermediate revealed by combining AFM, molecular dynamics simulations, NMR and protein engineering,” J. Mol. Biol. 322(4), 841–849 (2002).
[PubMed]

M. Carrion-Vazquez, A. F. Oberhauser, S. B. Fowler, P. E. Marszalek, S. E. Broedel, J. Clarke, and J. M. Fernandez, “Mechanical and chemical unfolding of a single protein: a comparison,” Proc. Natl. Acad. Sci. U.S.A. 96(7), 3694–3699 (1999).
[PubMed]

Frommer, W. B.

S. Okumoto, A. Jones, and W. B. Frommer, “Quantitative imaging with fluorescent biosensors,” Annu. Rev. Plant Biol. 63, 663–706 (2012).
[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).
[PubMed]

Georget, V.

T. Zimmermann, J. Rietdorf, A. Girod, V. Georget, and R. Pepperkok, “Spectral imaging and linear un-mixing enables improved FRET efficiency with a novel GFP2-YFP FRET pair,” FEBS Lett. 531(2), 245–249 (2002).
[PubMed]

Girod, A.

T. Zimmermann, J. Rietdorf, A. Girod, V. Georget, and R. Pepperkok, “Spectral imaging and linear un-mixing enables improved FRET efficiency with a novel GFP2-YFP FRET pair,” FEBS Lett. 531(2), 245–249 (2002).
[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).
[PubMed]

Gordon, G. W.

G. W. Gordon, G. Berry, X. H. Liang, B. Levine, and B. Herman, “Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy,” Biophys. J. 74(5), 2702–2713 (1998).
[PubMed]

Herman, B.

G. W. Gordon, G. Berry, X. H. Liang, B. Levine, and B. Herman, “Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy,” Biophys. J. 74(5), 2702–2713 (1998).
[PubMed]

Hirata, E.

K. Aoki, N. Komatsu, E. Hirata, Y. Kamioka, and M. Matsuda, “Stable expression of FRET biosensors: a new light in cancer research,” Cancer Sci. 103(4), 614–619 (2012).
[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).
[PubMed]

Hunter, J. J.

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).
[PubMed]

Johny, M. B

M. B Johny, D. N Yue, and D. T Yue,“Detecting stoichiometry of macromolecular complexes in live cells using FRET,” Nat. Commun. 7, 13709 (2016).

Jones, A.

S. Okumoto, A. Jones, and W. B. Frommer, “Quantitative imaging with fluorescent biosensors,” Annu. Rev. Plant Biol. 63, 663–706 (2012).
[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).
[PubMed]

Kaminski, C. F.

A. D. Elder, A. Domin, G. S. Kaminski Schierle, C. Lindon, J. Pines, A. Esponsito, and C. F. Kaminski, “A quantitative protocol for dynamic measurements of protein interactions by Forster resonance energy transfer-sensitized fluorescence emission,” J. R. Soc. Interface 6(S1), S59–S81 (2009).

Kaminski Schierle, G. S.

A. D. Elder, A. Domin, G. S. Kaminski Schierle, C. Lindon, J. Pines, A. Esponsito, and C. F. Kaminski, “A quantitative protocol for dynamic measurements of protein interactions by Forster resonance energy transfer-sensitized fluorescence emission,” J. R. Soc. Interface 6(S1), S59–S81 (2009).

Kamioka, Y.

K. Aoki, N. Komatsu, E. Hirata, Y. Kamioka, and M. Matsuda, “Stable expression of FRET biosensors: a new light in cancer research,” Cancer Sci. 103(4), 614–619 (2012).
[PubMed]

Karplus, M.

S. B. Fowler, R. B. Best, J. L. Toca Herrera, T. J. Rutherford, A. Steward, E. Paci, M. Karplus, and J. Clarke, “Mechanical unfolding of a titin Ig domain: structure of unfolding intermediate revealed by combining AFM, molecular dynamics simulations, NMR and protein engineering,” J. Mol. Biol. 322(4), 841–849 (2002).
[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).
[PubMed]

Komatsu, N.

K. Aoki, N. Komatsu, E. Hirata, Y. Kamioka, and M. Matsuda, “Stable expression of FRET biosensors: a new light in cancer research,” Cancer Sci. 103(4), 614–619 (2012).
[PubMed]

Koushik, S. V.

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).
[PubMed]

Levine, B.

G. W. Gordon, G. Berry, X. H. Liang, B. Levine, and B. Herman, “Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy,” Biophys. J. 74(5), 2702–2713 (1998).
[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).
[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).
[PubMed]

Li, I. T.

I. T. Li, E. Pham, and K. Truong, “Protein biosensors based on the principle of fluorescence resonance energy transfer for monitoring cellular dynamics,” Biotechnol. Lett. 28(24), 1971–1982 (2006).
[PubMed]

Liang, X. H.

G. W. Gordon, G. Berry, X. H. Liang, B. Levine, and B. Herman, “Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy,” Biophys. J. 74(5), 2702–2713 (1998).
[PubMed]

Lindon, C.

A. D. Elder, A. Domin, G. S. Kaminski Schierle, C. Lindon, J. Pines, A. Esponsito, and C. F. Kaminski, “A quantitative protocol for dynamic measurements of protein interactions by Forster resonance energy transfer-sensitized fluorescence emission,” J. R. Soc. Interface 6(S1), S59–S81 (2009).

Marszalek, P. E.

M. Carrion-Vazquez, A. F. Oberhauser, S. B. Fowler, P. E. Marszalek, S. E. Broedel, J. Clarke, and J. M. Fernandez, “Mechanical and chemical unfolding of a single protein: a comparison,” Proc. Natl. Acad. Sci. U.S.A. 96(7), 3694–3699 (1999).
[PubMed]

Matsuda, M.

K. Aoki, N. Komatsu, E. Hirata, Y. Kamioka, and M. Matsuda, “Stable expression of FRET biosensors: a new light in cancer research,” Cancer Sci. 103(4), 614–619 (2012).
[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).
[PubMed]

Newman, R. H.

R. H. Newman, M. D. Fosbrink, and J. Zhang, “Genetically encodable fluorescent biosensors for tracking signaling dynamics in living cells,” Chem. Rev. 111(5), 3614–3666 (2011).
[PubMed]

Oberhauser, A. F.

M. Carrion-Vazquez, A. F. Oberhauser, S. B. Fowler, P. E. Marszalek, S. E. Broedel, J. Clarke, and J. M. Fernandez, “Mechanical and chemical unfolding of a single protein: a comparison,” Proc. Natl. Acad. Sci. U.S.A. 96(7), 3694–3699 (1999).
[PubMed]

Okumoto, S.

S. Okumoto, A. Jones, and W. B. Frommer, “Quantitative imaging with fluorescent biosensors,” Annu. Rev. Plant Biol. 63, 663–706 (2012).
[PubMed]

Olmsted, P. D.

D. J. Brockwell, G. S. Beddard, J. Clarkson, R. C. Zinober, A. W. Blake, J. Trinick, P. D. Olmsted, D. A. Smith, and S. E. Radford, “The effect of core destabilization on the mechanical resistance of I27,” Biophys. J. 83(1), 458–472 (2002).
[PubMed]

Paci, E.

S. B. Fowler, R. B. Best, J. L. Toca Herrera, T. J. Rutherford, A. Steward, E. Paci, M. Karplus, and J. Clarke, “Mechanical unfolding of a titin Ig domain: structure of unfolding intermediate revealed by combining AFM, molecular dynamics simulations, NMR and protein engineering,” J. Mol. Biol. 322(4), 841–849 (2002).
[PubMed]

Pepperkok, R.

T. Zimmermann, J. Rietdorf, A. Girod, V. Georget, and R. Pepperkok, “Spectral imaging and linear un-mixing enables improved FRET efficiency with a novel GFP2-YFP FRET pair,” FEBS Lett. 531(2), 245–249 (2002).
[PubMed]

Pham, E.

I. T. Li, E. Pham, and K. Truong, “Protein biosensors based on the principle of fluorescence resonance energy transfer for monitoring cellular dynamics,” Biotechnol. Lett. 28(24), 1971–1982 (2006).
[PubMed]

Pines, J.

A. D. Elder, A. Domin, G. S. Kaminski Schierle, C. Lindon, J. Pines, A. Esponsito, and C. F. Kaminski, “A quantitative protocol for dynamic measurements of protein interactions by Forster resonance energy transfer-sensitized fluorescence emission,” J. R. Soc. Interface 6(S1), S59–S81 (2009).

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).
[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).
[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).
[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), 86011 (2015).
[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).
[PubMed]

Radford, S. E.

D. J. Brockwell, G. S. Beddard, J. Clarkson, R. C. Zinober, A. W. Blake, J. Trinick, P. D. Olmsted, D. A. Smith, and S. E. Radford, “The effect of core destabilization on the mechanical resistance of I27,” Biophys. J. 83(1), 458–472 (2002).
[PubMed]

Rappaz, B.

J. A. Broussard, B. Rappaz, D. J. Webb, and C. M. Brown, “Fluorescence resonance energy transfer microscopy as demonstrated by measuring the activation of the serine/threonine kinase Akt,” Nat. Protoc. 8(2), 265–281 (2013).
[PubMed]

Rietdorf, J.

T. Zimmermann, J. Rietdorf, A. Girod, V. Georget, and R. Pepperkok, “Spectral imaging and linear un-mixing enables improved FRET efficiency with a novel GFP2-YFP FRET pair,” FEBS Lett. 531(2), 245–249 (2002).
[PubMed]

Rutherford, T. J.

S. B. Fowler, R. B. Best, J. L. Toca Herrera, T. J. Rutherford, A. Steward, E. Paci, M. Karplus, and J. Clarke, “Mechanical unfolding of a titin Ig domain: structure of unfolding intermediate revealed by combining AFM, molecular dynamics simulations, NMR and protein engineering,” J. Mol. Biol. 322(4), 841–849 (2002).
[PubMed]

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).
[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).
[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).
[PubMed]

Smith, D. A.

D. J. Brockwell, G. S. Beddard, J. Clarkson, R. C. Zinober, A. W. Blake, J. Trinick, P. D. Olmsted, D. A. Smith, and S. E. Radford, “The effect of core destabilization on the mechanical resistance of I27,” Biophys. J. 83(1), 458–472 (2002).
[PubMed]

Steward, A.

S. B. Fowler, R. B. Best, J. L. Toca Herrera, T. J. Rutherford, A. Steward, E. Paci, M. Karplus, and J. Clarke, “Mechanical unfolding of a titin Ig domain: structure of unfolding intermediate revealed by combining AFM, molecular dynamics simulations, NMR and protein engineering,” J. Mol. Biol. 322(4), 841–849 (2002).
[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).
[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).
[PubMed]

Tao, W.

R. N. Day, W. Tao, and K. W. Dunn, “A simple approach for measuring FRET in fluorescent biosensors using two-photon microscopy,” Nat. Protoc. 11(11), 2066–2080 (2016).
[PubMed]

Toca Herrera, J. L.

S. B. Fowler, R. B. Best, J. L. Toca Herrera, T. J. Rutherford, A. Steward, E. Paci, M. Karplus, and J. Clarke, “Mechanical unfolding of a titin Ig domain: structure of unfolding intermediate revealed by combining AFM, molecular dynamics simulations, NMR and protein engineering,” J. Mol. Biol. 322(4), 841–849 (2002).
[PubMed]

Trinick, J.

D. J. Brockwell, G. S. Beddard, J. Clarkson, R. C. Zinober, A. W. Blake, J. Trinick, P. D. Olmsted, D. A. Smith, and S. E. Radford, “The effect of core destabilization on the mechanical resistance of I27,” Biophys. J. 83(1), 458–472 (2002).
[PubMed]

Truong, K.

I. T. Li, E. Pham, and K. Truong, “Protein biosensors based on the principle of fluorescence resonance energy transfer for monitoring cellular dynamics,” Biotechnol. Lett. 28(24), 1971–1982 (2006).
[PubMed]

Vogel, S. S.

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).
[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).
[PubMed]

Webb, D. J.

J. A. Broussard, B. Rappaz, D. J. Webb, and C. M. Brown, “Fluorescence resonance energy transfer microscopy as demonstrated by measuring the activation of the serine/threonine kinase Akt,” Nat. Protoc. 8(2), 265–281 (2013).
[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).
[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).
[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).
[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).
[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), 86011 (2015).
[PubMed]

Yang, F.

J. Zhang, L. Zhang, L. Chai, F. Yang, M. Du, and T. Chen, “Reliable measurement of the FRET sensitized-quenching transition factor for FRET quantification in living cells,” Micron 88, 7–15 (2016).
[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), 86011 (2015).
[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), 86011 (2015).
[PubMed]

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).
[PubMed]

Yue, D. N

M. B Johny, D. N Yue, and D. T Yue,“Detecting stoichiometry of macromolecular complexes in live cells using FRET,” Nat. Commun. 7, 13709 (2016).

Yue, D. T

M. B Johny, D. N Yue, and D. T Yue,“Detecting stoichiometry of macromolecular complexes in live cells using FRET,” Nat. Commun. 7, 13709 (2016).

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).
[PubMed]

Zal, T.

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

Zhang, J.

J. Zhang, L. Zhang, L. Chai, F. Yang, M. Du, and T. Chen, “Reliable measurement of the FRET sensitized-quenching transition factor for FRET quantification in living cells,” Micron 88, 7–15 (2016).
[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), 86011 (2015).
[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).
[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).
[PubMed]

R. H. Newman, M. D. Fosbrink, and J. Zhang, “Genetically encodable fluorescent biosensors for tracking signaling dynamics in living cells,” Chem. Rev. 111(5), 3614–3666 (2011).
[PubMed]

Zhang, L.

J. Zhang, L. Zhang, L. Chai, F. Yang, M. Du, and T. Chen, “Reliable measurement of the FRET sensitized-quenching transition factor for FRET quantification in living cells,” Micron 88, 7–15 (2016).
[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), 86011 (2015).
[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).
[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).
[PubMed]

Zimmermann, T.

T. Zimmermann, J. Rietdorf, A. Girod, V. Georget, and R. Pepperkok, “Spectral imaging and linear un-mixing enables improved FRET efficiency with a novel GFP2-YFP FRET pair,” FEBS Lett. 531(2), 245–249 (2002).
[PubMed]

Zinober, R. C.

D. J. Brockwell, G. S. Beddard, J. Clarkson, R. C. Zinober, A. W. Blake, J. Trinick, P. D. Olmsted, D. A. Smith, and S. E. Radford, “The effect of core destabilization on the mechanical resistance of I27,” Biophys. J. 83(1), 458–472 (2002).
[PubMed]

Ann Phys-Berlin. (1)

T. H. Förster, “Zwischenmolekulare energiewanderung und fluoreszenz,” Ann Phys-Berlin. 437(1‐2), 55–75 (1948).

Annu. Rev. Plant Biol. (1)

S. Okumoto, A. Jones, and W. B. Frommer, “Quantitative imaging with fluorescent biosensors,” Annu. Rev. Plant Biol. 63, 663–706 (2012).
[PubMed]

Biomed. Opt. Express (1)

Biophys. J. (5)

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).
[PubMed]

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

G. W. Gordon, G. Berry, X. H. Liang, B. Levine, and B. Herman, “Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy,” Biophys. J. 74(5), 2702–2713 (1998).
[PubMed]

D. J. Brockwell, G. S. Beddard, J. Clarkson, R. C. Zinober, A. W. Blake, J. Trinick, P. D. Olmsted, D. A. Smith, and S. E. Radford, “The effect of core destabilization on the mechanical resistance of I27,” Biophys. J. 83(1), 458–472 (2002).
[PubMed]

Biotechnol. Lett. (1)

I. T. Li, E. Pham, and K. Truong, “Protein biosensors based on the principle of fluorescence resonance energy transfer for monitoring cellular dynamics,” Biotechnol. Lett. 28(24), 1971–1982 (2006).
[PubMed]

Cancer Sci. (1)

K. Aoki, N. Komatsu, E. Hirata, Y. Kamioka, and M. Matsuda, “Stable expression of FRET biosensors: a new light in cancer research,” Cancer Sci. 103(4), 614–619 (2012).
[PubMed]

Chem. Rev. (1)

R. H. Newman, M. D. Fosbrink, and J. Zhang, “Genetically encodable fluorescent biosensors for tracking signaling dynamics in living cells,” Chem. Rev. 111(5), 3614–3666 (2011).
[PubMed]

FEBS Lett. (1)

T. Zimmermann, J. Rietdorf, A. Girod, V. Georget, and R. Pepperkok, “Spectral imaging and linear un-mixing enables improved FRET efficiency with a novel GFP2-YFP FRET pair,” FEBS Lett. 531(2), 245–249 (2002).
[PubMed]

J. Biomed. Opt. (2)

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), 86011 (2015).
[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).
[PubMed]

J. Microsc. (1)

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).
[PubMed]

J. Mol. Biol. (1)

S. B. Fowler, R. B. Best, J. L. Toca Herrera, T. J. Rutherford, A. Steward, E. Paci, M. Karplus, and J. Clarke, “Mechanical unfolding of a titin Ig domain: structure of unfolding intermediate revealed by combining AFM, molecular dynamics simulations, NMR and protein engineering,” J. Mol. Biol. 322(4), 841–849 (2002).
[PubMed]

J. R. Soc. Interface (1)

A. D. Elder, A. Domin, G. S. Kaminski Schierle, C. Lindon, J. Pines, A. Esponsito, and C. F. Kaminski, “A quantitative protocol for dynamic measurements of protein interactions by Forster resonance energy transfer-sensitized fluorescence emission,” J. R. Soc. Interface 6(S1), S59–S81 (2009).

Micron (1)

J. Zhang, L. Zhang, L. Chai, F. Yang, M. Du, and T. Chen, “Reliable measurement of the FRET sensitized-quenching transition factor for FRET quantification in living cells,” Micron 88, 7–15 (2016).
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Microsc. Microanal. (1)

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Figures (6)

Fig. 1
Fig. 1

Process of quantitative Lux-E-FRET measurement. Superscript i( = 1, 2) denotes each of two different excitation wavelengths; Tandem reference: a tandem construct with 1:1 acceptor-donor concentration ratio.

Fig. 2
Fig. 2

Illustration on the dual-channel wide-field microscopic FRET imaging system.

Fig. 3
Fig. 3

Emission distribution coefficient (uD) and excitation absorption ratio coefficient (vD) of donor. (a) Fluorescence images of living HepG2 cells expressing Cerulean in 510 nm and 537 nm emission detection channels with 430 nm and 470 nm excitation wavelengths respectively. (b and c) Pixel-to-pixel pseudocolor images (left) and histograms (right) of uD corresponding to (a) with 430 nm (b) and 470 nm (c) excitation wavelengths, respectively. (d and e) Pixel-to-pixel pseudocolor images (left) and histograms (right) of vD corresponding to (a) by using 510 nm (d) and 537 nm (e) emission detection channels, respectively. (f) Statistical values of uD from 27 cells in independent 12 frames with 430 nm and 470 nm excitation wavelengths, respectively. (g) Statistical values of vD from the same 27 cells by using 510 nm and 537 nm emission detection channels, respectively.

Fig. 4
Fig. 4

Emission distribution coefficient (uA) and excitation absorption ratio coefficient (vA) of acceptor. (a) Fluorescence images of living HepG2 cells expressing Venus in 510 nm and 537 nm emission detection channels with 430 nm and 470 nm excitation wavelengths respectively. (b and c) Pixel-to-pixel pseudocolor images (left) and histograms (right) of uA corresponding to (a) with 430 nm (b) and 470 nm (c) excitation wavelengths, respectively (d and e) Pixel-to-pixel pseudocolor images (left) and histograms (right) of vA corresponding to (a) in 510 nm (d) and 537 nm (e) emission detection channels, respectively. (f) Statistical values of uA from 15 cells in independent 8 frames with 430 nm and 470 nm excitation wavelengths, respectively. (g) Statistical values of vA from the same 15 cells measured by using 510 nm and 537 nm emission detection channels, respectively.

Fig. 5
Fig. 5

g and γi factors. (a) Fluorescence images of living HepG2 cells expressing C32V (up) and CTV (down) in 510 nm and 537 nm emission detection channels with 430 nm and 470 nm excitation wavelengths, respectively. (b) Pixel-to-pixel pseudocolor images (left) and histograms (right) of u1 DA (with 430 nm excitation) and u2 DA (with 470 nm excitation) for C32V (up) and CTV (down) corresponding to (a). (c) Pixel-to-pixel pseudocolor images (left) and histograms (right) of δ1 (with 430 nm excitation) and δ2 (with 470 nm excitation) for C32V (up) and CTV (down) corresponding to (a). (d) Statistical values of g and γi from 20 living cells expressing C32V and 22 living cells expressing CTV.

Fig. 6
Fig. 6

FRET efficiencies (E) of C + V,C32V,CVC,VCV and VCVV constructs. (a) Fluorescence images of living HepG2 cells expressing C + V,C32V,CVC,VCV and VCVV constructs. (b and c) Pixel-to-pixel pseudocolor images of u1 DA ((b) with 430 nm excitation) and u2 DA ((c) with 470 nm excitation) corresponding to (a). (d and e) Pixel-to-pixel pseudocolor images of δ1 ((d) with 430 nm excitation) and δ2 ((e) with 470 nm excitation) corresponding to (a). (f and g) Pixel-to-pixel pseudocolor images (f) and histograms (g) of E with 430 nm and 470 nm excitation wavelengths corresponding to (a). (h) Statistical E values of C + V,C32V,CVC,VCV and VCVV constructs with 430 nm and 470 nm excitations, or with single 430 nm excitation.

Tables (1)

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Table 1 Symbols and notations in this article

Equations (27)

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u D = e D (λ) η 1 (λ)dλ e D (λ) η 2 (λ)dλ = I ex i ε D i Q D [ D ref ] e D (λ) η 1 (λ)dλ I ex i ε D i Q D [ D ref ] e D (λ) η 2 (λ)dλ = I 1 i (D) I 2 i (D) ,
u A = e A (λ) η 1 (λ)dλ e A (λ) η 2 (λ)dλ = I ex i ε A i Q A [ A ref ] e A (λ) η 1 (λ)dλ I ex i ε A i Q A [ A ref ] e A (λ) η 2 (λ)dλ = I 1 i (A) I 2 i (A) .
C DCH1 = e D (λ) η 1 (λ)dλ C DCH2 = e D (λ) η 2 (λ)dλ C ACH1 = e A (λ) η 1 (λ)dλ C ACH2 = e A (λ) η 2 (λ)dλ.
v D = I ex 1 ε D 1 I ex 2 ε D 2 = I ex 1 ε D 1 Q D [ D ref ] e D (λ) η j (λ)dλ I ex 2 ε D 2 Q D [ D ref ] e D (λ) η j (λ)dλ = I j 1 (D) I j 2 (D) ,
v A = I ex 1 ε A 1 I ex 2 ε A 2 = I ex 1 ε A 1 Q A [ A ref ] e A (λ) η j (λ)dλ I ex 2 ε A 2 Q A [ A ref ] e A (λ) η j (λ)dλ = I j 1 (A) I j 2 (A) .
I j i (DA)= I ex i ε D i Q D [D](1E) C DCHj + I ex i ε D i Q A [D]E C ACHj + I ex i ε A i Q A [A] C ACHj + I ex i ε D i Q D [d] C DCHj + I ex i ε A i Q A [a] C ACHj = I ex i ε A i Q D { γ i [[D]+[d][D]E]× C DCHj + Q A / Q D [ γ i [D]E+([A]+[a])] × C ACHj }.
γ i = ε D i ε A i .
u DA i = I 1 i (DA) I 2 i (DA) = I ex i ε A i Q D { γ i [[D](1E)+[d]]× C DCH1 + Q A / Q D [ γ i [D]E+([A]+[a])]× C ACH1 } I ex i ε A i Q D { γ i [[D](1E)+[d]]× C DCH2 + Q A / Q D [ γ i [D]E+([A]+[a])] × C ACH2 } .
u DA i = 1 1+ γ i E [D] [D]+[d] + [A]+[a] [D]+[d] γ i γ i E [D] [D]+[d] × Q A Q D × C ACH2 C DCH2 × u D + γ i E [D] [D]+[d] + [A]+[a] [D]+[d] γ i γ i E [D] [D]+[d] × Q A Q D × C ACH2 C DCH2 1+ γ i E [D] [D]+[d] + [A]+[a] [D]+[d] γ i γ i E [D] [D]+[d] × Q A Q D × C ACH2 C DCH2 × u A .
g= Q A Q D × C ACH2 C DCH2 .
u DA i = w D i u D + w A i u A ,
w D i = 1 1+ δ i ,
w A i = δ i 1+ δ i
δ i = R t + γ i E f D γ i γ i E f D ×g.
δ i = u D u DA i u DA i u A .
δ 1 = R C + γ 1 E f D γ 1 γ 1 E f D ×g δ 2 = R C + γ 2 E f D γ 2 γ 2 E f D ×g.
v D γ 2 = v A γ 1 .
E f D = δ 1 v D δ 2 v A δ 1 v D δ 2 v A +( v D v A )×g ,
R t = ( δ 2 δ 1 ) v A γ 1 δ 1 v D δ 2 v A +( v D v A )×g .
g= δ 1 v D δ 2 v A v D v A × 1E E ,
γ 1 = δ 1 v D δ 2 v A ( δ 2 δ 1 )E v A ,
γ 2 = δ 1 v D δ 2 v A ( δ 2 δ 1 )E v D .
( δ 1 2 δ 1 1 ) v A γ 1 δ 1 1 v D δ 1 2 v A +( v D v A )×g = ( δ 2 2 δ 2 1 ) v A γ 1 δ 2 1 v D δ 2 2 v A +( v D v A )×g =1.
g= ( δ 2 2 δ 2 1 )( δ 1 1 v D δ 1 2 v A )( δ 1 2 δ 1 1 )( δ 2 1 v D δ 2 2 v A ) [( δ 1 2 δ 1 1 )( δ 2 2 δ 2 1 )]×( v D v A ) ,
γ 1 = ( δ 1 1 v D δ 1 2 v A )( δ 2 1 v D δ 2 2 v A ) [( δ 1 2 δ 1 1 )( δ 2 2 δ 2 1 )] v A ,
γ 2 = ( δ 1 1 v D δ 1 2 v A )( δ 2 1 v D δ 2 2 v A ) [( δ 1 2 δ 1 1 )( δ 2 2 δ 2 1 )] v D .
E= δγg R t δγ+gγ .