Abstract

Förster resonance energy transfer (FRET) is an important method in studying biochemistry reactions. But quantifying FRET rapidly is difficult to do because of crosstalk between free donor, free acceptor and FRET fluorescent signals when only excitation or emission property of a FRET sample is measured. If FRET is studied with excitation-emission matrix (EEM) measurements, because the fluorescence intensity maxima of donor, acceptor, and FRET emissions occupy different regions within the EEM, FRET fluorescence can be easily separated out by linear unmixing. In this paper, we report a novel high-speed Fourier Fluorescence Excitation Emission spectrometer, which simultaneously measures three projections of EEM from a FRET sample, which are excitation, emission and excitation-emission cross-correlation spectra. We demonstrate that these three EEM projections can be measured and unmixed in approximately 1 ms to provide rapid quantitative FRET in the presence of free donors and acceptors. The system can be utilized to enable real-time biochemistry reaction studies.

© 2010 OSA

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2009 (1)

A. Muñoz-Losa, C. Curutchet, B. P. Krueger, L. R. Hartsell, and B. Mennucci, “Fretting about FRET: failure of the ideal dipole approximation,” Biophys. J. 96(12), 4779–4788 (2009).
[CrossRef] [PubMed]

2008 (5)

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]

S. Padilla-Parra, N. Audugé, M. Coppey-Moisan, and M. Tramier, “Quantitative FRET analysis by fast acquisition time domain FLIM at high spatial resolution in living cells,” Biophys. J. 95(6), 2976–2988 (2008).
[CrossRef] [PubMed]

R. Roy, S. Hohng, and T. Ha, “A practical guide to single-molecule FRET,” Nat. Methods 5(6), 507–516 (2008).
[CrossRef] [PubMed]

L. Peng, J. A. Gardecki, B. E. Bouma, and G. J. Tearney, “Fourier fluorescence spectrometer for excitation emission matrix measurement,” Opt. Express 16(14), 10493–10500 (2008).
[CrossRef] [PubMed]

A. D. Hoppe, S. L. Shorte, J. A. Swanson, and R. Heintzmann, “Three-dimensional FRET reconstruction microscopy for analysis of dynamic molecular interactions in live cells,” Biophys. J. 95(1), 400–418 (2008).
[CrossRef] [PubMed]

2007 (4)

L. Peng, J. T. Motz, R. W. Redmond, B. E. Bouma, and G. J. Tearney, “Fourier transform emission lifetime spectrometer,” Opt. Lett. 32(4), 421–423 (2007).
[CrossRef] [PubMed]

D. W. Piston and G. J. Kremers, “Fluorescent protein FRET: the good, the bad and the ugly,” Trends Biochem. Sci. 32(9), 407–414 (2007).
[CrossRef] [PubMed]

R. Seidel and C. Dekker, “Single-molecule studies of nucleic acid motors,” Curr. Opin. Struct. Biol. 17(1), 80–86 (2007).
[CrossRef] [PubMed]

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 (3)

X. Michalet, S. Weiss, and M. Jäger, “Single-Molecule Fluorescence Studies of Protein Folding and Conformational Dynamics,” Chem. Rev. 106(5), 1785–1813 (2006).
[CrossRef] [PubMed]

R. D. Smiley and G. G. Hammes, “Single molecule studies of enzyme mechanisms,” Chem. Rev. 106(8), 3080–3094 (2006).
[CrossRef] [PubMed]

E. A. Jares-Erijman and T. M. Jovin, “Imaging molecular interactions in living cells by FRET microscopy,” Curr. Opin. Chem. Biol. 10(5), 409–416 (2006).
[CrossRef] [PubMed]

2005 (3)

X. Zhuang, “Single-molecule RNA science,” Annu. Rev. Biophys. Biomol. Struct. 34(1), 399–414 (2005).
[CrossRef] [PubMed]

E. B. Van Munster, G. J. Kremers, M. J. Adjobo-Hermans, and T. W. Gadella., “Fluorescence resonance energy transfer (FRET) measurement by gradual acceptor photobleaching,” J. Microsc. 218(3), 253–262 (2005).
[CrossRef] [PubMed]

G. Valentin, C. Verheggen, T. Piolot, H. Neel, M. Coppey-Moisan, and E. Bertrand, “Photoconversion of YFP into a CFP-like species during acceptor photobleaching FRET experiments,” Nat. Methods 2(11), 801 (2005).
[CrossRef] [PubMed]

2004 (4)

2003 (4)

M. Margittai, J. Widengren, E. Schweinberger, G. F. Schröder, S. Felekyan, E. Haustein, M. König, D. Fasshauer, H. Grubmüller, R. Jahn, and C. A. M. Seidel, “Single-molecule fluorescence resonance energy transfer reveals a dynamic equilibrium between closed and open conformations of syntaxin 1,” Proc. Natl. Acad. Sci. U.S.A. 100(26), 15516–15521 (2003).
[CrossRef] [PubMed]

E. A. Lipman, B. Schuler, O. Bakajin, and W. A. Eaton, “Single-molecule measurement of protein folding kinetics,” Science 301(5637), 1233–1235 (2003).
[CrossRef] [PubMed]

A. Miyawaki, “Visualization of the Spatial and Temporal Dynamics of Intracellular Signaling,” Dev. Cell 4(3), 295–305 (2003).
[CrossRef] [PubMed]

C. Berney and G. Danuser, “FRET or no FRET: a Quantitative Comparison,” Biophys. J. 84(6), 3992–4010 (2003).
[CrossRef] [PubMed]

2002 (1)

M. G. Müller, A. Wax, I. Georgakoudi, R. R. Dasari, and M. S. Feld, “A reflectance spectrofluorimeter for real-time spectral diagnosis of disease,” Rev. Sci. Instrum. 73(11), 3933–3937 (2002).
[CrossRef]

2001 (3)

A. K. Kenworthy, “Imaging Protein-Protein Interactions Using Fluorescence Resonance Energy Transfer Microscopy,” Methods 24(3), 289–296 (2001).
[CrossRef] [PubMed]

K. Truong and M. Ikura, “The use of FRET imaging microscopy to detect protein-protein interactions and protein conformational changes in vivo,” Curr. Opin. Struct. Biol. 11(5), 573–578 (2001).
[CrossRef]

M. G. Erickson, B. A. Alseikhan, B. Z. Peterson, and D. T. Yue, “Preassociation of Calmodulin with Voltage-Gated Ca2+ Channels Revealed by FRET in Single Living Cells,” Neuron 31(6), 973–985 (2001).
[CrossRef] [PubMed]

1999 (4)

1998 (2)

J. G. Hirschberg, G. Vereb, C. K. Meyer, A. K. Kirsch, E. Kohen, and T. M. Jovin, “Interferometric measurement of fluorescence excitation spectra,” Appl. Opt. 37(10), 1953–1957 (1998).
[CrossRef]

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).
[CrossRef] [PubMed]

1997 (2)

V. M. Mekler, A. Z. Averbakh, A. B. Sudarikov, and O. V. Kharitonova, “Fluorescence energy transfer-sensitized photobleaching of a fluorescent label as a tool to study donor-acceptor distance distributions and dynamics in protein assemblies: studies of a complex of biotinylated IgM with streptavidin and aggregates of concanavalin A,” J. Photochem. Photobiol. B 40(3), 278–287 (1997).
[CrossRef]

G. J. Tearney, B. E. Bouma, and J. G. Fujimoto, “High-speed phase- and group-delay scanning with a grating-based phase control delay line,” Opt. Lett. 22(23), 1811–1813 (1997).
[CrossRef]

1996 (3)

A. R. Muroski, K. S. Booksh, and M. L. Myrick, “Single-Measurement Excitation/Emission Matrix Spectrofluorometer for Determination of Hydrocarbons in Ocean Water. 1. Instrumentation and Background Correction,” Anal. Chem. 68(20), 3534–3538 (1996).
[CrossRef]

R. A. Zângaro, L. Silveira, R. Manoharan, G. Zonios, I. Itzkan, R. R. Dasari, J. Van Dam, and M. S. Feld, “Rapid multiexcitation fluorescence spectroscopy system for in vivo tissue diagnosis,” Appl. Opt. 35(25), 5211–5219 (1996).
[CrossRef] [PubMed]

R. D. Mitra, C. M. Silva, and D. C. Youvan, “Fluorescence resonance energy transfer between blue-emitting and red-shifted excitation derivatives of the green fluorescent protein,” Gene 173(11 Spec No), 13–17 (1996).
[CrossRef] [PubMed]

1995 (1)

R. M. Clegg, “Fluorescence resonance energy transfer,” Curr. Opin. Biotechnol. 6(1), 103–110 (1995).
[CrossRef] [PubMed]

1993 (1)

C. D. Tran and R. J. Furlan, “Spectrofluorometer based on acousto-optic tunable filters for rapid scanning and multicomponent sample analyses,” Anal. Chem. 65(13), 1675–1681 (1993).
[CrossRef] [PubMed]

1948 (1)

T. Förster, “Zwischenmolekulare energiewanderung und fluoreszenz,” Ann. Phys. 437(1-2), 55–75 (1948).
[CrossRef]

Adjobo-Hermans, M. J.

E. B. Van Munster, G. J. Kremers, M. J. Adjobo-Hermans, and T. W. Gadella., “Fluorescence resonance energy transfer (FRET) measurement by gradual acceptor photobleaching,” J. Microsc. 218(3), 253–262 (2005).
[CrossRef] [PubMed]

Alseikhan, B. A.

M. G. Erickson, B. A. Alseikhan, B. Z. Peterson, and D. T. Yue, “Preassociation of Calmodulin with Voltage-Gated Ca2+ Channels Revealed by FRET in Single Living Cells,” Neuron 31(6), 973–985 (2001).
[CrossRef] [PubMed]

Audugé, N.

S. Padilla-Parra, N. Audugé, M. Coppey-Moisan, and M. Tramier, “Quantitative FRET analysis by fast acquisition time domain FLIM at high spatial resolution in living cells,” Biophys. J. 95(6), 2976–2988 (2008).
[CrossRef] [PubMed]

Averbakh, A. Z.

V. M. Mekler, A. Z. Averbakh, A. B. Sudarikov, and O. V. Kharitonova, “Fluorescence energy transfer-sensitized photobleaching of a fluorescent label as a tool to study donor-acceptor distance distributions and dynamics in protein assemblies: studies of a complex of biotinylated IgM with streptavidin and aggregates of concanavalin A,” J. Photochem. Photobiol. B 40(3), 278–287 (1997).
[CrossRef]

Bakajin, O.

E. A. Lipman, B. Schuler, O. Bakajin, and W. A. Eaton, “Single-molecule measurement of protein folding kinetics,” Science 301(5637), 1233–1235 (2003).
[CrossRef] [PubMed]

Berney, C.

C. Berney and G. Danuser, “FRET or no FRET: a Quantitative Comparison,” Biophys. J. 84(6), 3992–4010 (2003).
[CrossRef] [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).
[CrossRef] [PubMed]

Bertrand, E.

G. Valentin, C. Verheggen, T. Piolot, H. Neel, M. Coppey-Moisan, and E. Bertrand, “Photoconversion of YFP into a CFP-like species during acceptor photobleaching FRET experiments,” Nat. Methods 2(11), 801 (2005).
[CrossRef] [PubMed]

Booksh, K. S.

A. R. Muroski, K. S. Booksh, and M. L. Myrick, “Single-Measurement Excitation/Emission Matrix Spectrofluorometer for Determination of Hydrocarbons in Ocean Water. 1. Instrumentation and Background Correction,” Anal. Chem. 68(20), 3534–3538 (1996).
[CrossRef]

Bouma, B. E.

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]

Clegg, R. M.

R. M. Clegg, “Fluorescence resonance energy transfer,” Curr. Opin. Biotechnol. 6(1), 103–110 (1995).
[CrossRef] [PubMed]

Cobb, M. J.

Coppey-Moisan, M.

S. Padilla-Parra, N. Audugé, M. Coppey-Moisan, and M. Tramier, “Quantitative FRET analysis by fast acquisition time domain FLIM at high spatial resolution in living cells,” Biophys. J. 95(6), 2976–2988 (2008).
[CrossRef] [PubMed]

G. Valentin, C. Verheggen, T. Piolot, H. Neel, M. Coppey-Moisan, and E. Bertrand, “Photoconversion of YFP into a CFP-like species during acceptor photobleaching FRET experiments,” Nat. Methods 2(11), 801 (2005).
[CrossRef] [PubMed]

Curutchet, C.

A. Muñoz-Losa, C. Curutchet, B. P. Krueger, L. R. Hartsell, and B. Mennucci, “Fretting about FRET: failure of the ideal dipole approximation,” Biophys. J. 96(12), 4779–4788 (2009).
[CrossRef] [PubMed]

Danuser, G.

C. Berney and G. Danuser, “FRET or no FRET: a Quantitative Comparison,” Biophys. J. 84(6), 3992–4010 (2003).
[CrossRef] [PubMed]

Dasari, R. R.

M. G. Müller, A. Wax, I. Georgakoudi, R. R. Dasari, and M. S. Feld, “A reflectance spectrofluorimeter for real-time spectral diagnosis of disease,” Rev. Sci. Instrum. 73(11), 3933–3937 (2002).
[CrossRef]

R. A. Zângaro, L. Silveira, R. Manoharan, G. Zonios, I. Itzkan, R. R. Dasari, J. Van Dam, and M. S. Feld, “Rapid multiexcitation fluorescence spectroscopy system for in vivo tissue diagnosis,” Appl. Opt. 35(25), 5211–5219 (1996).
[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]

Dekker, C.

R. Seidel and C. Dekker, “Single-molecule studies of nucleic acid motors,” Curr. Opin. Struct. Biol. 17(1), 80–86 (2007).
[CrossRef] [PubMed]

Durkin, A.

Eaton, W. A.

E. A. Lipman, B. Schuler, O. Bakajin, and W. A. Eaton, “Single-molecule measurement of protein folding kinetics,” Science 301(5637), 1233–1235 (2003).
[CrossRef] [PubMed]

Erickson, M. G.

M. G. Erickson, B. A. Alseikhan, B. Z. Peterson, and D. T. Yue, “Preassociation of Calmodulin with Voltage-Gated Ca2+ Channels Revealed by FRET in Single Living Cells,” Neuron 31(6), 973–985 (2001).
[CrossRef] [PubMed]

Fan, J.

Fasshauer, D.

M. Margittai, J. Widengren, E. Schweinberger, G. F. Schröder, S. Felekyan, E. Haustein, M. König, D. Fasshauer, H. Grubmüller, R. Jahn, and C. A. M. Seidel, “Single-molecule fluorescence resonance energy transfer reveals a dynamic equilibrium between closed and open conformations of syntaxin 1,” Proc. Natl. Acad. Sci. U.S.A. 100(26), 15516–15521 (2003).
[CrossRef] [PubMed]

Feld, M. S.

M. G. Müller, A. Wax, I. Georgakoudi, R. R. Dasari, and M. S. Feld, “A reflectance spectrofluorimeter for real-time spectral diagnosis of disease,” Rev. Sci. Instrum. 73(11), 3933–3937 (2002).
[CrossRef]

R. A. Zângaro, L. Silveira, R. Manoharan, G. Zonios, I. Itzkan, R. R. Dasari, J. Van Dam, and M. S. Feld, “Rapid multiexcitation fluorescence spectroscopy system for in vivo tissue diagnosis,” Appl. Opt. 35(25), 5211–5219 (1996).
[CrossRef] [PubMed]

Felekyan, S.

M. Margittai, J. Widengren, E. Schweinberger, G. F. Schröder, S. Felekyan, E. Haustein, M. König, D. Fasshauer, H. Grubmüller, R. Jahn, and C. A. M. Seidel, “Single-molecule fluorescence resonance energy transfer reveals a dynamic equilibrium between closed and open conformations of syntaxin 1,” Proc. Natl. Acad. Sci. U.S.A. 100(26), 15516–15521 (2003).
[CrossRef] [PubMed]

Förster, T.

T. Förster, “Zwischenmolekulare energiewanderung und fluoreszenz,” Ann. Phys. 437(1-2), 55–75 (1948).
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E. B. Van Munster, G. J. Kremers, M. J. Adjobo-Hermans, and T. W. Gadella., “Fluorescence resonance energy transfer (FRET) measurement by gradual acceptor photobleaching,” J. Microsc. 218(3), 253–262 (2005).
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T. Zal and N. R. J. Gascoigne, “Photobleaching-Corrected FRET Efficiency Imaging of Live Cells,” Biophys. J. 86(6), 3923–3939 (2004).
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Georgakoudi, I.

M. G. Müller, A. Wax, I. Georgakoudi, R. R. Dasari, and M. S. Feld, “A reflectance spectrofluorimeter for real-time spectral diagnosis of disease,” Rev. Sci. Instrum. 73(11), 3933–3937 (2002).
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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).
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P. R. Griffiths, B. L. Hirsche, and C. J. Manning, “Ultra-rapid-scanning Fourier transform infrared spectrometry,” Vib. Spectrosc. 19(1), 165–176 (1999).
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R. Roy, S. Hohng, and T. Ha, “A practical guide to single-molecule FRET,” Nat. Methods 5(6), 507–516 (2008).
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A. Muñoz-Losa, C. Curutchet, B. P. Krueger, L. R. Hartsell, and B. Mennucci, “Fretting about FRET: failure of the ideal dipole approximation,” Biophys. J. 96(12), 4779–4788 (2009).
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M. Margittai, J. Widengren, E. Schweinberger, G. F. Schröder, S. Felekyan, E. Haustein, M. König, D. Fasshauer, H. Grubmüller, R. Jahn, and C. A. M. Seidel, “Single-molecule fluorescence resonance energy transfer reveals a dynamic equilibrium between closed and open conformations of syntaxin 1,” Proc. Natl. Acad. Sci. U.S.A. 100(26), 15516–15521 (2003).
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B. A. Pollok and R. Heim, “Using GFP in FRET-based applications,” Trends Cell Biol. 9(2), 57–60 (1999).
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A. D. Hoppe, S. L. Shorte, J. A. Swanson, and R. Heintzmann, “Three-dimensional FRET reconstruction microscopy for analysis of dynamic molecular interactions in live cells,” Biophys. J. 95(1), 400–418 (2008).
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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).
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P. R. Griffiths, B. L. Hirsche, and C. J. Manning, “Ultra-rapid-scanning Fourier transform infrared spectrometry,” Vib. Spectrosc. 19(1), 165–176 (1999).
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R. Roy, S. Hohng, and T. Ha, “A practical guide to single-molecule FRET,” Nat. Methods 5(6), 507–516 (2008).
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A. D. Hoppe, S. L. Shorte, J. A. Swanson, and R. Heintzmann, “Three-dimensional FRET reconstruction microscopy for analysis of dynamic molecular interactions in live cells,” Biophys. J. 95(1), 400–418 (2008).
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K. Truong and M. Ikura, “The use of FRET imaging microscopy to detect protein-protein interactions and protein conformational changes in vivo,” Curr. Opin. Struct. Biol. 11(5), 573–578 (2001).
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X. Michalet, S. Weiss, and M. Jäger, “Single-Molecule Fluorescence Studies of Protein Folding and Conformational Dynamics,” Chem. Rev. 106(5), 1785–1813 (2006).
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M. Margittai, J. Widengren, E. Schweinberger, G. F. Schröder, S. Felekyan, E. Haustein, M. König, D. Fasshauer, H. Grubmüller, R. Jahn, and C. A. M. Seidel, “Single-molecule fluorescence resonance energy transfer reveals a dynamic equilibrium between closed and open conformations of syntaxin 1,” Proc. Natl. Acad. Sci. U.S.A. 100(26), 15516–15521 (2003).
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E. A. Jares-Erijman and T. M. Jovin, “Imaging molecular interactions in living cells by FRET microscopy,” Curr. Opin. Chem. Biol. 10(5), 409–416 (2006).
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V. M. Mekler, A. Z. Averbakh, A. B. Sudarikov, and O. V. Kharitonova, “Fluorescence energy transfer-sensitized photobleaching of a fluorescent label as a tool to study donor-acceptor distance distributions and dynamics in protein assemblies: studies of a complex of biotinylated IgM with streptavidin and aggregates of concanavalin A,” J. Photochem. Photobiol. B 40(3), 278–287 (1997).
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Kirsch, A. K.

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).
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König, M.

M. Margittai, J. Widengren, E. Schweinberger, G. F. Schröder, S. Felekyan, E. Haustein, M. König, D. Fasshauer, H. Grubmüller, R. Jahn, and C. A. M. Seidel, “Single-molecule fluorescence resonance energy transfer reveals a dynamic equilibrium between closed and open conformations of syntaxin 1,” Proc. Natl. Acad. Sci. U.S.A. 100(26), 15516–15521 (2003).
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D. W. Piston and G. J. Kremers, “Fluorescent protein FRET: the good, the bad and the ugly,” Trends Biochem. Sci. 32(9), 407–414 (2007).
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E. B. Van Munster, G. J. Kremers, M. J. Adjobo-Hermans, and T. W. Gadella., “Fluorescence resonance energy transfer (FRET) measurement by gradual acceptor photobleaching,” J. Microsc. 218(3), 253–262 (2005).
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A. Muñoz-Losa, C. Curutchet, B. P. Krueger, L. R. Hartsell, and B. Mennucci, “Fretting about FRET: failure of the ideal dipole approximation,” Biophys. J. 96(12), 4779–4788 (2009).
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Kuzminskiy, V.

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).
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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).
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E. A. Lipman, B. Schuler, O. Bakajin, and W. A. Eaton, “Single-molecule measurement of protein folding kinetics,” Science 301(5637), 1233–1235 (2003).
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Luryi, S.

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P. R. Griffiths, B. L. Hirsche, and C. J. Manning, “Ultra-rapid-scanning Fourier transform infrared spectrometry,” Vib. Spectrosc. 19(1), 165–176 (1999).
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Margittai, M.

M. Margittai, J. Widengren, E. Schweinberger, G. F. Schröder, S. Felekyan, E. Haustein, M. König, D. Fasshauer, H. Grubmüller, R. Jahn, and C. A. M. Seidel, “Single-molecule fluorescence resonance energy transfer reveals a dynamic equilibrium between closed and open conformations of syntaxin 1,” Proc. Natl. Acad. Sci. U.S.A. 100(26), 15516–15521 (2003).
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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).
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V. M. Mekler, A. Z. Averbakh, A. B. Sudarikov, and O. V. Kharitonova, “Fluorescence energy transfer-sensitized photobleaching of a fluorescent label as a tool to study donor-acceptor distance distributions and dynamics in protein assemblies: studies of a complex of biotinylated IgM with streptavidin and aggregates of concanavalin A,” J. Photochem. Photobiol. B 40(3), 278–287 (1997).
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A. Muñoz-Losa, C. Curutchet, B. P. Krueger, L. R. Hartsell, and B. Mennucci, “Fretting about FRET: failure of the ideal dipole approximation,” Biophys. J. 96(12), 4779–4788 (2009).
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Michalet, X.

X. Michalet, S. Weiss, and M. Jäger, “Single-Molecule Fluorescence Studies of Protein Folding and Conformational Dynamics,” Chem. Rev. 106(5), 1785–1813 (2006).
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R. D. Mitra, C. M. Silva, and D. C. Youvan, “Fluorescence resonance energy transfer between blue-emitting and red-shifted excitation derivatives of the green fluorescent protein,” Gene 173(11 Spec No), 13–17 (1996).
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M. G. Müller, A. Wax, I. Georgakoudi, R. R. Dasari, and M. S. Feld, “A reflectance spectrofluorimeter for real-time spectral diagnosis of disease,” Rev. Sci. Instrum. 73(11), 3933–3937 (2002).
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A. Muñoz-Losa, C. Curutchet, B. P. Krueger, L. R. Hartsell, and B. Mennucci, “Fretting about FRET: failure of the ideal dipole approximation,” Biophys. J. 96(12), 4779–4788 (2009).
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A. R. Muroski, K. S. Booksh, and M. L. Myrick, “Single-Measurement Excitation/Emission Matrix Spectrofluorometer for Determination of Hydrocarbons in Ocean Water. 1. Instrumentation and Background Correction,” Anal. Chem. 68(20), 3534–3538 (1996).
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A. R. Muroski, K. S. Booksh, and M. L. Myrick, “Single-Measurement Excitation/Emission Matrix Spectrofluorometer for Determination of Hydrocarbons in Ocean Water. 1. Instrumentation and Background Correction,” Anal. Chem. 68(20), 3534–3538 (1996).
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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).
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S. Padilla-Parra, N. Audugé, M. Coppey-Moisan, and M. Tramier, “Quantitative FRET analysis by fast acquisition time domain FLIM at high spatial resolution in living cells,” Biophys. J. 95(6), 2976–2988 (2008).
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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).
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M. G. Erickson, B. A. Alseikhan, B. Z. Peterson, and D. T. Yue, “Preassociation of Calmodulin with Voltage-Gated Ca2+ Channels Revealed by FRET in Single Living Cells,” Neuron 31(6), 973–985 (2001).
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G. Valentin, C. Verheggen, T. Piolot, H. Neel, M. Coppey-Moisan, and E. Bertrand, “Photoconversion of YFP into a CFP-like species during acceptor photobleaching FRET experiments,” Nat. Methods 2(11), 801 (2005).
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D. W. Piston and G. J. Kremers, “Fluorescent protein FRET: the good, the bad and the ugly,” Trends Biochem. Sci. 32(9), 407–414 (2007).
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B. A. Pollok and R. Heim, “Using GFP in FRET-based applications,” Trends Cell Biol. 9(2), 57–60 (1999).
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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).
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Richards-Kortum, R.

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R. Roy, S. Hohng, and T. Ha, “A practical guide to single-molecule FRET,” Nat. Methods 5(6), 507–516 (2008).
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M. Margittai, J. Widengren, E. Schweinberger, G. F. Schröder, S. Felekyan, E. Haustein, M. König, D. Fasshauer, H. Grubmüller, R. Jahn, and C. A. M. Seidel, “Single-molecule fluorescence resonance energy transfer reveals a dynamic equilibrium between closed and open conformations of syntaxin 1,” Proc. Natl. Acad. Sci. U.S.A. 100(26), 15516–15521 (2003).
[CrossRef] [PubMed]

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E. A. Lipman, B. Schuler, O. Bakajin, and W. A. Eaton, “Single-molecule measurement of protein folding kinetics,” Science 301(5637), 1233–1235 (2003).
[CrossRef] [PubMed]

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M. Margittai, J. Widengren, E. Schweinberger, G. F. Schröder, S. Felekyan, E. Haustein, M. König, D. Fasshauer, H. Grubmüller, R. Jahn, and C. A. M. Seidel, “Single-molecule fluorescence resonance energy transfer reveals a dynamic equilibrium between closed and open conformations of syntaxin 1,” Proc. Natl. Acad. Sci. U.S.A. 100(26), 15516–15521 (2003).
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M. Margittai, J. Widengren, E. Schweinberger, G. F. Schröder, S. Felekyan, E. Haustein, M. König, D. Fasshauer, H. Grubmüller, R. Jahn, and C. A. M. Seidel, “Single-molecule fluorescence resonance energy transfer reveals a dynamic equilibrium between closed and open conformations of syntaxin 1,” Proc. Natl. Acad. Sci. U.S.A. 100(26), 15516–15521 (2003).
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A. D. Hoppe, S. L. Shorte, J. A. Swanson, and R. Heintzmann, “Three-dimensional FRET reconstruction microscopy for analysis of dynamic molecular interactions in live cells,” Biophys. J. 95(1), 400–418 (2008).
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R. D. Mitra, C. M. Silva, and D. C. Youvan, “Fluorescence resonance energy transfer between blue-emitting and red-shifted excitation derivatives of the green fluorescent protein,” Gene 173(11 Spec No), 13–17 (1996).
[CrossRef] [PubMed]

Silveira, L.

Smiley, R. D.

R. D. Smiley and G. G. Hammes, “Single molecule studies of enzyme mechanisms,” Chem. Rev. 106(8), 3080–3094 (2006).
[CrossRef] [PubMed]

Sudarikov, A. B.

V. M. Mekler, A. Z. Averbakh, A. B. Sudarikov, and O. V. Kharitonova, “Fluorescence energy transfer-sensitized photobleaching of a fluorescent label as a tool to study donor-acceptor distance distributions and dynamics in protein assemblies: studies of a complex of biotinylated IgM with streptavidin and aggregates of concanavalin A,” J. Photochem. Photobiol. B 40(3), 278–287 (1997).
[CrossRef]

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A. D. Hoppe, S. L. Shorte, J. A. Swanson, and R. Heintzmann, “Three-dimensional FRET reconstruction microscopy for analysis of dynamic molecular interactions in live cells,” Biophys. J. 95(1), 400–418 (2008).
[CrossRef] [PubMed]

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Tramier, M.

S. Padilla-Parra, N. Audugé, M. Coppey-Moisan, and M. Tramier, “Quantitative FRET analysis by fast acquisition time domain FLIM at high spatial resolution in living cells,” Biophys. J. 95(6), 2976–2988 (2008).
[CrossRef] [PubMed]

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C. D. Tran and R. J. Furlan, “Spectrofluorometer based on acousto-optic tunable filters for rapid scanning and multicomponent sample analyses,” Anal. Chem. 65(13), 1675–1681 (1993).
[CrossRef] [PubMed]

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K. Truong and M. Ikura, “The use of FRET imaging microscopy to detect protein-protein interactions and protein conformational changes in vivo,” Curr. Opin. Struct. Biol. 11(5), 573–578 (2001).
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Valentin, G.

G. Valentin, C. Verheggen, T. Piolot, H. Neel, M. Coppey-Moisan, and E. Bertrand, “Photoconversion of YFP into a CFP-like species during acceptor photobleaching FRET experiments,” Nat. Methods 2(11), 801 (2005).
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Van Munster, E. B.

E. B. Van Munster, G. J. Kremers, M. J. Adjobo-Hermans, and T. W. Gadella., “Fluorescence resonance energy transfer (FRET) measurement by gradual acceptor photobleaching,” J. Microsc. 218(3), 253–262 (2005).
[CrossRef] [PubMed]

Vereb, G.

Verheggen, C.

G. Valentin, C. Verheggen, T. Piolot, H. Neel, M. Coppey-Moisan, and E. Bertrand, “Photoconversion of YFP into a CFP-like species during acceptor photobleaching FRET experiments,” Nat. Methods 2(11), 801 (2005).
[CrossRef] [PubMed]

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M. G. Müller, A. Wax, I. Georgakoudi, R. R. Dasari, and M. S. Feld, “A reflectance spectrofluorimeter for real-time spectral diagnosis of disease,” Rev. Sci. Instrum. 73(11), 3933–3937 (2002).
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X. Michalet, S. Weiss, and M. Jäger, “Single-Molecule Fluorescence Studies of Protein Folding and Conformational Dynamics,” Chem. Rev. 106(5), 1785–1813 (2006).
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M. Margittai, J. Widengren, E. Schweinberger, G. F. Schröder, S. Felekyan, E. Haustein, M. König, D. Fasshauer, H. Grubmüller, R. Jahn, and C. A. M. Seidel, “Single-molecule fluorescence resonance energy transfer reveals a dynamic equilibrium between closed and open conformations of syntaxin 1,” Proc. Natl. Acad. Sci. U.S.A. 100(26), 15516–15521 (2003).
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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).
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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).
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R. D. Mitra, C. M. Silva, and D. C. Youvan, “Fluorescence resonance energy transfer between blue-emitting and red-shifted excitation derivatives of the green fluorescent protein,” Gene 173(11 Spec No), 13–17 (1996).
[CrossRef] [PubMed]

Yue, D. T.

M. G. Erickson, B. A. Alseikhan, B. Z. Peterson, and D. T. Yue, “Preassociation of Calmodulin with Voltage-Gated Ca2+ Channels Revealed by FRET in Single Living Cells,” Neuron 31(6), 973–985 (2001).
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T. Zal and N. R. J. Gascoigne, “Photobleaching-Corrected FRET Efficiency Imaging of Live Cells,” Biophys. J. 86(6), 3923–3939 (2004).
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Zângaro, R. A.

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).
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X. Zhuang, “Single-molecule RNA science,” Annu. Rev. Biophys. Biomol. Struct. 34(1), 399–414 (2005).
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Zuluaga, A. F.

Anal. Chem. (2)

A. R. Muroski, K. S. Booksh, and M. L. Myrick, “Single-Measurement Excitation/Emission Matrix Spectrofluorometer for Determination of Hydrocarbons in Ocean Water. 1. Instrumentation and Background Correction,” Anal. Chem. 68(20), 3534–3538 (1996).
[CrossRef]

C. D. Tran and R. J. Furlan, “Spectrofluorometer based on acousto-optic tunable filters for rapid scanning and multicomponent sample analyses,” Anal. Chem. 65(13), 1675–1681 (1993).
[CrossRef] [PubMed]

Ann. Phys. (1)

T. Förster, “Zwischenmolekulare energiewanderung und fluoreszenz,” Ann. Phys. 437(1-2), 55–75 (1948).
[CrossRef]

Annu. Rev. Biophys. Biomol. Struct. (1)

X. Zhuang, “Single-molecule RNA science,” Annu. Rev. Biophys. Biomol. Struct. 34(1), 399–414 (2005).
[CrossRef] [PubMed]

Appl. Opt. (4)

Appl. Spectrosc. (1)

Biophys. J. (7)

A. Muñoz-Losa, C. Curutchet, B. P. Krueger, L. R. Hartsell, and B. Mennucci, “Fretting about FRET: failure of the ideal dipole approximation,” Biophys. J. 96(12), 4779–4788 (2009).
[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]

S. Padilla-Parra, N. Audugé, M. Coppey-Moisan, and M. Tramier, “Quantitative FRET analysis by fast acquisition time domain FLIM at high spatial resolution in living cells,” Biophys. J. 95(6), 2976–2988 (2008).
[CrossRef] [PubMed]

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Figures (4)

Fig. 1
Fig. 1

High speed Fourier fluorescence excitation emission spectrometer. The system consisted of a broadband light source and a double-pass Michelson interferometer. SP: short pass filter; LP: long pass filter; BS: beam splitter; BP: beam pickup; DM: dichroic mirror; L: lens; Obj: Objective

Fig. 2
Fig. 2

Normalized EEM’s projections for a mixture of Alexa Fluor 514 goat anti-rabbit IgG (donor) and Alexa Fluor 568 rabbit anti-mouse IgG (acceptor) secondary antibodies. EEM projections were measured at 600 sets per second. (a) Excitation spectra (0 degree EEM projection), (b) Emission spectra (90 degree EEM projection), and (c) Excitation-emission cross-correlation (45 degree diagonal projection of EEM). Blue dashed, green dotted and purple solid lines present donor, acceptor and their mixture, respectively.

Fig. 3
Fig. 3

Quantitative FRET analysis by EEM spectroscopy of the mixtures of Alexa Fluor 514 goat anti-rabbit IgG (donor) and Alexa Fluor 568 rabbit anti-mouse IgG (acceptor) secondary antibodies in the group of acceptor titration experiments. EEM diagonal projection amplitudes from the donor emission, direct acceptor excitation and FRET channels obtained by linear unmixing (a) Excitation spectra (0 degree diagonal projection of EEM), (b) Emission spectra (90 degree diagonal projection of EEM) and (c) Excitation-emission cross-correlation (45 degree diagonal projection of EEM). The spectral amplitudes from donor emission, direct acceptor excitation and FRET channels are presented in blue, green and purple, respectively. Fitting curves are shown as dashed lines. Error bars and circular points represent standard deviation and mean value of 5 experiments, respectively.

Fig. 4
Fig. 4

Quantitative FRET analysis by EEM spectroscopy of the mixtures of Alexa Fluor 514 goat anti-rabbit IgG (donor) and Alexa Fluor 568 rabbit anti-mouse IgG (acceptor) secondary antibodies in the group of donor titration experiments. EEM projection amplitudes contributed by the donor emission and acceptor emission channels obtained by linear unmixing (a) Excitation spectra (0 degree EEM projection), (b) Emission spectra (90 degree EEM projection) and (c) Excitation-emission cross-correlation (45 degree diagonal projection of EEM). The spectral amplitudes from donor emission, direct acceptor excitation and FRET channels are presented in blue, green and purple, respectively. Fitting curves are shown as dashed lines. Error bars and circular points represent standard deviation and mean value of 5 experiments, respectively.

Equations (13)

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O P D ( ϕ ) = 4 h [ tan ( ϕ + θ ) tan θ ]
P I l l u m ( O P D ) P 0 [ 1 + cos ( 2 π σ 1 O P D ) ]
I E m ( O P D ) P 0 [ 1 + cos ( 2 π σ 1 O P D ) ] S E x ( σ 1 ) S E m ( σ 2 )
P E m ( O P D ) P 0 S E x ( σ 1 ) S E m ( σ 2 ) [ 1 + cos ( 2 π σ 1 O P D ) ] [ 1 + cos ( 2 π σ 2 O P D ) ] d σ 2
P E m ( O P D ) S 0 S E x ( σ 1 ) S E m ( σ 2 ) [ 1 + cos ( 2 π σ 1 O P D ) ] [ 1 + cos ( 2 π σ 2 O P D ) ] d σ 1 d σ 2
I ( σ ) F [ P E m ( O P D ) ] O P D = S 0 ( σ 1 ) S E x ( σ 1 ) S E m ( σ 2 ) d σ 1 d σ 2 + S 0 ( σ ) S E x ( σ ) S E m ( σ 2 ) d σ 2 + S 0 ( σ 1 ) S E x ( σ 1 ) S E m ( σ ) d σ 1 + S 0 ( σ 1 ) S E x ( σ 1 ) S E m ( σ σ 1 ) d σ 1
I ( σ ) = A D 0 S D 0 ( σ ) + A A 0 S A 0 ( σ ) + A D 90 S D 90 ( σ ) + A A 90 S A 90 ( σ ) + A D 45 S D 45 ( σ ) + A A 45 S A 45 ( σ ) + A F R E T 45 S F R E T 45 ( σ )
( A D 0 A A 0 A D 90 A A 90 A D 45 A A 45 A F R E T 45 ) ( α D 0 α D ρ F R E T ( 1 + g ) 0 α A g 0 α D 0 α D ρ F R E T 0 α A g α D ρ F R E T g α D 0 α D ρ F R E T 0 α A g 0 0 0 α D ρ F R E T g ) ( C D C A C D A )
g = Q A η A Q D η D
Δ A F R E T 45 = g Δ A D 45
Δ A D 45 { A D 45 } B e f o r e = Δ A D 90 { A D 90 } B e f o r e = ρ F R E T
Δ A A 90 Δ C D A : Δ A D 90 Δ C D A = Δ A A 90 Δ C D : Δ A D 90 Δ C D = ( g ρ F R E T ) : ( 1 ρ F R E T )
Δ A A 45 Δ C D A : Δ A D 45 Δ C D A = Δ A A 45 Δ C D : Δ A D 45 Δ C D = ( g ρ F R E T ) : ( 1 ρ F R E T )

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