Abstract

We propose two-photon excited fluorescence (TPEF) microscopy employing a novel phase modulation technique of ultra-broadband laser pulses, which allows the relative excitation of an individual fluorophore with respect to other fluorophores. This technique is based on the generation of multi-wavelength pulse train, which independently interacts with each fluorophore. Our technique is applied to dual-color imaging of cells expressing two types of fluorescent proteins. We achieve the selective excitation of one over the other and vice versa. The product of the maximum contrast ratios exceeds 100. We also demonstrate yielded equal excitation rates in the simultaneous excitation. By the selective excitation of a donor fluorescent protein, fluorescence resonance energy transfer imaging is also achieved.

© 2009 OSA

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    [CrossRef]

2009

2008

A. A. Eilanlou, Y. Nabekawa, K. L. Ishikawa, H. Takahashi, and K. Midorikawa “Direct amplification of terawatt sub-10-fs pulses in a CPA system of Ti:sapphire laser,” Opt. Express 16, 13431–13438 (2008).
[CrossRef]

B. Vacano and M. Motzkus, “Time-resolving molecular vibration for microanalytics: single laser beam nonlinear Raman spectroscopy in simulation and experiment,” Phys. Chem. Chem. Phys. 10(5), 681–691 (2008).
[CrossRef]

K. Isobe, A. Suda, M. Tanaka, F. Kannari, H. Kawano, H. Mizuno, A. Miyawaki, and K. Midorikawa, “Fourier transform spectroscopy combined with 5-fs broadband pulse for multispectral nonlinear microscopy,” Phys. Rev. A . 77, 063832/1–13 (2008).
[CrossRef]

2006

M. A. Mena, T. P. Treynor, S. L. Mayo, and P. S. Daugherty, “Blue fluorescent proteins with enhanced brightness and photostability from a structurally targeted library,” Nat. Biotechnol. 24(12), 1569–1571 (2006).
[CrossRef] [PubMed]

V. V. Lozovoy, B. Xu, J. C. Shane, and M. Dantus “Selective nonlinear optical excitation with pulses shaped by pseudorandom Galois fields,” Phys. Rev. A 74, 041805(R) (2006).
[CrossRef]

L. T. Schelhas, J. C. Shane, and M. Dantus, “Advantages of ultrashort phase-shaped pulses for selective two-photon activation and biomedical imaging,” Nanomedicine 2(3), 177–181 (2006).
[CrossRef]

J. P. Ogilvie, D. Débarre, X. Solinas, J.-L. Martin, E. Beaurepaire, and M. Joffre, “Use of coherent control for selective two-photon fluorescence microscopy in live organisms,” Opt. Express 14(2), 759–766 (2006).
[CrossRef] [PubMed]

2005

2004

T. Nagai, S. Yamada, T. Tominaga, M. Ichikawa, and A. Miyawaki, “Expanded dynamic range of fluorescent indicators for Ca(2+) by circularly permuted yellow fluorescent proteins,” Proc. Natl. Acad. Sci. U.S.A. 101(29), 10554–10559 (2004).
[CrossRef] [PubMed]

M. Comstock, V. V. Lozovoy, I. Pastirk, and M. Dantus, “Multiphoton intrapulse interference 6; binary phase shaping,” Opt. Express 12(6), 1061–1066 (2004).
[CrossRef] [PubMed]

2003

V. V. Lozovoy, I. Pastirk, K. A. Walowicz, and M. Dantus, “Multiphoton intrapulse interference. II. Control of two- and three-photon laser induced fluorescence with shaped pulses,” J. Chem. Phys. 118(7), 3187–3196 (2003).
[CrossRef]

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

2002

K. A. Walowicz, I. Pastirk, V. V. Lozovoy, and M. Dantus, “Multiphoton intrapulse interference. 1. Control of multiphoton processes in condensed phases,” J. Phys. Chem. A 106(41), 9369–9373 (2002).
[CrossRef]

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]

2000

K. König, “Multiphoton microscopy in life sciences,” J. Microsc. 200(Pt 2), 83–104 (2000).
[CrossRef] [PubMed]

K. G. Heinze, A. Koltermann, and P. Schwille, “Simultaneous two-photon excitation of distinct labels for dual-color fluorescence crosscorrelation analysis,” Proc. Natl. Acad. Sci. U.S.A. 97(19), 10377–10382 (2000).
[CrossRef] [PubMed]

1999

G. Y. Fan, H. Fujisaki, A. Miyawaki, R. K. Tsay, R. Y. Tsien, and M. H. Ellisman, “Video-rate scanning two-photon excitation fluorescence microscopy and ratio imaging with cameleons,” Biophys. J. 76(5), 2412–2420 (1999).
[CrossRef] [PubMed]

E. Zeek, K. Maginnis, S. Backus, U. Russek, M. Murnane, G. Mourou, H. Kapteyn, and G. Vdovin, “Pulse compression by use of deformable mirrors,” Opt. Lett. 24(7), 493–495 (1999).
[CrossRef]

K. Fujita, O. Nakamura, T. Kaneko, S. Kawata, M. Oyamada, and T. Takamatsu, “Real-time imaging of two-photon-induced fluorescence with a microlens-array scanner and a regenerative amplifier,” J. Microsc. 194(Pt 2-3), 528–531 (1999).
[CrossRef]

1998

A. H. Buist, M. Müller, J. Squier, and G. J. Brakenhoff, “Real time two-photon absorption microscopy using multi point excitation,” J. Microsc. 192(2), 217–226 (1998).
[CrossRef]

P. Allcock and D. L. Andrews, “Two-photon fluorescence: Resonance energy transfer,” J. Chem. Phys. 108(8), 3089–3095 (1998).
[CrossRef]

D. Meshulach and Y. Silberberg, “Coherent quantum control of two-photon transitions by a femtosecond laser pulse,” Nature 396(6708), 239–242 (1998).
[CrossRef]

1997

D. C. Youvan, C. M. Silva, E. J. Bylina, W. J. Coleman, M. R. Dilworth, and M. M. Yang, “Calibration of fluorescence resonance energy transfer in microscopy using genetically engineered GFP derivatives on nickel chelating beads,” Biotechnology. 3, 1–18 (1997).

A. Miyawaki, J. Llopis, R. Heim, J. M. McCaffery, J. A. Adams, M. Ikura, and R. Y. Tsien, “Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin,” Nature 388(6645), 882–887 (1997).
[CrossRef] [PubMed]

1996

C. Xu, W. Zipfel, J. B. Shear, R. M. Williams, and W. W. Webb, “Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy,” Proc. Natl. Acad. Sci. U.S.A. 93(20), 10763–10768 (1996).
[CrossRef] [PubMed]

1994

1990

Adams, J. A.

A. Miyawaki, J. Llopis, R. Heim, J. M. McCaffery, J. A. Adams, M. Ikura, and R. Y. Tsien, “Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin,” Nature 388(6645), 882–887 (1997).
[CrossRef] [PubMed]

Allcock, P.

P. Allcock and D. L. Andrews, “Two-photon fluorescence: Resonance energy transfer,” J. Chem. Phys. 108(8), 3089–3095 (1998).
[CrossRef]

Andrews, D. L.

P. Allcock and D. L. Andrews, “Two-photon fluorescence: Resonance energy transfer,” J. Chem. Phys. 108(8), 3089–3095 (1998).
[CrossRef]

Angelow, G.

Backus, S.

Beaurepaire, E.

Birge, J. R.

Boutou, V.

M. Roth, L. Guyon, J. Roslund, V. Boutou, F. Courvoisier, J.-P. Wolf, and H. Rabitz, “Quantum control of tightly competitive product channels,” Phys. Rev. Lett. 102, 253001 (2009).
[CrossRef] [PubMed]

Brakenhoff, G. J.

A. H. Buist, M. Müller, J. Squier, and G. J. Brakenhoff, “Real time two-photon absorption microscopy using multi point excitation,” J. Microsc. 192(2), 217–226 (1998).
[CrossRef]

Buist, A. H.

A. H. Buist, M. Müller, J. Squier, and G. J. Brakenhoff, “Real time two-photon absorption microscopy using multi point excitation,” J. Microsc. 192(2), 217–226 (1998).
[CrossRef]

Bylina, E. J.

D. C. Youvan, C. M. Silva, E. J. Bylina, W. J. Coleman, M. R. Dilworth, and M. M. Yang, “Calibration of fluorescence resonance energy transfer in microscopy using genetically engineered GFP derivatives on nickel chelating beads,” Biotechnology. 3, 1–18 (1997).

Coleman, W. J.

D. C. Youvan, C. M. Silva, E. J. Bylina, W. J. Coleman, M. R. Dilworth, and M. M. Yang, “Calibration of fluorescence resonance energy transfer in microscopy using genetically engineered GFP derivatives on nickel chelating beads,” Biotechnology. 3, 1–18 (1997).

Comstock, M.

Courvoisier, F.

M. Roth, L. Guyon, J. Roslund, V. Boutou, F. Courvoisier, J.-P. Wolf, and H. Rabitz, “Quantum control of tightly competitive product channels,” Phys. Rev. Lett. 102, 253001 (2009).
[CrossRef] [PubMed]

Dantus, M.

V. V. Lozovoy, B. Xu, J. C. Shane, and M. Dantus “Selective nonlinear optical excitation with pulses shaped by pseudorandom Galois fields,” Phys. Rev. A 74, 041805(R) (2006).
[CrossRef]

L. T. Schelhas, J. C. Shane, and M. Dantus, “Advantages of ultrashort phase-shaped pulses for selective two-photon activation and biomedical imaging,” Nanomedicine 2(3), 177–181 (2006).
[CrossRef]

M. Comstock, V. V. Lozovoy, I. Pastirk, and M. Dantus, “Multiphoton intrapulse interference 6; binary phase shaping,” Opt. Express 12(6), 1061–1066 (2004).
[CrossRef] [PubMed]

V. V. Lozovoy, I. Pastirk, K. A. Walowicz, and M. Dantus, “Multiphoton intrapulse interference. II. Control of two- and three-photon laser induced fluorescence with shaped pulses,” J. Chem. Phys. 118(7), 3187–3196 (2003).
[CrossRef]

K. A. Walowicz, I. Pastirk, V. V. Lozovoy, and M. Dantus, “Multiphoton intrapulse interference. 1. Control of multiphoton processes in condensed phases,” J. Phys. Chem. A 106(41), 9369–9373 (2002).
[CrossRef]

Daugherty, P. S.

M. A. Mena, T. P. Treynor, S. L. Mayo, and P. S. Daugherty, “Blue fluorescent proteins with enhanced brightness and photostability from a structurally targeted library,” Nat. Biotechnol. 24(12), 1569–1571 (2006).
[CrossRef] [PubMed]

de Boeij, W. P.

Débarre, D.

Denk, W.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Dilworth, M. R.

D. C. Youvan, C. M. Silva, E. J. Bylina, W. J. Coleman, M. R. Dilworth, and M. M. Yang, “Calibration of fluorescence resonance energy transfer in microscopy using genetically engineered GFP derivatives on nickel chelating beads,” Biotechnology. 3, 1–18 (1997).

Eilanlou, A. A.

Ellisman, M. H.

G. Y. Fan, H. Fujisaki, A. Miyawaki, R. K. Tsay, R. Y. Tsien, and M. H. Ellisman, “Video-rate scanning two-photon excitation fluorescence microscopy and ratio imaging with cameleons,” Biophys. J. 76(5), 2412–2420 (1999).
[CrossRef] [PubMed]

Fan, G. Y.

G. Y. Fan, H. Fujisaki, A. Miyawaki, R. K. Tsay, R. Y. Tsien, and M. H. Ellisman, “Video-rate scanning two-photon excitation fluorescence microscopy and ratio imaging with cameleons,” Biophys. J. 76(5), 2412–2420 (1999).
[CrossRef] [PubMed]

Frumker, E.

Fujimoto, J. G.

Fujisaki, H.

G. Y. Fan, H. Fujisaki, A. Miyawaki, R. K. Tsay, R. Y. Tsien, and M. H. Ellisman, “Video-rate scanning two-photon excitation fluorescence microscopy and ratio imaging with cameleons,” Biophys. J. 76(5), 2412–2420 (1999).
[CrossRef] [PubMed]

Fujita, K.

K. Fujita, O. Nakamura, T. Kaneko, S. Kawata, M. Oyamada, and T. Takamatsu, “Real-time imaging of two-photon-induced fluorescence with a microlens-array scanner and a regenerative amplifier,” J. Microsc. 194(Pt 2-3), 528–531 (1999).
[CrossRef]

Fukui, K.

K. Isobe, W. Watanabe, S. Matsunaga, T. Higashi, K. Fukui, and K. Itoh, “Multi-spectral two-photon excited fluorescence microscopy using supercontinuum light source,” Jpn. J. Appl. Phys. 44(4), L167–L169 (2005).
[CrossRef]

Goswami, D.

Guyon, L.

M. Roth, L. Guyon, J. Roslund, V. Boutou, F. Courvoisier, J.-P. Wolf, and H. Rabitz, “Quantum control of tightly competitive product channels,” Phys. Rev. Lett. 102, 253001 (2009).
[CrossRef] [PubMed]

Hashimoto, H.

Heim, R.

A. Miyawaki, J. Llopis, R. Heim, J. M. McCaffery, J. A. Adams, M. Ikura, and R. Y. Tsien, “Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin,” Nature 388(6645), 882–887 (1997).
[CrossRef] [PubMed]

Heinze, K. G.

K. G. Heinze, A. Koltermann, and P. Schwille, “Simultaneous two-photon excitation of distinct labels for dual-color fluorescence crosscorrelation analysis,” Proc. Natl. Acad. Sci. U.S.A. 97(19), 10377–10382 (2000).
[CrossRef] [PubMed]

Higashi, T.

K. Isobe, W. Watanabe, S. Matsunaga, T. Higashi, K. Fukui, and K. Itoh, “Multi-spectral two-photon excited fluorescence microscopy using supercontinuum light source,” Jpn. J. Appl. Phys. 44(4), L167–L169 (2005).
[CrossRef]

Hillegas, C. W.

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]

Ichikawa, M.

T. Nagai, S. Yamada, T. Tominaga, M. Ichikawa, and A. Miyawaki, “Expanded dynamic range of fluorescent indicators for Ca(2+) by circularly permuted yellow fluorescent proteins,” Proc. Natl. Acad. Sci. U.S.A. 101(29), 10554–10559 (2004).
[CrossRef] [PubMed]

Ikura, M.

A. Miyawaki, J. Llopis, R. Heim, J. M. McCaffery, J. A. Adams, M. Ikura, and R. Y. Tsien, “Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin,” Nature 388(6645), 882–887 (1997).
[CrossRef] [PubMed]

Ishikawa, K. L.

Isobe, K.

K. Isobe, A. Suda, M. Tanaka, H. Hashimoto, F. Kannari, H. Kawano, H. Mizuno, A. Miyawaki, and K. Midorikawa, “Single-pulse coherent anti-Stokes Raman scattering microscopy employing an octave spanning pulse,” Opt. Express 17(14), 11259–11266 (2009).
[CrossRef] [PubMed]

K. Isobe, A. Suda, M. Tanaka, F. Kannari, H. Kawano, H. Mizuno, A. Miyawaki, and K. Midorikawa, “Fourier transform spectroscopy combined with 5-fs broadband pulse for multispectral nonlinear microscopy,” Phys. Rev. A . 77, 063832/1–13 (2008).
[CrossRef]

K. Isobe, W. Watanabe, S. Matsunaga, T. Higashi, K. Fukui, and K. Itoh, “Multi-spectral two-photon excited fluorescence microscopy using supercontinuum light source,” Jpn. J. Appl. Phys. 44(4), L167–L169 (2005).
[CrossRef]

Itoh, K.

K. Isobe, W. Watanabe, S. Matsunaga, T. Higashi, K. Fukui, and K. Itoh, “Multi-spectral two-photon excited fluorescence microscopy using supercontinuum light source,” Jpn. J. Appl. Phys. 44(4), L167–L169 (2005).
[CrossRef]

Joffre, M.

Kaneko, T.

K. Fujita, O. Nakamura, T. Kaneko, S. Kawata, M. Oyamada, and T. Takamatsu, “Real-time imaging of two-photon-induced fluorescence with a microlens-array scanner and a regenerative amplifier,” J. Microsc. 194(Pt 2-3), 528–531 (1999).
[CrossRef]

Kannari, F.

K. Isobe, A. Suda, M. Tanaka, H. Hashimoto, F. Kannari, H. Kawano, H. Mizuno, A. Miyawaki, and K. Midorikawa, “Single-pulse coherent anti-Stokes Raman scattering microscopy employing an octave spanning pulse,” Opt. Express 17(14), 11259–11266 (2009).
[CrossRef] [PubMed]

K. Isobe, A. Suda, M. Tanaka, F. Kannari, H. Kawano, H. Mizuno, A. Miyawaki, and K. Midorikawa, “Fourier transform spectroscopy combined with 5-fs broadband pulse for multispectral nonlinear microscopy,” Phys. Rev. A . 77, 063832/1–13 (2008).
[CrossRef]

Kapteyn, H.

Kärtner, E. X.

Kawano, H.

K. Isobe, A. Suda, M. Tanaka, H. Hashimoto, F. Kannari, H. Kawano, H. Mizuno, A. Miyawaki, and K. Midorikawa, “Single-pulse coherent anti-Stokes Raman scattering microscopy employing an octave spanning pulse,” Opt. Express 17(14), 11259–11266 (2009).
[CrossRef] [PubMed]

K. Isobe, A. Suda, M. Tanaka, F. Kannari, H. Kawano, H. Mizuno, A. Miyawaki, and K. Midorikawa, “Fourier transform spectroscopy combined with 5-fs broadband pulse for multispectral nonlinear microscopy,” Phys. Rev. A . 77, 063832/1–13 (2008).
[CrossRef]

Kawata, S.

K. Fujita, O. Nakamura, T. Kaneko, S. Kawata, M. Oyamada, and T. Takamatsu, “Real-time imaging of two-photon-induced fluorescence with a microlens-array scanner and a regenerative amplifier,” J. Microsc. 194(Pt 2-3), 528–531 (1999).
[CrossRef]

Kim, J.

Koltermann, A.

K. G. Heinze, A. Koltermann, and P. Schwille, “Simultaneous two-photon excitation of distinct labels for dual-color fluorescence crosscorrelation analysis,” Proc. Natl. Acad. Sci. U.S.A. 97(19), 10377–10382 (2000).
[CrossRef] [PubMed]

König, K.

K. König, “Multiphoton microscopy in life sciences,” J. Microsc. 200(Pt 2), 83–104 (2000).
[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]

Leaird, D. E.

Llopis, J.

A. Miyawaki, J. Llopis, R. Heim, J. M. McCaffery, J. A. Adams, M. Ikura, and R. Y. Tsien, “Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin,” Nature 388(6645), 882–887 (1997).
[CrossRef] [PubMed]

Lozovoy, V. V.

V. V. Lozovoy, B. Xu, J. C. Shane, and M. Dantus “Selective nonlinear optical excitation with pulses shaped by pseudorandom Galois fields,” Phys. Rev. A 74, 041805(R) (2006).
[CrossRef]

M. Comstock, V. V. Lozovoy, I. Pastirk, and M. Dantus, “Multiphoton intrapulse interference 6; binary phase shaping,” Opt. Express 12(6), 1061–1066 (2004).
[CrossRef] [PubMed]

V. V. Lozovoy, I. Pastirk, K. A. Walowicz, and M. Dantus, “Multiphoton intrapulse interference. II. Control of two- and three-photon laser induced fluorescence with shaped pulses,” J. Chem. Phys. 118(7), 3187–3196 (2003).
[CrossRef]

K. A. Walowicz, I. Pastirk, V. V. Lozovoy, and M. Dantus, “Multiphoton intrapulse interference. 1. Control of multiphoton processes in condensed phases,” J. Phys. Chem. A 106(41), 9369–9373 (2002).
[CrossRef]

Maginnis, K.

Majer, D.

Martin, J.-L.

Matsunaga, S.

K. Isobe, W. Watanabe, S. Matsunaga, T. Higashi, K. Fukui, and K. Itoh, “Multi-spectral two-photon excited fluorescence microscopy using supercontinuum light source,” Jpn. J. Appl. Phys. 44(4), L167–L169 (2005).
[CrossRef]

Mayo, S. L.

M. A. Mena, T. P. Treynor, S. L. Mayo, and P. S. Daugherty, “Blue fluorescent proteins with enhanced brightness and photostability from a structurally targeted library,” Nat. Biotechnol. 24(12), 1569–1571 (2006).
[CrossRef] [PubMed]

McCaffery, J. M.

A. Miyawaki, J. Llopis, R. Heim, J. M. McCaffery, J. A. Adams, M. Ikura, and R. Y. Tsien, “Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin,” Nature 388(6645), 882–887 (1997).
[CrossRef] [PubMed]

Mena, M. A.

M. A. Mena, T. P. Treynor, S. L. Mayo, and P. S. Daugherty, “Blue fluorescent proteins with enhanced brightness and photostability from a structurally targeted library,” Nat. Biotechnol. 24(12), 1569–1571 (2006).
[CrossRef] [PubMed]

Meshulach, D.

D. Meshulach and Y. Silberberg, “Coherent quantum control of two-photon transitions by a femtosecond laser pulse,” Nature 396(6708), 239–242 (1998).
[CrossRef]

Midorikawa, K.

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]

Miyawaki, A.

K. Isobe, A. Suda, M. Tanaka, H. Hashimoto, F. Kannari, H. Kawano, H. Mizuno, A. Miyawaki, and K. Midorikawa, “Single-pulse coherent anti-Stokes Raman scattering microscopy employing an octave spanning pulse,” Opt. Express 17(14), 11259–11266 (2009).
[CrossRef] [PubMed]

K. Isobe, A. Suda, M. Tanaka, F. Kannari, H. Kawano, H. Mizuno, A. Miyawaki, and K. Midorikawa, “Fourier transform spectroscopy combined with 5-fs broadband pulse for multispectral nonlinear microscopy,” Phys. Rev. A . 77, 063832/1–13 (2008).
[CrossRef]

T. Nagai, S. Yamada, T. Tominaga, M. Ichikawa, and A. Miyawaki, “Expanded dynamic range of fluorescent indicators for Ca(2+) by circularly permuted yellow fluorescent proteins,” Proc. Natl. Acad. Sci. U.S.A. 101(29), 10554–10559 (2004).
[CrossRef] [PubMed]

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]

G. Y. Fan, H. Fujisaki, A. Miyawaki, R. K. Tsay, R. Y. Tsien, and M. H. Ellisman, “Video-rate scanning two-photon excitation fluorescence microscopy and ratio imaging with cameleons,” Biophys. J. 76(5), 2412–2420 (1999).
[CrossRef] [PubMed]

A. Miyawaki, J. Llopis, R. Heim, J. M. McCaffery, J. A. Adams, M. Ikura, and R. Y. Tsien, “Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin,” Nature 388(6645), 882–887 (1997).
[CrossRef] [PubMed]

Mizuno, H.

K. Isobe, A. Suda, M. Tanaka, H. Hashimoto, F. Kannari, H. Kawano, H. Mizuno, A. Miyawaki, and K. Midorikawa, “Single-pulse coherent anti-Stokes Raman scattering microscopy employing an octave spanning pulse,” Opt. Express 17(14), 11259–11266 (2009).
[CrossRef] [PubMed]

K. Isobe, A. Suda, M. Tanaka, F. Kannari, H. Kawano, H. Mizuno, A. Miyawaki, and K. Midorikawa, “Fourier transform spectroscopy combined with 5-fs broadband pulse for multispectral nonlinear microscopy,” Phys. Rev. A . 77, 063832/1–13 (2008).
[CrossRef]

Motzkus, M.

B. Vacano and M. Motzkus, “Time-resolving molecular vibration for microanalytics: single laser beam nonlinear Raman spectroscopy in simulation and experiment,” Phys. Chem. Chem. Phys. 10(5), 681–691 (2008).
[CrossRef]

Mourou, G.

Müller, M.

A. H. Buist, M. Müller, J. Squier, and G. J. Brakenhoff, “Real time two-photon absorption microscopy using multi point excitation,” J. Microsc. 192(2), 217–226 (1998).
[CrossRef]

Murnane, M.

Nabekawa, Y.

Nagai, T.

T. Nagai, S. Yamada, T. Tominaga, M. Ichikawa, and A. Miyawaki, “Expanded dynamic range of fluorescent indicators for Ca(2+) by circularly permuted yellow fluorescent proteins,” Proc. Natl. Acad. Sci. U.S.A. 101(29), 10554–10559 (2004).
[CrossRef] [PubMed]

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]

Nakamura, O.

K. Fujita, O. Nakamura, T. Kaneko, S. Kawata, M. Oyamada, and T. Takamatsu, “Real-time imaging of two-photon-induced fluorescence with a microlens-array scanner and a regenerative amplifier,” J. Microsc. 194(Pt 2-3), 528–531 (1999).
[CrossRef]

Ogilvie, J. P.

Oyamada, M.

K. Fujita, O. Nakamura, T. Kaneko, S. Kawata, M. Oyamada, and T. Takamatsu, “Real-time imaging of two-photon-induced fluorescence with a microlens-array scanner and a regenerative amplifier,” J. Microsc. 194(Pt 2-3), 528–531 (1999).
[CrossRef]

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]

Pastirk, I.

M. Comstock, V. V. Lozovoy, I. Pastirk, and M. Dantus, “Multiphoton intrapulse interference 6; binary phase shaping,” Opt. Express 12(6), 1061–1066 (2004).
[CrossRef] [PubMed]

V. V. Lozovoy, I. Pastirk, K. A. Walowicz, and M. Dantus, “Multiphoton intrapulse interference. II. Control of two- and three-photon laser induced fluorescence with shaped pulses,” J. Chem. Phys. 118(7), 3187–3196 (2003).
[CrossRef]

K. A. Walowicz, I. Pastirk, V. V. Lozovoy, and M. Dantus, “Multiphoton intrapulse interference. 1. Control of multiphoton processes in condensed phases,” J. Phys. Chem. A 106(41), 9369–9373 (2002).
[CrossRef]

Patel, J. S.

Pshenichnikov, M. S.

Rabitz, H.

M. Roth, L. Guyon, J. Roslund, V. Boutou, F. Courvoisier, J.-P. Wolf, and H. Rabitz, “Quantum control of tightly competitive product channels,” Phys. Rev. Lett. 102, 253001 (2009).
[CrossRef] [PubMed]

Roslund, J.

M. Roth, L. Guyon, J. Roslund, V. Boutou, F. Courvoisier, J.-P. Wolf, and H. Rabitz, “Quantum control of tightly competitive product channels,” Phys. Rev. Lett. 102, 253001 (2009).
[CrossRef] [PubMed]

Roth, M.

M. Roth, L. Guyon, J. Roslund, V. Boutou, F. Courvoisier, J.-P. Wolf, and H. Rabitz, “Quantum control of tightly competitive product channels,” Phys. Rev. Lett. 102, 253001 (2009).
[CrossRef] [PubMed]

Russek, U.

Schelhas, L. T.

L. T. Schelhas, J. C. Shane, and M. Dantus, “Advantages of ultrashort phase-shaped pulses for selective two-photon activation and biomedical imaging,” Nanomedicine 2(3), 177–181 (2006).
[CrossRef]

Scheuer, V.

Schwille, P.

K. G. Heinze, A. Koltermann, and P. Schwille, “Simultaneous two-photon excitation of distinct labels for dual-color fluorescence crosscorrelation analysis,” Proc. Natl. Acad. Sci. U.S.A. 97(19), 10377–10382 (2000).
[CrossRef] [PubMed]

Shane, J. C.

L. T. Schelhas, J. C. Shane, and M. Dantus, “Advantages of ultrashort phase-shaped pulses for selective two-photon activation and biomedical imaging,” Nanomedicine 2(3), 177–181 (2006).
[CrossRef]

V. V. Lozovoy, B. Xu, J. C. Shane, and M. Dantus “Selective nonlinear optical excitation with pulses shaped by pseudorandom Galois fields,” Phys. Rev. A 74, 041805(R) (2006).
[CrossRef]

Sharma, V.

Shear, J. B.

C. Xu, W. Zipfel, J. B. Shear, R. M. Williams, and W. W. Webb, “Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy,” Proc. Natl. Acad. Sci. U.S.A. 93(20), 10763–10768 (1996).
[CrossRef] [PubMed]

Silberberg, Y.

E. Frumker, E. Tal, Y. Silberberg, and D. Majer, “Femtosecond pulse-shape modulation at nanosecond rates,” Opt. Lett. 30(20), 2796–2798 (2005).
[CrossRef] [PubMed]

D. Meshulach and Y. Silberberg, “Coherent quantum control of two-photon transitions by a femtosecond laser pulse,” Nature 396(6708), 239–242 (1998).
[CrossRef]

Silva, C. M.

D. C. Youvan, C. M. Silva, E. J. Bylina, W. J. Coleman, M. R. Dilworth, and M. M. Yang, “Calibration of fluorescence resonance energy transfer in microscopy using genetically engineered GFP derivatives on nickel chelating beads,” Biotechnology. 3, 1–18 (1997).

Solinas, X.

Squier, J.

A. H. Buist, M. Müller, J. Squier, and G. J. Brakenhoff, “Real time two-photon absorption microscopy using multi point excitation,” J. Microsc. 192(2), 217–226 (1998).
[CrossRef]

Strickland, D.

Strickler, J. H.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Suda, A.

K. Isobe, A. Suda, M. Tanaka, H. Hashimoto, F. Kannari, H. Kawano, H. Mizuno, A. Miyawaki, and K. Midorikawa, “Single-pulse coherent anti-Stokes Raman scattering microscopy employing an octave spanning pulse,” Opt. Express 17(14), 11259–11266 (2009).
[CrossRef] [PubMed]

K. Isobe, A. Suda, M. Tanaka, F. Kannari, H. Kawano, H. Mizuno, A. Miyawaki, and K. Midorikawa, “Fourier transform spectroscopy combined with 5-fs broadband pulse for multispectral nonlinear microscopy,” Phys. Rev. A . 77, 063832/1–13 (2008).
[CrossRef]

Takahashi, H.

Takamatsu, T.

K. Fujita, O. Nakamura, T. Kaneko, S. Kawata, M. Oyamada, and T. Takamatsu, “Real-time imaging of two-photon-induced fluorescence with a microlens-array scanner and a regenerative amplifier,” J. Microsc. 194(Pt 2-3), 528–531 (1999).
[CrossRef]

Tal, E.

Tanaka, M.

K. Isobe, A. Suda, M. Tanaka, H. Hashimoto, F. Kannari, H. Kawano, H. Mizuno, A. Miyawaki, and K. Midorikawa, “Single-pulse coherent anti-Stokes Raman scattering microscopy employing an octave spanning pulse,” Opt. Express 17(14), 11259–11266 (2009).
[CrossRef] [PubMed]

K. Isobe, A. Suda, M. Tanaka, F. Kannari, H. Kawano, H. Mizuno, A. Miyawaki, and K. Midorikawa, “Fourier transform spectroscopy combined with 5-fs broadband pulse for multispectral nonlinear microscopy,” Phys. Rev. A . 77, 063832/1–13 (2008).
[CrossRef]

Tominaga, T.

T. Nagai, S. Yamada, T. Tominaga, M. Ichikawa, and A. Miyawaki, “Expanded dynamic range of fluorescent indicators for Ca(2+) by circularly permuted yellow fluorescent proteins,” Proc. Natl. Acad. Sci. U.S.A. 101(29), 10554–10559 (2004).
[CrossRef] [PubMed]

Treynor, T. P.

M. A. Mena, T. P. Treynor, S. L. Mayo, and P. S. Daugherty, “Blue fluorescent proteins with enhanced brightness and photostability from a structurally targeted library,” Nat. Biotechnol. 24(12), 1569–1571 (2006).
[CrossRef] [PubMed]

Tsay, R. K.

G. Y. Fan, H. Fujisaki, A. Miyawaki, R. K. Tsay, R. Y. Tsien, and M. H. Ellisman, “Video-rate scanning two-photon excitation fluorescence microscopy and ratio imaging with cameleons,” Biophys. J. 76(5), 2412–2420 (1999).
[CrossRef] [PubMed]

Tsien, R. Y.

G. Y. Fan, H. Fujisaki, A. Miyawaki, R. K. Tsay, R. Y. Tsien, and M. H. Ellisman, “Video-rate scanning two-photon excitation fluorescence microscopy and ratio imaging with cameleons,” Biophys. J. 76(5), 2412–2420 (1999).
[CrossRef] [PubMed]

A. Miyawaki, J. Llopis, R. Heim, J. M. McCaffery, J. A. Adams, M. Ikura, and R. Y. Tsien, “Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin,” Nature 388(6645), 882–887 (1997).
[CrossRef] [PubMed]

Tull, J. X.

Vacano, B.

B. Vacano and M. Motzkus, “Time-resolving molecular vibration for microanalytics: single laser beam nonlinear Raman spectroscopy in simulation and experiment,” Phys. Chem. Chem. Phys. 10(5), 681–691 (2008).
[CrossRef]

Vdovin, G.

Walowicz, K. A.

V. V. Lozovoy, I. Pastirk, K. A. Walowicz, and M. Dantus, “Multiphoton intrapulse interference. II. Control of two- and three-photon laser induced fluorescence with shaped pulses,” J. Chem. Phys. 118(7), 3187–3196 (2003).
[CrossRef]

K. A. Walowicz, I. Pastirk, V. V. Lozovoy, and M. Dantus, “Multiphoton intrapulse interference. 1. Control of multiphoton processes in condensed phases,” J. Phys. Chem. A 106(41), 9369–9373 (2002).
[CrossRef]

Warren, W. S.

Watanabe, W.

K. Isobe, W. Watanabe, S. Matsunaga, T. Higashi, K. Fukui, and K. Itoh, “Multi-spectral two-photon excited fluorescence microscopy using supercontinuum light source,” Jpn. J. Appl. Phys. 44(4), L167–L169 (2005).
[CrossRef]

Webb, W. W.

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

C. Xu, W. Zipfel, J. B. Shear, R. M. Williams, and W. W. Webb, “Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy,” Proc. Natl. Acad. Sci. U.S.A. 93(20), 10763–10768 (1996).
[CrossRef] [PubMed]

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Weiner, A. M.

Wiersma, D. A.

Williams, R. M.

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

C. Xu, W. Zipfel, J. B. Shear, R. M. Williams, and W. W. Webb, “Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy,” Proc. Natl. Acad. Sci. U.S.A. 93(20), 10763–10768 (1996).
[CrossRef] [PubMed]

Wolf, J.-P.

M. Roth, L. Guyon, J. Roslund, V. Boutou, F. Courvoisier, J.-P. Wolf, and H. Rabitz, “Quantum control of tightly competitive product channels,” Phys. Rev. Lett. 102, 253001 (2009).
[CrossRef] [PubMed]

Wullert, J. R.

Xu, B.

V. V. Lozovoy, B. Xu, J. C. Shane, and M. Dantus “Selective nonlinear optical excitation with pulses shaped by pseudorandom Galois fields,” Phys. Rev. A 74, 041805(R) (2006).
[CrossRef]

Xu, C.

C. Xu, W. Zipfel, J. B. Shear, R. M. Williams, and W. W. Webb, “Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy,” Proc. Natl. Acad. Sci. U.S.A. 93(20), 10763–10768 (1996).
[CrossRef] [PubMed]

Yamada, S.

T. Nagai, S. Yamada, T. Tominaga, M. Ichikawa, and A. Miyawaki, “Expanded dynamic range of fluorescent indicators for Ca(2+) by circularly permuted yellow fluorescent proteins,” Proc. Natl. Acad. Sci. U.S.A. 101(29), 10554–10559 (2004).
[CrossRef] [PubMed]

Yang, M. M.

D. C. Youvan, C. M. Silva, E. J. Bylina, W. J. Coleman, M. R. Dilworth, and M. M. Yang, “Calibration of fluorescence resonance energy transfer in microscopy using genetically engineered GFP derivatives on nickel chelating beads,” Biotechnology. 3, 1–18 (1997).

Youvan, D. C.

D. C. Youvan, C. M. Silva, E. J. Bylina, W. J. Coleman, M. R. Dilworth, and M. M. Yang, “Calibration of fluorescence resonance energy transfer in microscopy using genetically engineered GFP derivatives on nickel chelating beads,” Biotechnology. 3, 1–18 (1997).

Zeek, E.

Zipfel, W.

C. Xu, W. Zipfel, J. B. Shear, R. M. Williams, and W. W. Webb, “Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy,” Proc. Natl. Acad. Sci. U.S.A. 93(20), 10763–10768 (1996).
[CrossRef] [PubMed]

Zipfel, W. R.

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

Biophys. J.

G. Y. Fan, H. Fujisaki, A. Miyawaki, R. K. Tsay, R. Y. Tsien, and M. H. Ellisman, “Video-rate scanning two-photon excitation fluorescence microscopy and ratio imaging with cameleons,” Biophys. J. 76(5), 2412–2420 (1999).
[CrossRef] [PubMed]

Biotechnology.

D. C. Youvan, C. M. Silva, E. J. Bylina, W. J. Coleman, M. R. Dilworth, and M. M. Yang, “Calibration of fluorescence resonance energy transfer in microscopy using genetically engineered GFP derivatives on nickel chelating beads,” Biotechnology. 3, 1–18 (1997).

J. Chem. Phys.

P. Allcock and D. L. Andrews, “Two-photon fluorescence: Resonance energy transfer,” J. Chem. Phys. 108(8), 3089–3095 (1998).
[CrossRef]

V. V. Lozovoy, I. Pastirk, K. A. Walowicz, and M. Dantus, “Multiphoton intrapulse interference. II. Control of two- and three-photon laser induced fluorescence with shaped pulses,” J. Chem. Phys. 118(7), 3187–3196 (2003).
[CrossRef]

J. Microsc.

K. König, “Multiphoton microscopy in life sciences,” J. Microsc. 200(Pt 2), 83–104 (2000).
[CrossRef] [PubMed]

A. H. Buist, M. Müller, J. Squier, and G. J. Brakenhoff, “Real time two-photon absorption microscopy using multi point excitation,” J. Microsc. 192(2), 217–226 (1998).
[CrossRef]

K. Fujita, O. Nakamura, T. Kaneko, S. Kawata, M. Oyamada, and T. Takamatsu, “Real-time imaging of two-photon-induced fluorescence with a microlens-array scanner and a regenerative amplifier,” J. Microsc. 194(Pt 2-3), 528–531 (1999).
[CrossRef]

J. Phys. Chem. A

K. A. Walowicz, I. Pastirk, V. V. Lozovoy, and M. Dantus, “Multiphoton intrapulse interference. 1. Control of multiphoton processes in condensed phases,” J. Phys. Chem. A 106(41), 9369–9373 (2002).
[CrossRef]

Jpn. J. Appl. Phys.

K. Isobe, W. Watanabe, S. Matsunaga, T. Higashi, K. Fukui, and K. Itoh, “Multi-spectral two-photon excited fluorescence microscopy using supercontinuum light source,” Jpn. J. Appl. Phys. 44(4), L167–L169 (2005).
[CrossRef]

Nanomedicine

L. T. Schelhas, J. C. Shane, and M. Dantus, “Advantages of ultrashort phase-shaped pulses for selective two-photon activation and biomedical imaging,” Nanomedicine 2(3), 177–181 (2006).
[CrossRef]

Nat. Biotechnol.

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]

M. A. Mena, T. P. Treynor, S. L. Mayo, and P. S. Daugherty, “Blue fluorescent proteins with enhanced brightness and photostability from a structurally targeted library,” Nat. Biotechnol. 24(12), 1569–1571 (2006).
[CrossRef] [PubMed]

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

Nature

D. Meshulach and Y. Silberberg, “Coherent quantum control of two-photon transitions by a femtosecond laser pulse,” Nature 396(6708), 239–242 (1998).
[CrossRef]

A. Miyawaki, J. Llopis, R. Heim, J. M. McCaffery, J. A. Adams, M. Ikura, and R. Y. Tsien, “Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin,” Nature 388(6645), 882–887 (1997).
[CrossRef] [PubMed]

Opt. Express

Opt. Lett.

Phys. Chem. Chem. Phys.

B. Vacano and M. Motzkus, “Time-resolving molecular vibration for microanalytics: single laser beam nonlinear Raman spectroscopy in simulation and experiment,” Phys. Chem. Chem. Phys. 10(5), 681–691 (2008).
[CrossRef]

Phys. Rev. A

K. Isobe, A. Suda, M. Tanaka, F. Kannari, H. Kawano, H. Mizuno, A. Miyawaki, and K. Midorikawa, “Fourier transform spectroscopy combined with 5-fs broadband pulse for multispectral nonlinear microscopy,” Phys. Rev. A . 77, 063832/1–13 (2008).
[CrossRef]

V. V. Lozovoy, B. Xu, J. C. Shane, and M. Dantus “Selective nonlinear optical excitation with pulses shaped by pseudorandom Galois fields,” Phys. Rev. A 74, 041805(R) (2006).
[CrossRef]

Phys. Rev. Lett.

M. Roth, L. Guyon, J. Roslund, V. Boutou, F. Courvoisier, J.-P. Wolf, and H. Rabitz, “Quantum control of tightly competitive product channels,” Phys. Rev. Lett. 102, 253001 (2009).
[CrossRef] [PubMed]

Proc. Natl. Acad. Sci. U.S.A.

T. Nagai, S. Yamada, T. Tominaga, M. Ichikawa, and A. Miyawaki, “Expanded dynamic range of fluorescent indicators for Ca(2+) by circularly permuted yellow fluorescent proteins,” Proc. Natl. Acad. Sci. U.S.A. 101(29), 10554–10559 (2004).
[CrossRef] [PubMed]

C. Xu, W. Zipfel, J. B. Shear, R. M. Williams, and W. W. Webb, “Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy,” Proc. Natl. Acad. Sci. U.S.A. 93(20), 10763–10768 (1996).
[CrossRef] [PubMed]

K. G. Heinze, A. Koltermann, and P. Schwille, “Simultaneous two-photon excitation of distinct labels for dual-color fluorescence crosscorrelation analysis,” Proc. Natl. Acad. Sci. U.S.A. 97(19), 10377–10382 (2000).
[CrossRef] [PubMed]

Science

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

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Figures (5)

Fig. 1.
Fig. 1.

Experimental setup in vitro (a) and in vivo (b). (c) Spectra of broadband pulse at the focal point for a pair of BFP and GFP (black line) and a pair of CFP and YFP (pink line). SLM: spatial light modulator, BS: beam splitter, OB: objective lens, SP: short-pass filter, BP: band-pass filter, PMT: photomultiplier tube.

Fig. 2.
Fig. 2.

(a) SH spectra at constructive (black line) and destructive (red line) interference phases and spectral phases for selective excitations of GFP (green line) or BFP (blue line). (b) Constructive (black) and destructive (red) interference phases.

Fig. 3.
Fig. 3.

Control of TPEF intensities from two fluorescent proteins by spectral phase modulation. (a) TPEF intensities from GFP and BFP with selective excitations of GFP (green square) or BFP (blue square) and with simultaneous excitation together with control of TPEF intensity for only GFP (blue cross) or BFP (green cross). (b) Spectral phases at the highest contrast ratio in the selective excitation between a pair of BFP and GFP. (c) Spectral phases in the simultaneous excitation for controlling TPEF intensity of BFP while keeping that of GFP constant. (d) SH spectra at spectral phase for controlling TPEF intensity of BFP while keeping that of GFP constant. (e) SH spectra at spectral phase for controlling TPEF intensity of GFP while keeping that of BFP constant. (f) TPEF intensities from CFP and YFP with selective excitation of CFP (cyan square) or YFP (yellow square) and with simultaneous excitation together with control of TPEF intensity for only YFP (cyan cross) or CFP (yellow cross).

Fig. 4.
Fig. 4.

Dual-color images of a HeLa cell labeled with BFP (nucleus, top) and GFP (cytoplasm, bottom). The scale bar is 10 µm. (a) BFP-enhanced excitation (left) and GFP-enhanced excitation (right). (b, c) Simultaneous excitation with control of TPEF intensities. (b) BFP intensity is regulated, while GFP intensity is frozen. (c) GFP intensity is regulated, while BFP intensity is frozen.

Fig. 5.
Fig. 5.

Ca2+ response in cytoplasm of a HeLa cell loaded with yellow-cameleon (YC3.60). CFP (a, d), YFP (b, e) and FRET (c, f) images with CFP-enhanced excitation before (a, b, c) and after (d, e, f) Ca2+ stimulus excitation by treatment with 5 µM ionomycin. The scale bar is 15 µm. (g) Signal ratio of YFP channel to CFP channel before and after Ca2+ stimulus excitation by treatment with 20 µM histamine.

Equations (7)

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IF= g(2) (ω)E(2)(ω)2dω.
E(2)(ω)2=E(ω)E(ωω)exp{i[φ(ω)+φ(ωω)]}dω2,
S(2ωj)=kE(ωj+ωk)E(ωjωk)exp[i{φ(ωj+ωk)+φ(ωjωk)}]2,
ISH=jS(2ωj).
φ(ω)={φc(ω)(ωR1)φd(ω)(ωR2).
φ(ω)={φc(ω)+α{φd(ω)φc(ω)}+φ(ωω0)(ωR1)φc(ω)+β{φd(ω)φc(ω)}(ωR2).
nF=IFRETaICFPbIYFP,

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