Abstract

A challenge for nonlinear imaging in living tissue is to maximize the total fluorescent yield from each fluorophore. We investigated the emission rates of three fluorophores—rhodamine B, a red fluorescent protein, and CdSe quantum dots—while manipulating the phase of the laser excitation pulse at the focus. In all cases a transform-limited pulse maximized the total yield to insure the highest signal-to-noise ratio. Further, we find evidence of fluorescence antibleaching in quantum dot samples.

© 2010 OSA

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A. Mohanty, Y. Yang, A. Luo, A. W. Sylvester, and D. Jackson, “Methods for generation and analysis of fluorescent protein-tagged maize lines,” Methods Mol. Biol. 526, 71–89 (2009).
[CrossRef] [PubMed]

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[CrossRef] [PubMed]

2008

N. Ji, J. C. Magee, and E. Betzig, “High-speed, low-photodamage nonlinear imaging using passive pulse splitters,” Nat. Methods 5(2), 197–202 (2008).
[CrossRef] [PubMed]

P. Xi, Y. Andegeko, L. R. Weisel, V. V. Lozovoy, and M. Dantus, “Greater signal, increased depth, and less photobleaching in two-photon microscopy with 10 fs pulses,” Opt. Commun. 281(7), 1841–1849 (2008).
[CrossRef]

D. J. Kane, “Principal components generalized projections: a review [Invited],” J. Opt. Soc. Am. B 25(6), A120–A132 (2008).
[CrossRef]

R. Carriles, K. E. Sheetz, E. E. Hoover, J. A. Squier, and V. Barzda, “Simultaneous multifocal, multiphoton, photon counting microscopy,” Opt. Express 16(14), 10364–10371 (2008).
[CrossRef] [PubMed]

2007

J. J. Field, C. G. Durfee, J. A. Squier, and S. Kane, “Quartic-phase-limited grism-based ultrashort pulse shaper,” Opt. Lett. 32(21), 3101–3103 (2007).
[CrossRef] [PubMed]

G. Donnert, C. Eggeling, and S. W. Hell, “Major signal increase in fluorescence microscopy through dark-state relaxation,” Nat. Methods 4(1), 81–86 (2007).
[CrossRef]

J. J. Field, T. A. Planchon, W. Amir, C. G. Durfee, and J. A. Squier, “Characterization of a high efficiency, ultrashort pulse shaper incorporating a reflective 4096-element spatial light modulator,” Opt. Commun. 278(2), 368–376 (2007).
[CrossRef] [PubMed]

2006

2005

A. Shavel, N. Gaponik, and A. Eychmüller, “Covalent linking of CdTe nanocrystals to amino-functionalized surfaces,” ChemPhysChem 6(3), 449–451 (2005).
[CrossRef] [PubMed]

C. R. Dietrich, M. A. D. N. Perera, M. D. Yandeau-Nelson, R. B. Meeley, B. J. Nikolau, and P. S. Schnable, “Characterization of two GL8 paralogs reveals that the 3-ketoacyl reductase component of fatty acid elongase is essential for maize (Zea mays L.) development,” Plant J. 42(6), 844–861 (2005).
[CrossRef] [PubMed]

F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2(12), 932–940 (2005).
[CrossRef] [PubMed]

2004

A. J. Berglund, “Nonexponential statistics of fluorescence photobleaching,” J. Chem. Phys. 121(7), 2899–2903 (2004).
[CrossRef] [PubMed]

2003

H. Kawano, Y. Nabekawa, A. Suda, Y. Oishi, H. Mizuno, A. Miyawaki, and K. Midorikawa, “Attenuation of photobleaching in two-photon excitation fluorescence from green fluorescent protein with shaped excitation pulses,” Biochem. Biophys. Res. Commun. 311(3), 592–596 (2003).
[CrossRef] [PubMed]

2001

W. G. J. H. M. van Sark, P. L. T. M. Frederix, D. J. Van den Heuvel, H. C. Gerritsen, A. A. Bol, J. N. J. van Lingen, C. de Mello Donegá, and A. Meijerink, “Photooxidation and photobleaching of single CdSe/ZnS quantum dots probed by room-temperature time-resolved spectroscopy,” J. Phys. Chem. B 105(35), 8281–8284 (2001).
[CrossRef]

2000

G. H. Patterson and D. W. Piston, “Photobleaching in two-photon excitation microscopy,” Biophys. J. 78(4), 2159–2162 (2000).
[CrossRef] [PubMed]

1999

D. N. Fittinghoff, A. C. Millard, J. A. Squier, and M. Müller, “Frequency-resolved optical gating measurement of ultrashort pulses passing through a high numerical aperture objective,” IEEE J. Quantum Electron. 35(4), 479–486 (1999).
[CrossRef]

C. Bardeen, V. Yakovlev, J. Squier, K. R. Wilson, S. D. Carpenter, and P. M. Weber, “Effect of pulse shape on the efficiency of multiphoton processes: implications for biological microscopy,” J. Biomed. Opt. 4(3), 362–367 (1999).
[CrossRef]

1998

M. Müller, J. Squier, R. Wolleschensky, U. Simon, and G. J. Brakenhoff, “Dispersion pre-compensation of 15 femtosecond optical pulses for high-numerical-aperture objectives,” J. Microsc. 191(2), 141–150 (1998).
[CrossRef] [PubMed]

1997

1996

1990

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

Akturk, S.

Amir, W.

J. J. Field, T. A. Planchon, W. Amir, C. G. Durfee, and J. A. Squier, “Characterization of a high efficiency, ultrashort pulse shaper incorporating a reflective 4096-element spatial light modulator,” Opt. Commun. 278(2), 368–376 (2007).
[CrossRef] [PubMed]

W. Amir, T. A. Planchon, C. G. Durfee, J. A. Squier, P. Gabolde, R. Trebino, and M. Müller, “Simultaneous visualization of spatial and chromatic aberrations by two-dimensional Fourier transform spectral interferometry,” Opt. Lett. 31(19), 2927–2929 (2006).
[CrossRef] [PubMed]

Andegeko, Y.

P. Xi, Y. Andegeko, L. R. Weisel, V. V. Lozovoy, and M. Dantus, “Greater signal, increased depth, and less photobleaching in two-photon microscopy with 10 fs pulses,” Opt. Commun. 281(7), 1841–1849 (2008).
[CrossRef]

Bardeen, C.

C. Bardeen, V. Yakovlev, J. Squier, K. R. Wilson, S. D. Carpenter, and P. M. Weber, “Effect of pulse shape on the efficiency of multiphoton processes: implications for biological microscopy,” J. Biomed. Opt. 4(3), 362–367 (1999).
[CrossRef]

Barzda, V.

Berglund, A. J.

A. J. Berglund, “Nonexponential statistics of fluorescence photobleaching,” J. Chem. Phys. 121(7), 2899–2903 (2004).
[CrossRef] [PubMed]

Betzig, E.

N. Ji, J. C. Magee, and E. Betzig, “High-speed, low-photodamage nonlinear imaging using passive pulse splitters,” Nat. Methods 5(2), 197–202 (2008).
[CrossRef] [PubMed]

Bol, A. A.

W. G. J. H. M. van Sark, P. L. T. M. Frederix, D. J. Van den Heuvel, H. C. Gerritsen, A. A. Bol, J. N. J. van Lingen, C. de Mello Donegá, and A. Meijerink, “Photooxidation and photobleaching of single CdSe/ZnS quantum dots probed by room-temperature time-resolved spectroscopy,” J. Phys. Chem. B 105(35), 8281–8284 (2001).
[CrossRef]

Bowlan, P.

Brakenhoff, G. J.

M. Müller, J. Squier, R. Wolleschensky, U. Simon, and G. J. Brakenhoff, “Dispersion pre-compensation of 15 femtosecond optical pulses for high-numerical-aperture objectives,” J. Microsc. 191(2), 141–150 (1998).
[CrossRef] [PubMed]

Carpenter, S. D.

C. Bardeen, V. Yakovlev, J. Squier, K. R. Wilson, S. D. Carpenter, and P. M. Weber, “Effect of pulse shape on the efficiency of multiphoton processes: implications for biological microscopy,” J. Biomed. Opt. 4(3), 362–367 (1999).
[CrossRef]

Carriles, R.

Clement, T. S.

D. Yandeau-Nelson, M.

C. R. Dietrich, M. A. D. N. Perera, M. D. Yandeau-Nelson, R. B. Meeley, B. J. Nikolau, and P. S. Schnable, “Characterization of two GL8 paralogs reveals that the 3-ketoacyl reductase component of fatty acid elongase is essential for maize (Zea mays L.) development,” Plant J. 42(6), 844–861 (2005).
[CrossRef] [PubMed]

Dantus, M.

P. Xi, Y. Andegeko, L. R. Weisel, V. V. Lozovoy, and M. Dantus, “Greater signal, increased depth, and less photobleaching in two-photon microscopy with 10 fs pulses,” Opt. Commun. 281(7), 1841–1849 (2008).
[CrossRef]

de Mello Donegá, C.

W. G. J. H. M. van Sark, P. L. T. M. Frederix, D. J. Van den Heuvel, H. C. Gerritsen, A. A. Bol, J. N. J. van Lingen, C. de Mello Donegá, and A. Meijerink, “Photooxidation and photobleaching of single CdSe/ZnS quantum dots probed by room-temperature time-resolved spectroscopy,” J. Phys. Chem. B 105(35), 8281–8284 (2001).
[CrossRef]

Denk, W.

F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2(12), 932–940 (2005).
[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]

Dietrich, C. R.

C. R. Dietrich, M. A. D. N. Perera, M. D. Yandeau-Nelson, R. B. Meeley, B. J. Nikolau, and P. S. Schnable, “Characterization of two GL8 paralogs reveals that the 3-ketoacyl reductase component of fatty acid elongase is essential for maize (Zea mays L.) development,” Plant J. 42(6), 844–861 (2005).
[CrossRef] [PubMed]

Donnert, G.

G. Donnert, C. Eggeling, and S. W. Hell, “Major signal increase in fluorescence microscopy through dark-state relaxation,” Nat. Methods 4(1), 81–86 (2007).
[CrossRef]

Drobizhev, M.

M. Drobizhev, S. Tillo, N. S. Makarov, T. E. Hughes, and A. Rebane, “Absolute two-photon absorption spectra and two-photon brightness of orange and red fluorescent proteins,” J. Phys. Chem. B 113(4), 855–859 (2009).
[CrossRef] [PubMed]

Durfee, C. G.

Eggeling, C.

G. Donnert, C. Eggeling, and S. W. Hell, “Major signal increase in fluorescence microscopy through dark-state relaxation,” Nat. Methods 4(1), 81–86 (2007).
[CrossRef]

Eychmüller, A.

A. Shavel, N. Gaponik, and A. Eychmüller, “Covalent linking of CdTe nanocrystals to amino-functionalized surfaces,” ChemPhysChem 6(3), 449–451 (2005).
[CrossRef] [PubMed]

Field, J. J.

J. J. Field, T. A. Planchon, W. Amir, C. G. Durfee, and J. A. Squier, “Characterization of a high efficiency, ultrashort pulse shaper incorporating a reflective 4096-element spatial light modulator,” Opt. Commun. 278(2), 368–376 (2007).
[CrossRef] [PubMed]

J. J. Field, C. G. Durfee, J. A. Squier, and S. Kane, “Quartic-phase-limited grism-based ultrashort pulse shaper,” Opt. Lett. 32(21), 3101–3103 (2007).
[CrossRef] [PubMed]

Fittinghoff, D. N.

D. N. Fittinghoff, A. C. Millard, J. A. Squier, and M. Müller, “Frequency-resolved optical gating measurement of ultrashort pulses passing through a high numerical aperture objective,” IEEE J. Quantum Electron. 35(4), 479–486 (1999).
[CrossRef]

Frederix, P. L. T. M.

W. G. J. H. M. van Sark, P. L. T. M. Frederix, D. J. Van den Heuvel, H. C. Gerritsen, A. A. Bol, J. N. J. van Lingen, C. de Mello Donegá, and A. Meijerink, “Photooxidation and photobleaching of single CdSe/ZnS quantum dots probed by room-temperature time-resolved spectroscopy,” J. Phys. Chem. B 105(35), 8281–8284 (2001).
[CrossRef]

Gabolde, P.

Gaponik, N.

A. Shavel, N. Gaponik, and A. Eychmüller, “Covalent linking of CdTe nanocrystals to amino-functionalized surfaces,” ChemPhysChem 6(3), 449–451 (2005).
[CrossRef] [PubMed]

Gerritsen, H. C.

W. G. J. H. M. van Sark, P. L. T. M. Frederix, D. J. Van den Heuvel, H. C. Gerritsen, A. A. Bol, J. N. J. van Lingen, C. de Mello Donegá, and A. Meijerink, “Photooxidation and photobleaching of single CdSe/ZnS quantum dots probed by room-temperature time-resolved spectroscopy,” J. Phys. Chem. B 105(35), 8281–8284 (2001).
[CrossRef]

Hell, S. W.

G. Donnert, C. Eggeling, and S. W. Hell, “Major signal increase in fluorescence microscopy through dark-state relaxation,” Nat. Methods 4(1), 81–86 (2007).
[CrossRef]

Helmchen, F.

F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2(12), 932–940 (2005).
[CrossRef] [PubMed]

Holtom, G. R.

E. J. Sánchez, L. Novotny, G. R. Holtom, and X. S. Xie, “Room-temperature fluorescence imaging and spectroscopy of single molecules by two-photon excitation,” J. Phys. Chem. A 101(38), 7019–7023 (1997).
[CrossRef]

Hoover, E. E.

Hughes, T. E.

M. Drobizhev, S. Tillo, N. S. Makarov, T. E. Hughes, and A. Rebane, “Absolute two-photon absorption spectra and two-photon brightness of orange and red fluorescent proteins,” J. Phys. Chem. B 113(4), 855–859 (2009).
[CrossRef] [PubMed]

Jackson, D.

A. Mohanty, Y. Yang, A. Luo, A. W. Sylvester, and D. Jackson, “Methods for generation and analysis of fluorescent protein-tagged maize lines,” Methods Mol. Biol. 526, 71–89 (2009).
[CrossRef] [PubMed]

Ji, N.

N. Ji, J. C. Magee, and E. Betzig, “High-speed, low-photodamage nonlinear imaging using passive pulse splitters,” Nat. Methods 5(2), 197–202 (2008).
[CrossRef] [PubMed]

Kane, D. J.

Kane, S.

Kawano, H.

H. Kawano, Y. Nabekawa, A. Suda, Y. Oishi, H. Mizuno, A. Miyawaki, and K. Midorikawa, “Attenuation of photobleaching in two-photon excitation fluorescence from green fluorescent protein with shaped excitation pulses,” Biochem. Biophys. Res. Commun. 311(3), 592–596 (2003).
[CrossRef] [PubMed]

Lozovoy, V. V.

P. Xi, Y. Andegeko, L. R. Weisel, V. V. Lozovoy, and M. Dantus, “Greater signal, increased depth, and less photobleaching in two-photon microscopy with 10 fs pulses,” Opt. Commun. 281(7), 1841–1849 (2008).
[CrossRef]

Luo, A.

A. Mohanty, Y. Yang, A. Luo, A. W. Sylvester, and D. Jackson, “Methods for generation and analysis of fluorescent protein-tagged maize lines,” Methods Mol. Biol. 526, 71–89 (2009).
[CrossRef] [PubMed]

Magee, J. C.

N. Ji, J. C. Magee, and E. Betzig, “High-speed, low-photodamage nonlinear imaging using passive pulse splitters,” Nat. Methods 5(2), 197–202 (2008).
[CrossRef] [PubMed]

Makarov, N. S.

M. Drobizhev, S. Tillo, N. S. Makarov, T. E. Hughes, and A. Rebane, “Absolute two-photon absorption spectra and two-photon brightness of orange and red fluorescent proteins,” J. Phys. Chem. B 113(4), 855–859 (2009).
[CrossRef] [PubMed]

McGresham, K.

Meeley, R. B.

C. R. Dietrich, M. A. D. N. Perera, M. D. Yandeau-Nelson, R. B. Meeley, B. J. Nikolau, and P. S. Schnable, “Characterization of two GL8 paralogs reveals that the 3-ketoacyl reductase component of fatty acid elongase is essential for maize (Zea mays L.) development,” Plant J. 42(6), 844–861 (2005).
[CrossRef] [PubMed]

Meijerink, A.

W. G. J. H. M. van Sark, P. L. T. M. Frederix, D. J. Van den Heuvel, H. C. Gerritsen, A. A. Bol, J. N. J. van Lingen, C. de Mello Donegá, and A. Meijerink, “Photooxidation and photobleaching of single CdSe/ZnS quantum dots probed by room-temperature time-resolved spectroscopy,” J. Phys. Chem. B 105(35), 8281–8284 (2001).
[CrossRef]

Meshulach, D.

Midorikawa, K.

H. Kawano, Y. Nabekawa, A. Suda, Y. Oishi, H. Mizuno, A. Miyawaki, and K. Midorikawa, “Attenuation of photobleaching in two-photon excitation fluorescence from green fluorescent protein with shaped excitation pulses,” Biochem. Biophys. Res. Commun. 311(3), 592–596 (2003).
[CrossRef] [PubMed]

Millard, A. C.

D. N. Fittinghoff, A. C. Millard, J. A. Squier, and M. Müller, “Frequency-resolved optical gating measurement of ultrashort pulses passing through a high numerical aperture objective,” IEEE J. Quantum Electron. 35(4), 479–486 (1999).
[CrossRef]

Miyawaki, A.

H. Kawano, Y. Nabekawa, A. Suda, Y. Oishi, H. Mizuno, A. Miyawaki, and K. Midorikawa, “Attenuation of photobleaching in two-photon excitation fluorescence from green fluorescent protein with shaped excitation pulses,” Biochem. Biophys. Res. Commun. 311(3), 592–596 (2003).
[CrossRef] [PubMed]

Mizuno, H.

H. Kawano, Y. Nabekawa, A. Suda, Y. Oishi, H. Mizuno, A. Miyawaki, and K. Midorikawa, “Attenuation of photobleaching in two-photon excitation fluorescence from green fluorescent protein with shaped excitation pulses,” Biochem. Biophys. Res. Commun. 311(3), 592–596 (2003).
[CrossRef] [PubMed]

Mohanty, A.

A. Mohanty, Y. Yang, A. Luo, A. W. Sylvester, and D. Jackson, “Methods for generation and analysis of fluorescent protein-tagged maize lines,” Methods Mol. Biol. 526, 71–89 (2009).
[CrossRef] [PubMed]

Müller, M.

W. Amir, T. A. Planchon, C. G. Durfee, J. A. Squier, P. Gabolde, R. Trebino, and M. Müller, “Simultaneous visualization of spatial and chromatic aberrations by two-dimensional Fourier transform spectral interferometry,” Opt. Lett. 31(19), 2927–2929 (2006).
[CrossRef] [PubMed]

D. N. Fittinghoff, A. C. Millard, J. A. Squier, and M. Müller, “Frequency-resolved optical gating measurement of ultrashort pulses passing through a high numerical aperture objective,” IEEE J. Quantum Electron. 35(4), 479–486 (1999).
[CrossRef]

M. Müller, J. Squier, R. Wolleschensky, U. Simon, and G. J. Brakenhoff, “Dispersion pre-compensation of 15 femtosecond optical pulses for high-numerical-aperture objectives,” J. Microsc. 191(2), 141–150 (1998).
[CrossRef] [PubMed]

Nabekawa, Y.

H. Kawano, Y. Nabekawa, A. Suda, Y. Oishi, H. Mizuno, A. Miyawaki, and K. Midorikawa, “Attenuation of photobleaching in two-photon excitation fluorescence from green fluorescent protein with shaped excitation pulses,” Biochem. Biophys. Res. Commun. 311(3), 592–596 (2003).
[CrossRef] [PubMed]

Nikolau, B. J.

C. R. Dietrich, M. A. D. N. Perera, M. D. Yandeau-Nelson, R. B. Meeley, B. J. Nikolau, and P. S. Schnable, “Characterization of two GL8 paralogs reveals that the 3-ketoacyl reductase component of fatty acid elongase is essential for maize (Zea mays L.) development,” Plant J. 42(6), 844–861 (2005).
[CrossRef] [PubMed]

Novotny, L.

E. J. Sánchez, L. Novotny, G. R. Holtom, and X. S. Xie, “Room-temperature fluorescence imaging and spectroscopy of single molecules by two-photon excitation,” J. Phys. Chem. A 101(38), 7019–7023 (1997).
[CrossRef]

Oishi, Y.

H. Kawano, Y. Nabekawa, A. Suda, Y. Oishi, H. Mizuno, A. Miyawaki, and K. Midorikawa, “Attenuation of photobleaching in two-photon excitation fluorescence from green fluorescent protein with shaped excitation pulses,” Biochem. Biophys. Res. Commun. 311(3), 592–596 (2003).
[CrossRef] [PubMed]

Patterson, G. H.

G. H. Patterson and D. W. Piston, “Photobleaching in two-photon excitation microscopy,” Biophys. J. 78(4), 2159–2162 (2000).
[CrossRef] [PubMed]

Perera, M. A. D. N.

C. R. Dietrich, M. A. D. N. Perera, M. D. Yandeau-Nelson, R. B. Meeley, B. J. Nikolau, and P. S. Schnable, “Characterization of two GL8 paralogs reveals that the 3-ketoacyl reductase component of fatty acid elongase is essential for maize (Zea mays L.) development,” Plant J. 42(6), 844–861 (2005).
[CrossRef] [PubMed]

Piston, D. W.

G. H. Patterson and D. W. Piston, “Photobleaching in two-photon excitation microscopy,” Biophys. J. 78(4), 2159–2162 (2000).
[CrossRef] [PubMed]

Planchon, T. A.

J. J. Field, T. A. Planchon, W. Amir, C. G. Durfee, and J. A. Squier, “Characterization of a high efficiency, ultrashort pulse shaper incorporating a reflective 4096-element spatial light modulator,” Opt. Commun. 278(2), 368–376 (2007).
[CrossRef] [PubMed]

W. Amir, T. A. Planchon, C. G. Durfee, J. A. Squier, P. Gabolde, R. Trebino, and M. Müller, “Simultaneous visualization of spatial and chromatic aberrations by two-dimensional Fourier transform spectral interferometry,” Opt. Lett. 31(19), 2927–2929 (2006).
[CrossRef] [PubMed]

Rebane, A.

M. Drobizhev, S. Tillo, N. S. Makarov, T. E. Hughes, and A. Rebane, “Absolute two-photon absorption spectra and two-photon brightness of orange and red fluorescent proteins,” J. Phys. Chem. B 113(4), 855–859 (2009).
[CrossRef] [PubMed]

Rodriguez, G.

Sánchez, E. J.

E. J. Sánchez, L. Novotny, G. R. Holtom, and X. S. Xie, “Room-temperature fluorescence imaging and spectroscopy of single molecules by two-photon excitation,” J. Phys. Chem. A 101(38), 7019–7023 (1997).
[CrossRef]

Schnable, P. S.

C. R. Dietrich, M. A. D. N. Perera, M. D. Yandeau-Nelson, R. B. Meeley, B. J. Nikolau, and P. S. Schnable, “Characterization of two GL8 paralogs reveals that the 3-ketoacyl reductase component of fatty acid elongase is essential for maize (Zea mays L.) development,” Plant J. 42(6), 844–861 (2005).
[CrossRef] [PubMed]

Shavel, A.

A. Shavel, N. Gaponik, and A. Eychmüller, “Covalent linking of CdTe nanocrystals to amino-functionalized surfaces,” ChemPhysChem 6(3), 449–451 (2005).
[CrossRef] [PubMed]

Sheetz, K. E.

Shreenath, A.

Silberberg, Y.

Simon, U.

M. Müller, J. Squier, R. Wolleschensky, U. Simon, and G. J. Brakenhoff, “Dispersion pre-compensation of 15 femtosecond optical pulses for high-numerical-aperture objectives,” J. Microsc. 191(2), 141–150 (1998).
[CrossRef] [PubMed]

Squier, J.

C. Bardeen, V. Yakovlev, J. Squier, K. R. Wilson, S. D. Carpenter, and P. M. Weber, “Effect of pulse shape on the efficiency of multiphoton processes: implications for biological microscopy,” J. Biomed. Opt. 4(3), 362–367 (1999).
[CrossRef]

M. Müller, J. Squier, R. Wolleschensky, U. Simon, and G. J. Brakenhoff, “Dispersion pre-compensation of 15 femtosecond optical pulses for high-numerical-aperture objectives,” J. Microsc. 191(2), 141–150 (1998).
[CrossRef] [PubMed]

Squier, J. A.

R. Carriles, K. E. Sheetz, E. E. Hoover, J. A. Squier, and V. Barzda, “Simultaneous multifocal, multiphoton, photon counting microscopy,” Opt. Express 16(14), 10364–10371 (2008).
[CrossRef] [PubMed]

J. J. Field, T. A. Planchon, W. Amir, C. G. Durfee, and J. A. Squier, “Characterization of a high efficiency, ultrashort pulse shaper incorporating a reflective 4096-element spatial light modulator,” Opt. Commun. 278(2), 368–376 (2007).
[CrossRef] [PubMed]

J. J. Field, C. G. Durfee, J. A. Squier, and S. Kane, “Quartic-phase-limited grism-based ultrashort pulse shaper,” Opt. Lett. 32(21), 3101–3103 (2007).
[CrossRef] [PubMed]

W. Amir, T. A. Planchon, C. G. Durfee, J. A. Squier, P. Gabolde, R. Trebino, and M. Müller, “Simultaneous visualization of spatial and chromatic aberrations by two-dimensional Fourier transform spectral interferometry,” Opt. Lett. 31(19), 2927–2929 (2006).
[CrossRef] [PubMed]

D. N. Fittinghoff, A. C. Millard, J. A. Squier, and M. Müller, “Frequency-resolved optical gating measurement of ultrashort pulses passing through a high numerical aperture objective,” IEEE J. Quantum Electron. 35(4), 479–486 (1999).
[CrossRef]

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.

H. Kawano, Y. Nabekawa, A. Suda, Y. Oishi, H. Mizuno, A. Miyawaki, and K. Midorikawa, “Attenuation of photobleaching in two-photon excitation fluorescence from green fluorescent protein with shaped excitation pulses,” Biochem. Biophys. Res. Commun. 311(3), 592–596 (2003).
[CrossRef] [PubMed]

Sylvester, A. W.

A. Mohanty, Y. Yang, A. Luo, A. W. Sylvester, and D. Jackson, “Methods for generation and analysis of fluorescent protein-tagged maize lines,” Methods Mol. Biol. 526, 71–89 (2009).
[CrossRef] [PubMed]

Taylor, A. J.

Tillo, S.

M. Drobizhev, S. Tillo, N. S. Makarov, T. E. Hughes, and A. Rebane, “Absolute two-photon absorption spectra and two-photon brightness of orange and red fluorescent proteins,” J. Phys. Chem. B 113(4), 855–859 (2009).
[CrossRef] [PubMed]

Trebino, R.

Van den Heuvel, D. J.

W. G. J. H. M. van Sark, P. L. T. M. Frederix, D. J. Van den Heuvel, H. C. Gerritsen, A. A. Bol, J. N. J. van Lingen, C. de Mello Donegá, and A. Meijerink, “Photooxidation and photobleaching of single CdSe/ZnS quantum dots probed by room-temperature time-resolved spectroscopy,” J. Phys. Chem. B 105(35), 8281–8284 (2001).
[CrossRef]

van Lingen, J. N. J.

W. G. J. H. M. van Sark, P. L. T. M. Frederix, D. J. Van den Heuvel, H. C. Gerritsen, A. A. Bol, J. N. J. van Lingen, C. de Mello Donegá, and A. Meijerink, “Photooxidation and photobleaching of single CdSe/ZnS quantum dots probed by room-temperature time-resolved spectroscopy,” J. Phys. Chem. B 105(35), 8281–8284 (2001).
[CrossRef]

van Sark, W. G. J. H. M.

W. G. J. H. M. van Sark, P. L. T. M. Frederix, D. J. Van den Heuvel, H. C. Gerritsen, A. A. Bol, J. N. J. van Lingen, C. de Mello Donegá, and A. Meijerink, “Photooxidation and photobleaching of single CdSe/ZnS quantum dots probed by room-temperature time-resolved spectroscopy,” J. Phys. Chem. B 105(35), 8281–8284 (2001).
[CrossRef]

Webb, W. W.

Weber, P. M.

C. Bardeen, V. Yakovlev, J. Squier, K. R. Wilson, S. D. Carpenter, and P. M. Weber, “Effect of pulse shape on the efficiency of multiphoton processes: implications for biological microscopy,” J. Biomed. Opt. 4(3), 362–367 (1999).
[CrossRef]

Weisel, L. R.

P. Xi, Y. Andegeko, L. R. Weisel, V. V. Lozovoy, and M. Dantus, “Greater signal, increased depth, and less photobleaching in two-photon microscopy with 10 fs pulses,” Opt. Commun. 281(7), 1841–1849 (2008).
[CrossRef]

Wilson, K. R.

C. Bardeen, V. Yakovlev, J. Squier, K. R. Wilson, S. D. Carpenter, and P. M. Weber, “Effect of pulse shape on the efficiency of multiphoton processes: implications for biological microscopy,” J. Biomed. Opt. 4(3), 362–367 (1999).
[CrossRef]

Wolleschensky, R.

M. Müller, J. Squier, R. Wolleschensky, U. Simon, and G. J. Brakenhoff, “Dispersion pre-compensation of 15 femtosecond optical pulses for high-numerical-aperture objectives,” J. Microsc. 191(2), 141–150 (1998).
[CrossRef] [PubMed]

Xi, P.

P. Xi, Y. Andegeko, L. R. Weisel, V. V. Lozovoy, and M. Dantus, “Greater signal, increased depth, and less photobleaching in two-photon microscopy with 10 fs pulses,” Opt. Commun. 281(7), 1841–1849 (2008).
[CrossRef]

Xie, X. S.

E. J. Sánchez, L. Novotny, G. R. Holtom, and X. S. Xie, “Room-temperature fluorescence imaging and spectroscopy of single molecules by two-photon excitation,” J. Phys. Chem. A 101(38), 7019–7023 (1997).
[CrossRef]

Xu, C.

Yakovlev, V.

C. Bardeen, V. Yakovlev, J. Squier, K. R. Wilson, S. D. Carpenter, and P. M. Weber, “Effect of pulse shape on the efficiency of multiphoton processes: implications for biological microscopy,” J. Biomed. Opt. 4(3), 362–367 (1999).
[CrossRef]

Yang, Y.

A. Mohanty, Y. Yang, A. Luo, A. W. Sylvester, and D. Jackson, “Methods for generation and analysis of fluorescent protein-tagged maize lines,” Methods Mol. Biol. 526, 71–89 (2009).
[CrossRef] [PubMed]

Yelin, D.

Biochem. Biophys. Res. Commun.

H. Kawano, Y. Nabekawa, A. Suda, Y. Oishi, H. Mizuno, A. Miyawaki, and K. Midorikawa, “Attenuation of photobleaching in two-photon excitation fluorescence from green fluorescent protein with shaped excitation pulses,” Biochem. Biophys. Res. Commun. 311(3), 592–596 (2003).
[CrossRef] [PubMed]

Biophys. J.

G. H. Patterson and D. W. Piston, “Photobleaching in two-photon excitation microscopy,” Biophys. J. 78(4), 2159–2162 (2000).
[CrossRef] [PubMed]

ChemPhysChem

A. Shavel, N. Gaponik, and A. Eychmüller, “Covalent linking of CdTe nanocrystals to amino-functionalized surfaces,” ChemPhysChem 6(3), 449–451 (2005).
[CrossRef] [PubMed]

IEEE J. Quantum Electron.

D. N. Fittinghoff, A. C. Millard, J. A. Squier, and M. Müller, “Frequency-resolved optical gating measurement of ultrashort pulses passing through a high numerical aperture objective,” IEEE J. Quantum Electron. 35(4), 479–486 (1999).
[CrossRef]

J. Biomed. Opt.

C. Bardeen, V. Yakovlev, J. Squier, K. R. Wilson, S. D. Carpenter, and P. M. Weber, “Effect of pulse shape on the efficiency of multiphoton processes: implications for biological microscopy,” J. Biomed. Opt. 4(3), 362–367 (1999).
[CrossRef]

J. Chem. Phys.

A. J. Berglund, “Nonexponential statistics of fluorescence photobleaching,” J. Chem. Phys. 121(7), 2899–2903 (2004).
[CrossRef] [PubMed]

J. Microsc.

M. Müller, J. Squier, R. Wolleschensky, U. Simon, and G. J. Brakenhoff, “Dispersion pre-compensation of 15 femtosecond optical pulses for high-numerical-aperture objectives,” J. Microsc. 191(2), 141–150 (1998).
[CrossRef] [PubMed]

J. Opt. Soc. Am. B

J. Phys. Chem. A

E. J. Sánchez, L. Novotny, G. R. Holtom, and X. S. Xie, “Room-temperature fluorescence imaging and spectroscopy of single molecules by two-photon excitation,” J. Phys. Chem. A 101(38), 7019–7023 (1997).
[CrossRef]

J. Phys. Chem. B

W. G. J. H. M. van Sark, P. L. T. M. Frederix, D. J. Van den Heuvel, H. C. Gerritsen, A. A. Bol, J. N. J. van Lingen, C. de Mello Donegá, and A. Meijerink, “Photooxidation and photobleaching of single CdSe/ZnS quantum dots probed by room-temperature time-resolved spectroscopy,” J. Phys. Chem. B 105(35), 8281–8284 (2001).
[CrossRef]

M. Drobizhev, S. Tillo, N. S. Makarov, T. E. Hughes, and A. Rebane, “Absolute two-photon absorption spectra and two-photon brightness of orange and red fluorescent proteins,” J. Phys. Chem. B 113(4), 855–859 (2009).
[CrossRef] [PubMed]

Methods Mol. Biol.

A. Mohanty, Y. Yang, A. Luo, A. W. Sylvester, and D. Jackson, “Methods for generation and analysis of fluorescent protein-tagged maize lines,” Methods Mol. Biol. 526, 71–89 (2009).
[CrossRef] [PubMed]

Nat. Methods

G. Donnert, C. Eggeling, and S. W. Hell, “Major signal increase in fluorescence microscopy through dark-state relaxation,” Nat. Methods 4(1), 81–86 (2007).
[CrossRef]

N. Ji, J. C. Magee, and E. Betzig, “High-speed, low-photodamage nonlinear imaging using passive pulse splitters,” Nat. Methods 5(2), 197–202 (2008).
[CrossRef] [PubMed]

F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2(12), 932–940 (2005).
[CrossRef] [PubMed]

Opt. Commun.

P. Xi, Y. Andegeko, L. R. Weisel, V. V. Lozovoy, and M. Dantus, “Greater signal, increased depth, and less photobleaching in two-photon microscopy with 10 fs pulses,” Opt. Commun. 281(7), 1841–1849 (2008).
[CrossRef]

J. J. Field, T. A. Planchon, W. Amir, C. G. Durfee, and J. A. Squier, “Characterization of a high efficiency, ultrashort pulse shaper incorporating a reflective 4096-element spatial light modulator,” Opt. Commun. 278(2), 368–376 (2007).
[CrossRef] [PubMed]

Opt. Express

Opt. Lett.

Plant J.

C. R. Dietrich, M. A. D. N. Perera, M. D. Yandeau-Nelson, R. B. Meeley, B. J. Nikolau, and P. S. Schnable, “Characterization of two GL8 paralogs reveals that the 3-ketoacyl reductase component of fatty acid elongase is essential for maize (Zea mays L.) development,” Plant J. 42(6), 844–861 (2005).
[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]

Other

A. Diaspro, G. Chirico, C. Usai, P. Ramoino, and J. Dobrucki, in Handbook of Biological Confocal Microscopy, 3rd. Ed., (ed. James B. Pawley) Ch. 39, 690–702 (Springer Science + Business Media, 2006).

R. Trebino, Frequency-Resolved Optical Gating: The Measurement of Ultrashort Laser Pulses, (Kluwer Academic Publishers, Norwell, MA, 2000).

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Figures (5)

Fig. 1
Fig. 1

Method for quantifying photobleaching in the non-scanning mode. (a) Image of a RhB-ITC sample prior to bleaching, taken with the INP pulse. (b) Image of the sample after photobleaching measurements, imaged with the OBJ pulse. Note the increase in photons/pixel with the OBJ pulse. (c) Bleaching data is stored in a two-dimensional matrix during the bleaching measurement. (d) Averaged photon-counted photobleaching curves for each pulse shape. Note that this figure is intended to be an example only, and data obtained in this figure are not part of the experimental results discussed below.

Fig. 2
Fig. 2

Measured pulse shapes in the focal plane of the excitation objective. Blind FROG traces for (a) INP, (b) TOL, (c) FOL, and (d) OBJ pulses obtained in focus. Extracted pulse shapes with calculated peak intensities (see text) are shown on a (e) linear and (f) logarithmic scale. FROG errors for the reconstructed traces were (a) 0.019, (b) 0.019, (c) 0.012, and (d) 0.014, each on a 80x80 point grid.

Fig. 3
Fig. 3

Photobleaching data corresponding to the pulse shapes displayed in Fig. 2. Bleaching curves for (a) RhB-ITC, (b) TagRFP, crosslinked in porcine gelatin, and (c) QD samples.

Fig. 4
Fig. 4

Photobleaching curves with broadband femtosecond pulses. Bleaching in non-scanning mode for (a) Rhodamine B and (b) TagRFP in agarose, and scanning mode for (c) Rhodamine B and (d) TagRFP. Second order correction improves the number of emitted photons in all cases. The distinction between TOL and FOL pulses yields little or no improvement for (a) & (c), and is essentially within the signal to noise of the measurement.

Fig. 5
Fig. 5

Improvement of TPEF signal by shortening excitation pulse in epidermal cells of a maize plant expressing the GL8 protein tagged with mRFP. Images were taken with (a) INP and (b) OBJ pulses respectively. In the OBJ image, the arrow points to mRFP concentration near the nucleus, while the arrowheads indicate cytoplasmic strands in the cell. Note that the only post-processing of these images is the false color intensity map, and that the contour numbers in the calibration bar represent photon count. The improvement in imaging with a TL pulse at the sample plane is clear from the histogram of the photon counts in each image shown in (c). Scanning photobleaching tests were performed in vivo using the INP and OBJ pulses, as shown in (d) and (e) respectively. Each image was acquired in 2 seconds (0.5 Hz, 256x256-pixel) for 50 consecutive images. The mean photon count as a function of imaging time was calculated, and is displayed in (f). The photobleaching rates and intensities follow the same pattern as that observed in homogeneous TagRFP samples. Scale bars are 10 µm.

Tables (1)

Tables Icon

Table 1 Photobleaching rates and fluorescence yield in all three fluorophores and corresponding measurements of the second-order temporal coherence obtained from Blind FROG

Equations (2)

Equations on this page are rendered with MathJax. Learn more.

I peak = g P ave A = g p τ f P ave π r 0 2
g d t f I 2 ( t ) [ d t f I ( t ) ] 2

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