Abstract

We report quantitative measurements of two-, three-, and four-photon excitation action cross sections of several commonly used fluorophores and fluorescent proteins at three different excitation wavelengths of 800 nm, 1300 nm, and 1680 nm. The measured cross section values are consistent with simple quantum mechanic estimations. These values indicate that the optimum repetition rate for deep tissue 3-photon microscopy is approximately 1 to 2 MHz. We further demonstrate that it is feasible to perform 4-photon fluorescence microscopy of GFP labeled microglia in mouse brain in vivo at 1700 nm. 4-photon excitation increases the accessibility of fluorophores at the long wavelength spectral window of 1700 nm.

© 2014 Optical Society of America

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  1. D. Kobat, M. E. Durst, N. Nishimura, A. W. Wong, C. B. Schaffer, and C. Xu, “Deep tissue multiphoton microscopy using longer wavelength excitation,” Opt. Express 17(16), 13354–13364 (2009).
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    [Crossref] [PubMed]
  4. V. Andresen, S. Alexander, W. M. Heupel, M. Hirschberg, R. M. Hoffman, and P. Friedl, “Infrared multiphoton microscopy: subcellular-resolved deep tissue imaging,” Curr. Opin. Biotechnol. 20(1), 54–62 (2009).
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  6. P. Theer and W. Denk, “On the fundamental imaging-depth limit in two-photon microscopy,” J. Opt. Soc. Am. A 23(12), 3139–3149 (2006).
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    [Crossref]
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    [Crossref] [PubMed]
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    [Crossref] [PubMed]
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    [Crossref] [PubMed]
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2013 (1)

N. G. Horton, K. Wang, D. Kobat, C. G. Clark, F. W. Wise, C. B. Schaffer, and C. Xu, “In vivo three-photon microscopy of subcortical structures within an intact mouse brain,” Nat. Photonics 7(3), 205–209 (2013).
[Crossref] [PubMed]

2012 (1)

J. Mütze, V. Iyer, J. J. Macklin, J. Colonell, B. Karsh, Z. Petrášek, P. Schwille, L. L. Looger, L. D. Lavis, and T. D. Harris, “Excitation spectra and brightness optimization of two-photon excited probes,” Biophys. J. 102(4), 934–944 (2012).
[Crossref] [PubMed]

2011 (4)

D. Kobat, N. G. Horton, and C. Xu, “In vivo two-photon microscopy to 1.6-mm depth in mouse cortex,” J. Biomed. Opt. 16(10), 106014 (2011).
[Crossref] [PubMed]

M. Drobizhev, N. S. Makarov, S. E. Tillo, T. E. Hughes, and A. Rebane, “Two-photon absorption properties of fluorescent proteins,” Nat. Methods 8(5), 393–399 (2011).
[Crossref] [PubMed]

K. Wang and C. Xu, “Tunable high-energy soliton pulse generation from a large mode- area fiber and its application to third harmonic generation microscopy,” Appl. Phys. Lett. 99(7), 071112 (2011).
[Crossref]

W. Mittmann, D. J. Wallace, U. Czubayko, J. T. Herb, A. T. Schaefer, L. L. Looger, W. Denk, and J. N. D. Kerr, “Two-photon calcium imaging of evoked activity from L5 somatosensory neurons in vivo,” Nat. Neurosci. 14(8), 1089–1093 (2011).
[Crossref] [PubMed]

2009 (4)

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]

M. Balu, T. Baldacchini, J. Carter, T. B. Krasieva, R. Zadoyan, and B. J. Tromberg, “Effect of excitation wavelength on penetration depth in nonlinear optical microscopy of turbid media,” J. Biomed. Opt. 14(1), 010508 (2009).
[Crossref] [PubMed]

V. Andresen, S. Alexander, W. M. Heupel, M. Hirschberg, R. M. Hoffman, and P. Friedl, “Infrared multiphoton microscopy: subcellular-resolved deep tissue imaging,” Curr. Opin. Biotechnol. 20(1), 54–62 (2009).
[Crossref] [PubMed]

D. Kobat, M. E. Durst, N. Nishimura, A. W. Wong, C. B. Schaffer, and C. Xu, “Deep tissue multiphoton microscopy using longer wavelength excitation,” Opt. Express 17(16), 13354–13364 (2009).
[Crossref] [PubMed]

2008 (3)

2006 (2)

2002 (1)

I.-H. Chen, S.-W. Chu, C.-K. Sun, P.-C. Cheng, and B.-L. Lin, “Wavelength dependent damage in biological multi-photon confocal microscopy: a micro-spectroscopic comparison between femtosecond Ti:sapphire and Cr:forsterite laser sources,” Opt. Quantum Electron. 34(12), 1251–1266 (2002).
[Crossref]

2001 (1)

G. A. Blab, P. H. M. Lommerse, L. Cognet, G. S. Harms, and T. Schmidt, “Two-photon excitation action cross-sections of the autofluorescent proteins,” Chem. Phys. Lett. 350(1-2), 71–77 (2001).
[Crossref]

1998 (1)

1996 (2)

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]

C. Xu and W. W. Webb, “Measurement of two-photon excitation cross sections of molecular fluorophores with data from 690 to 1050 nm,” J. Opt. Soc. Am. B 13(3), 481–491 (1996).
[Crossref]

1987 (1)

B. Zysset, P. Beaud, and W. Hodel, “Generation of optical solitons in the wavelength region 1.37–1.49 mm,” Appl. Phys. Lett. 50(16), 1027–1029 (1987).
[Crossref]

Albota, M. A.

Alexander, S.

V. Andresen, S. Alexander, W. M. Heupel, M. Hirschberg, R. M. Hoffman, and P. Friedl, “Infrared multiphoton microscopy: subcellular-resolved deep tissue imaging,” Curr. Opin. Biotechnol. 20(1), 54–62 (2009).
[Crossref] [PubMed]

Andresen, V.

V. Andresen, S. Alexander, W. M. Heupel, M. Hirschberg, R. M. Hoffman, and P. Friedl, “Infrared multiphoton microscopy: subcellular-resolved deep tissue imaging,” Curr. Opin. Biotechnol. 20(1), 54–62 (2009).
[Crossref] [PubMed]

Baldacchini, T.

M. Balu, T. Baldacchini, J. Carter, T. B. Krasieva, R. Zadoyan, and B. J. Tromberg, “Effect of excitation wavelength on penetration depth in nonlinear optical microscopy of turbid media,” J. Biomed. Opt. 14(1), 010508 (2009).
[Crossref] [PubMed]

Balu, M.

M. Balu, T. Baldacchini, J. Carter, T. B. Krasieva, R. Zadoyan, and B. J. Tromberg, “Effect of excitation wavelength on penetration depth in nonlinear optical microscopy of turbid media,” J. Biomed. Opt. 14(1), 010508 (2009).
[Crossref] [PubMed]

Beaud, P.

B. Zysset, P. Beaud, and W. Hodel, “Generation of optical solitons in the wavelength region 1.37–1.49 mm,” Appl. Phys. Lett. 50(16), 1027–1029 (1987).
[Crossref]

Blab, G. A.

G. A. Blab, P. H. M. Lommerse, L. Cognet, G. S. Harms, and T. Schmidt, “Two-photon excitation action cross-sections of the autofluorescent proteins,” Chem. Phys. Lett. 350(1-2), 71–77 (2001).
[Crossref]

Bolmont, T.

T. Bolmont, F. Haiss, D. Eicke, R. Radde, C. A. Mathis, W. E. Klunk, S. Kohsaka, M. Jucker, and M. E. Calhoun, “Dynamics of the microglial/amyloid interaction indicate a role in plaque maintenance,” J. Neurosci. 28(16), 4283–4292 (2008).
[Crossref] [PubMed]

Brunette, I.

Calhoun, M. E.

T. Bolmont, F. Haiss, D. Eicke, R. Radde, C. A. Mathis, W. E. Klunk, S. Kohsaka, M. Jucker, and M. E. Calhoun, “Dynamics of the microglial/amyloid interaction indicate a role in plaque maintenance,” J. Neurosci. 28(16), 4283–4292 (2008).
[Crossref] [PubMed]

Carter, J.

M. Balu, T. Baldacchini, J. Carter, T. B. Krasieva, R. Zadoyan, and B. J. Tromberg, “Effect of excitation wavelength on penetration depth in nonlinear optical microscopy of turbid media,” J. Biomed. Opt. 14(1), 010508 (2009).
[Crossref] [PubMed]

Chen, I.-H.

I.-H. Chen, S.-W. Chu, C.-K. Sun, P.-C. Cheng, and B.-L. Lin, “Wavelength dependent damage in biological multi-photon confocal microscopy: a micro-spectroscopic comparison between femtosecond Ti:sapphire and Cr:forsterite laser sources,” Opt. Quantum Electron. 34(12), 1251–1266 (2002).
[Crossref]

Cheng, J.-X.

Cheng, P.-C.

I.-H. Chen, S.-W. Chu, C.-K. Sun, P.-C. Cheng, and B.-L. Lin, “Wavelength dependent damage in biological multi-photon confocal microscopy: a micro-spectroscopic comparison between femtosecond Ti:sapphire and Cr:forsterite laser sources,” Opt. Quantum Electron. 34(12), 1251–1266 (2002).
[Crossref]

Chu, S.-W.

I.-H. Chen, S.-W. Chu, C.-K. Sun, P.-C. Cheng, and B.-L. Lin, “Wavelength dependent damage in biological multi-photon confocal microscopy: a micro-spectroscopic comparison between femtosecond Ti:sapphire and Cr:forsterite laser sources,” Opt. Quantum Electron. 34(12), 1251–1266 (2002).
[Crossref]

Clark, C. G.

N. G. Horton, K. Wang, D. Kobat, C. G. Clark, F. W. Wise, C. B. Schaffer, and C. Xu, “In vivo three-photon microscopy of subcortical structures within an intact mouse brain,” Nat. Photonics 7(3), 205–209 (2013).
[Crossref] [PubMed]

Cognet, L.

G. A. Blab, P. H. M. Lommerse, L. Cognet, G. S. Harms, and T. Schmidt, “Two-photon excitation action cross-sections of the autofluorescent proteins,” Chem. Phys. Lett. 350(1-2), 71–77 (2001).
[Crossref]

Colonell, J.

J. Mütze, V. Iyer, J. J. Macklin, J. Colonell, B. Karsh, Z. Petrášek, P. Schwille, L. L. Looger, L. D. Lavis, and T. D. Harris, “Excitation spectra and brightness optimization of two-photon excited probes,” Biophys. J. 102(4), 934–944 (2012).
[Crossref] [PubMed]

Czubayko, U.

W. Mittmann, D. J. Wallace, U. Czubayko, J. T. Herb, A. T. Schaefer, L. L. Looger, W. Denk, and J. N. D. Kerr, “Two-photon calcium imaging of evoked activity from L5 somatosensory neurons in vivo,” Nat. Neurosci. 14(8), 1089–1093 (2011).
[Crossref] [PubMed]

Denk, W.

W. Mittmann, D. J. Wallace, U. Czubayko, J. T. Herb, A. T. Schaefer, L. L. Looger, W. Denk, and J. N. D. Kerr, “Two-photon calcium imaging of evoked activity from L5 somatosensory neurons in vivo,” Nat. Neurosci. 14(8), 1089–1093 (2011).
[Crossref] [PubMed]

P. Theer and W. Denk, “On the fundamental imaging-depth limit in two-photon microscopy,” J. Opt. Soc. Am. A 23(12), 3139–3149 (2006).
[Crossref] [PubMed]

Drobizhev, M.

M. Drobizhev, N. S. Makarov, S. E. Tillo, T. E. Hughes, and A. Rebane, “Two-photon absorption properties of fluorescent proteins,” Nat. Methods 8(5), 393–399 (2011).
[Crossref] [PubMed]

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]

N. S. Makarov, M. Drobizhev, and A. Rebane, “Two-photon absorption standards in the 550-1600 nm excitation wavelength range,” Opt. Express 16(6), 4029–4047 (2008).
[Crossref] [PubMed]

Durst, M. E.

Eicke, D.

T. Bolmont, F. Haiss, D. Eicke, R. Radde, C. A. Mathis, W. E. Klunk, S. Kohsaka, M. Jucker, and M. E. Calhoun, “Dynamics of the microglial/amyloid interaction indicate a role in plaque maintenance,” J. Neurosci. 28(16), 4283–4292 (2008).
[Crossref] [PubMed]

Friedl, P.

V. Andresen, S. Alexander, W. M. Heupel, M. Hirschberg, R. M. Hoffman, and P. Friedl, “Infrared multiphoton microscopy: subcellular-resolved deep tissue imaging,” Curr. Opin. Biotechnol. 20(1), 54–62 (2009).
[Crossref] [PubMed]

Fu, Y.

Giguère, D.

Haiss, F.

T. Bolmont, F. Haiss, D. Eicke, R. Radde, C. A. Mathis, W. E. Klunk, S. Kohsaka, M. Jucker, and M. E. Calhoun, “Dynamics of the microglial/amyloid interaction indicate a role in plaque maintenance,” J. Neurosci. 28(16), 4283–4292 (2008).
[Crossref] [PubMed]

Harms, G. S.

G. A. Blab, P. H. M. Lommerse, L. Cognet, G. S. Harms, and T. Schmidt, “Two-photon excitation action cross-sections of the autofluorescent proteins,” Chem. Phys. Lett. 350(1-2), 71–77 (2001).
[Crossref]

Harris, T. D.

J. Mütze, V. Iyer, J. J. Macklin, J. Colonell, B. Karsh, Z. Petrášek, P. Schwille, L. L. Looger, L. D. Lavis, and T. D. Harris, “Excitation spectra and brightness optimization of two-photon excited probes,” Biophys. J. 102(4), 934–944 (2012).
[Crossref] [PubMed]

Herb, J. T.

W. Mittmann, D. J. Wallace, U. Czubayko, J. T. Herb, A. T. Schaefer, L. L. Looger, W. Denk, and J. N. D. Kerr, “Two-photon calcium imaging of evoked activity from L5 somatosensory neurons in vivo,” Nat. Neurosci. 14(8), 1089–1093 (2011).
[Crossref] [PubMed]

Heupel, W. M.

V. Andresen, S. Alexander, W. M. Heupel, M. Hirschberg, R. M. Hoffman, and P. Friedl, “Infrared multiphoton microscopy: subcellular-resolved deep tissue imaging,” Curr. Opin. Biotechnol. 20(1), 54–62 (2009).
[Crossref] [PubMed]

Hirschberg, M.

V. Andresen, S. Alexander, W. M. Heupel, M. Hirschberg, R. M. Hoffman, and P. Friedl, “Infrared multiphoton microscopy: subcellular-resolved deep tissue imaging,” Curr. Opin. Biotechnol. 20(1), 54–62 (2009).
[Crossref] [PubMed]

Hodel, W.

B. Zysset, P. Beaud, and W. Hodel, “Generation of optical solitons in the wavelength region 1.37–1.49 mm,” Appl. Phys. Lett. 50(16), 1027–1029 (1987).
[Crossref]

Hoffman, R. M.

V. Andresen, S. Alexander, W. M. Heupel, M. Hirschberg, R. M. Hoffman, and P. Friedl, “Infrared multiphoton microscopy: subcellular-resolved deep tissue imaging,” Curr. Opin. Biotechnol. 20(1), 54–62 (2009).
[Crossref] [PubMed]

Horton, N. G.

N. G. Horton, K. Wang, D. Kobat, C. G. Clark, F. W. Wise, C. B. Schaffer, and C. Xu, “In vivo three-photon microscopy of subcortical structures within an intact mouse brain,” Nat. Photonics 7(3), 205–209 (2013).
[Crossref] [PubMed]

D. Kobat, N. G. Horton, and C. Xu, “In vivo two-photon microscopy to 1.6-mm depth in mouse cortex,” J. Biomed. Opt. 16(10), 106014 (2011).
[Crossref] [PubMed]

Hughes, T. E.

M. Drobizhev, N. S. Makarov, S. E. Tillo, T. E. Hughes, and A. Rebane, “Two-photon absorption properties of fluorescent proteins,” Nat. Methods 8(5), 393–399 (2011).
[Crossref] [PubMed]

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]

Iyer, V.

J. Mütze, V. Iyer, J. J. Macklin, J. Colonell, B. Karsh, Z. Petrášek, P. Schwille, L. L. Looger, L. D. Lavis, and T. D. Harris, “Excitation spectra and brightness optimization of two-photon excited probes,” Biophys. J. 102(4), 934–944 (2012).
[Crossref] [PubMed]

Jucker, M.

T. Bolmont, F. Haiss, D. Eicke, R. Radde, C. A. Mathis, W. E. Klunk, S. Kohsaka, M. Jucker, and M. E. Calhoun, “Dynamics of the microglial/amyloid interaction indicate a role in plaque maintenance,” J. Neurosci. 28(16), 4283–4292 (2008).
[Crossref] [PubMed]

Karsh, B.

J. Mütze, V. Iyer, J. J. Macklin, J. Colonell, B. Karsh, Z. Petrášek, P. Schwille, L. L. Looger, L. D. Lavis, and T. D. Harris, “Excitation spectra and brightness optimization of two-photon excited probes,” Biophys. J. 102(4), 934–944 (2012).
[Crossref] [PubMed]

Kerr, J. N. D.

W. Mittmann, D. J. Wallace, U. Czubayko, J. T. Herb, A. T. Schaefer, L. L. Looger, W. Denk, and J. N. D. Kerr, “Two-photon calcium imaging of evoked activity from L5 somatosensory neurons in vivo,” Nat. Neurosci. 14(8), 1089–1093 (2011).
[Crossref] [PubMed]

Kieffer, J.-C.

Klunk, W. E.

T. Bolmont, F. Haiss, D. Eicke, R. Radde, C. A. Mathis, W. E. Klunk, S. Kohsaka, M. Jucker, and M. E. Calhoun, “Dynamics of the microglial/amyloid interaction indicate a role in plaque maintenance,” J. Neurosci. 28(16), 4283–4292 (2008).
[Crossref] [PubMed]

Kobat, D.

N. G. Horton, K. Wang, D. Kobat, C. G. Clark, F. W. Wise, C. B. Schaffer, and C. Xu, “In vivo three-photon microscopy of subcortical structures within an intact mouse brain,” Nat. Photonics 7(3), 205–209 (2013).
[Crossref] [PubMed]

D. Kobat, N. G. Horton, and C. Xu, “In vivo two-photon microscopy to 1.6-mm depth in mouse cortex,” J. Biomed. Opt. 16(10), 106014 (2011).
[Crossref] [PubMed]

D. Kobat, M. E. Durst, N. Nishimura, A. W. Wong, C. B. Schaffer, and C. Xu, “Deep tissue multiphoton microscopy using longer wavelength excitation,” Opt. Express 17(16), 13354–13364 (2009).
[Crossref] [PubMed]

Kohsaka, S.

T. Bolmont, F. Haiss, D. Eicke, R. Radde, C. A. Mathis, W. E. Klunk, S. Kohsaka, M. Jucker, and M. E. Calhoun, “Dynamics of the microglial/amyloid interaction indicate a role in plaque maintenance,” J. Neurosci. 28(16), 4283–4292 (2008).
[Crossref] [PubMed]

Krasieva, T. B.

M. Balu, T. Baldacchini, J. Carter, T. B. Krasieva, R. Zadoyan, and B. J. Tromberg, “Effect of excitation wavelength on penetration depth in nonlinear optical microscopy of turbid media,” J. Biomed. Opt. 14(1), 010508 (2009).
[Crossref] [PubMed]

Lavis, L. D.

J. Mütze, V. Iyer, J. J. Macklin, J. Colonell, B. Karsh, Z. Petrášek, P. Schwille, L. L. Looger, L. D. Lavis, and T. D. Harris, “Excitation spectra and brightness optimization of two-photon excited probes,” Biophys. J. 102(4), 934–944 (2012).
[Crossref] [PubMed]

Lin, B.-L.

I.-H. Chen, S.-W. Chu, C.-K. Sun, P.-C. Cheng, and B.-L. Lin, “Wavelength dependent damage in biological multi-photon confocal microscopy: a micro-spectroscopic comparison between femtosecond Ti:sapphire and Cr:forsterite laser sources,” Opt. Quantum Electron. 34(12), 1251–1266 (2002).
[Crossref]

Lommerse, P. H. M.

G. A. Blab, P. H. M. Lommerse, L. Cognet, G. S. Harms, and T. Schmidt, “Two-photon excitation action cross-sections of the autofluorescent proteins,” Chem. Phys. Lett. 350(1-2), 71–77 (2001).
[Crossref]

Looger, L. L.

J. Mütze, V. Iyer, J. J. Macklin, J. Colonell, B. Karsh, Z. Petrášek, P. Schwille, L. L. Looger, L. D. Lavis, and T. D. Harris, “Excitation spectra and brightness optimization of two-photon excited probes,” Biophys. J. 102(4), 934–944 (2012).
[Crossref] [PubMed]

W. Mittmann, D. J. Wallace, U. Czubayko, J. T. Herb, A. T. Schaefer, L. L. Looger, W. Denk, and J. N. D. Kerr, “Two-photon calcium imaging of evoked activity from L5 somatosensory neurons in vivo,” Nat. Neurosci. 14(8), 1089–1093 (2011).
[Crossref] [PubMed]

Macklin, J. J.

J. Mütze, V. Iyer, J. J. Macklin, J. Colonell, B. Karsh, Z. Petrášek, P. Schwille, L. L. Looger, L. D. Lavis, and T. D. Harris, “Excitation spectra and brightness optimization of two-photon excited probes,” Biophys. J. 102(4), 934–944 (2012).
[Crossref] [PubMed]

Makarov, N. S.

M. Drobizhev, N. S. Makarov, S. E. Tillo, T. E. Hughes, and A. Rebane, “Two-photon absorption properties of fluorescent proteins,” Nat. Methods 8(5), 393–399 (2011).
[Crossref] [PubMed]

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]

N. S. Makarov, M. Drobizhev, and A. Rebane, “Two-photon absorption standards in the 550-1600 nm excitation wavelength range,” Opt. Express 16(6), 4029–4047 (2008).
[Crossref] [PubMed]

Mathis, C. A.

T. Bolmont, F. Haiss, D. Eicke, R. Radde, C. A. Mathis, W. E. Klunk, S. Kohsaka, M. Jucker, and M. E. Calhoun, “Dynamics of the microglial/amyloid interaction indicate a role in plaque maintenance,” J. Neurosci. 28(16), 4283–4292 (2008).
[Crossref] [PubMed]

Mittmann, W.

W. Mittmann, D. J. Wallace, U. Czubayko, J. T. Herb, A. T. Schaefer, L. L. Looger, W. Denk, and J. N. D. Kerr, “Two-photon calcium imaging of evoked activity from L5 somatosensory neurons in vivo,” Nat. Neurosci. 14(8), 1089–1093 (2011).
[Crossref] [PubMed]

Mütze, J.

J. Mütze, V. Iyer, J. J. Macklin, J. Colonell, B. Karsh, Z. Petrášek, P. Schwille, L. L. Looger, L. D. Lavis, and T. D. Harris, “Excitation spectra and brightness optimization of two-photon excited probes,” Biophys. J. 102(4), 934–944 (2012).
[Crossref] [PubMed]

Nada, O.

Nishimura, N.

Olivié, G.

Ozaki, T.

Petrášek, Z.

J. Mütze, V. Iyer, J. J. Macklin, J. Colonell, B. Karsh, Z. Petrášek, P. Schwille, L. L. Looger, L. D. Lavis, and T. D. Harris, “Excitation spectra and brightness optimization of two-photon excited probes,” Biophys. J. 102(4), 934–944 (2012).
[Crossref] [PubMed]

Radde, R.

T. Bolmont, F. Haiss, D. Eicke, R. Radde, C. A. Mathis, W. E. Klunk, S. Kohsaka, M. Jucker, and M. E. Calhoun, “Dynamics of the microglial/amyloid interaction indicate a role in plaque maintenance,” J. Neurosci. 28(16), 4283–4292 (2008).
[Crossref] [PubMed]

Rebane, A.

M. Drobizhev, N. S. Makarov, S. E. Tillo, T. E. Hughes, and A. Rebane, “Two-photon absorption properties of fluorescent proteins,” Nat. Methods 8(5), 393–399 (2011).
[Crossref] [PubMed]

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]

N. S. Makarov, M. Drobizhev, and A. Rebane, “Two-photon absorption standards in the 550-1600 nm excitation wavelength range,” Opt. Express 16(6), 4029–4047 (2008).
[Crossref] [PubMed]

Schaefer, A. T.

W. Mittmann, D. J. Wallace, U. Czubayko, J. T. Herb, A. T. Schaefer, L. L. Looger, W. Denk, and J. N. D. Kerr, “Two-photon calcium imaging of evoked activity from L5 somatosensory neurons in vivo,” Nat. Neurosci. 14(8), 1089–1093 (2011).
[Crossref] [PubMed]

Schaffer, C. B.

N. G. Horton, K. Wang, D. Kobat, C. G. Clark, F. W. Wise, C. B. Schaffer, and C. Xu, “In vivo three-photon microscopy of subcortical structures within an intact mouse brain,” Nat. Photonics 7(3), 205–209 (2013).
[Crossref] [PubMed]

D. Kobat, M. E. Durst, N. Nishimura, A. W. Wong, C. B. Schaffer, and C. Xu, “Deep tissue multiphoton microscopy using longer wavelength excitation,” Opt. Express 17(16), 13354–13364 (2009).
[Crossref] [PubMed]

Schmidt, T.

G. A. Blab, P. H. M. Lommerse, L. Cognet, G. S. Harms, and T. Schmidt, “Two-photon excitation action cross-sections of the autofluorescent proteins,” Chem. Phys. Lett. 350(1-2), 71–77 (2001).
[Crossref]

Schwille, P.

J. Mütze, V. Iyer, J. J. Macklin, J. Colonell, B. Karsh, Z. Petrášek, P. Schwille, L. L. Looger, L. D. Lavis, and T. D. Harris, “Excitation spectra and brightness optimization of two-photon excited probes,” Biophys. J. 102(4), 934–944 (2012).
[Crossref] [PubMed]

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]

Shi, R.

Sun, C.-K.

I.-H. Chen, S.-W. Chu, C.-K. Sun, P.-C. Cheng, and B.-L. Lin, “Wavelength dependent damage in biological multi-photon confocal microscopy: a micro-spectroscopic comparison between femtosecond Ti:sapphire and Cr:forsterite laser sources,” Opt. Quantum Electron. 34(12), 1251–1266 (2002).
[Crossref]

Theer, P.

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]

Tillo, S. E.

M. Drobizhev, N. S. Makarov, S. E. Tillo, T. E. Hughes, and A. Rebane, “Two-photon absorption properties of fluorescent proteins,” Nat. Methods 8(5), 393–399 (2011).
[Crossref] [PubMed]

Tromberg, B. J.

M. Balu, T. Baldacchini, J. Carter, T. B. Krasieva, R. Zadoyan, and B. J. Tromberg, “Effect of excitation wavelength on penetration depth in nonlinear optical microscopy of turbid media,” J. Biomed. Opt. 14(1), 010508 (2009).
[Crossref] [PubMed]

Vidal, F.

Wallace, D. J.

W. Mittmann, D. J. Wallace, U. Czubayko, J. T. Herb, A. T. Schaefer, L. L. Looger, W. Denk, and J. N. D. Kerr, “Two-photon calcium imaging of evoked activity from L5 somatosensory neurons in vivo,” Nat. Neurosci. 14(8), 1089–1093 (2011).
[Crossref] [PubMed]

Wang, H.

Wang, K.

N. G. Horton, K. Wang, D. Kobat, C. G. Clark, F. W. Wise, C. B. Schaffer, and C. Xu, “In vivo three-photon microscopy of subcortical structures within an intact mouse brain,” Nat. Photonics 7(3), 205–209 (2013).
[Crossref] [PubMed]

K. Wang and C. Xu, “Tunable high-energy soliton pulse generation from a large mode- area fiber and its application to third harmonic generation microscopy,” Appl. Phys. Lett. 99(7), 071112 (2011).
[Crossref]

Webb, W. W.

Williams, R. M.

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]

Wise, F. W.

N. G. Horton, K. Wang, D. Kobat, C. G. Clark, F. W. Wise, C. B. Schaffer, and C. Xu, “In vivo three-photon microscopy of subcortical structures within an intact mouse brain,” Nat. Photonics 7(3), 205–209 (2013).
[Crossref] [PubMed]

Wong, A. W.

Xu, C.

N. G. Horton, K. Wang, D. Kobat, C. G. Clark, F. W. Wise, C. B. Schaffer, and C. Xu, “In vivo three-photon microscopy of subcortical structures within an intact mouse brain,” Nat. Photonics 7(3), 205–209 (2013).
[Crossref] [PubMed]

D. Kobat, N. G. Horton, and C. Xu, “In vivo two-photon microscopy to 1.6-mm depth in mouse cortex,” J. Biomed. Opt. 16(10), 106014 (2011).
[Crossref] [PubMed]

K. Wang and C. Xu, “Tunable high-energy soliton pulse generation from a large mode- area fiber and its application to third harmonic generation microscopy,” Appl. Phys. Lett. 99(7), 071112 (2011).
[Crossref]

D. Kobat, M. E. Durst, N. Nishimura, A. W. Wong, C. B. Schaffer, and C. Xu, “Deep tissue multiphoton microscopy using longer wavelength excitation,” Opt. Express 17(16), 13354–13364 (2009).
[Crossref] [PubMed]

M. A. Albota, C. Xu, and W. W. Webb, “Two-photon fluorescence excitation cross sections of biomolecular probes from 690 to 960 nm,” Appl. Opt. 37(31), 7352–7356 (1998).
[Crossref] [PubMed]

C. Xu and W. W. Webb, “Measurement of two-photon excitation cross sections of molecular fluorophores with data from 690 to 1050 nm,” J. Opt. Soc. Am. B 13(3), 481–491 (1996).
[Crossref]

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]

Zadoyan, R.

M. Balu, T. Baldacchini, J. Carter, T. B. Krasieva, R. Zadoyan, and B. J. Tromberg, “Effect of excitation wavelength on penetration depth in nonlinear optical microscopy of turbid media,” J. Biomed. Opt. 14(1), 010508 (2009).
[Crossref] [PubMed]

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).
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Zysset, B.

B. Zysset, P. Beaud, and W. Hodel, “Generation of optical solitons in the wavelength region 1.37–1.49 mm,” Appl. Phys. Lett. 50(16), 1027–1029 (1987).
[Crossref]

Appl. Opt. (1)

Appl. Phys. Lett. (2)

B. Zysset, P. Beaud, and W. Hodel, “Generation of optical solitons in the wavelength region 1.37–1.49 mm,” Appl. Phys. Lett. 50(16), 1027–1029 (1987).
[Crossref]

K. Wang and C. Xu, “Tunable high-energy soliton pulse generation from a large mode- area fiber and its application to third harmonic generation microscopy,” Appl. Phys. Lett. 99(7), 071112 (2011).
[Crossref]

Biophys. J. (1)

J. Mütze, V. Iyer, J. J. Macklin, J. Colonell, B. Karsh, Z. Petrášek, P. Schwille, L. L. Looger, L. D. Lavis, and T. D. Harris, “Excitation spectra and brightness optimization of two-photon excited probes,” Biophys. J. 102(4), 934–944 (2012).
[Crossref] [PubMed]

Chem. Phys. Lett. (1)

G. A. Blab, P. H. M. Lommerse, L. Cognet, G. S. Harms, and T. Schmidt, “Two-photon excitation action cross-sections of the autofluorescent proteins,” Chem. Phys. Lett. 350(1-2), 71–77 (2001).
[Crossref]

Curr. Opin. Biotechnol. (1)

V. Andresen, S. Alexander, W. M. Heupel, M. Hirschberg, R. M. Hoffman, and P. Friedl, “Infrared multiphoton microscopy: subcellular-resolved deep tissue imaging,” Curr. Opin. Biotechnol. 20(1), 54–62 (2009).
[Crossref] [PubMed]

J. Biomed. Opt. (2)

D. Kobat, N. G. Horton, and C. Xu, “In vivo two-photon microscopy to 1.6-mm depth in mouse cortex,” J. Biomed. Opt. 16(10), 106014 (2011).
[Crossref] [PubMed]

M. Balu, T. Baldacchini, J. Carter, T. B. Krasieva, R. Zadoyan, and B. J. Tromberg, “Effect of excitation wavelength on penetration depth in nonlinear optical microscopy of turbid media,” J. Biomed. Opt. 14(1), 010508 (2009).
[Crossref] [PubMed]

J. Neurosci. (1)

T. Bolmont, F. Haiss, D. Eicke, R. Radde, C. A. Mathis, W. E. Klunk, S. Kohsaka, M. Jucker, and M. E. Calhoun, “Dynamics of the microglial/amyloid interaction indicate a role in plaque maintenance,” J. Neurosci. 28(16), 4283–4292 (2008).
[Crossref] [PubMed]

J. Opt. Soc. Am. A (1)

J. Opt. Soc. Am. B (1)

J. Phys. Chem. B (1)

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]

Nat. Methods (1)

M. Drobizhev, N. S. Makarov, S. E. Tillo, T. E. Hughes, and A. Rebane, “Two-photon absorption properties of fluorescent proteins,” Nat. Methods 8(5), 393–399 (2011).
[Crossref] [PubMed]

Nat. Neurosci. (1)

W. Mittmann, D. J. Wallace, U. Czubayko, J. T. Herb, A. T. Schaefer, L. L. Looger, W. Denk, and J. N. D. Kerr, “Two-photon calcium imaging of evoked activity from L5 somatosensory neurons in vivo,” Nat. Neurosci. 14(8), 1089–1093 (2011).
[Crossref] [PubMed]

Nat. Photonics (1)

N. G. Horton, K. Wang, D. Kobat, C. G. Clark, F. W. Wise, C. B. Schaffer, and C. Xu, “In vivo three-photon microscopy of subcortical structures within an intact mouse brain,” Nat. Photonics 7(3), 205–209 (2013).
[Crossref] [PubMed]

Opt. Express (4)

Opt. Quantum Electron. (1)

I.-H. Chen, S.-W. Chu, C.-K. Sun, P.-C. Cheng, and B.-L. Lin, “Wavelength dependent damage in biological multi-photon confocal microscopy: a micro-spectroscopic comparison between femtosecond Ti:sapphire and Cr:forsterite laser sources,” Opt. Quantum Electron. 34(12), 1251–1266 (2002).
[Crossref]

Proc. Natl. Acad. Sci. U.S.A. (1)

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]

Other (3)

D. Ouzounov, N. Horton, T. Wang, D. Feng, N. Nishimura, C. Xu, “In Vivo Three-photon Calcium Imaging of Brain Activity from Layer 6 Neurons in Mouse Brain,” CLEO, postdeadline paper, STh4C.2 (2014).

D. Kobat, N.G. Horton, and C. Xu, “In vivo two-photon imaging of cortical vasculature in mice to 1.5-mm depth with 1280-nm excitation,” CLEO, postdeadline paper, PDPB3(2011).

C. Xu and W. W. Webb, Topics in Fluorescence Spectroscopy, Vol. 5 (Springer, 1997), Chap. 11.

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Figures (3)

Fig. 1
Fig. 1 Experiment setup for measuring two-, three-, and four-photon excitation cross section. HP: half-wave plate. PBS: polarization beam splitter. M: mirror. BE: beam expander. DM: dichroic mirror. OL: objective lens. S: sample. F: emission filter. PMT: photomultiplier tube.
Fig. 2
Fig. 2 Logarithmic plots of the dependence of (a) two-, (b) three-, and (c) four-photon-excited fluorescence on excitation intensity for fluorescein (red square), wtGFP (blue square), and SR101 (black square). The slopes are indicated in the lower-right corner of each figure.
Fig. 3
Fig. 3 4PM and THG images of a mouse brain in vivo. (a-c): 2-frame averaging at a depth of 472 μm below the surface of the brain. (d-f): Average intensity of a 32 μm stack (2 frames per depth, 4 μm step size) ranging from 456 to 484 μm below the surface of the brain. The acquisition time was 4 seconds per frame, and the average power was 23 mW (repetition rate 1.3 MHz) on the brain surface. The scale bar is 50 μm.

Tables (1)

Tables Icon

Table 1 Two-, three-, and four-photon action cross sections of fluorescein, wtGFP and SR101

Equations (2)

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

F (n) (t) = 1 n g p (n) (fτ) n1 ϕη σ n C n 0 a n (NA) 2n4 P( t ) n 8 π 3n λ 2n3 ,
g p (n) = τ n1 T 2 T 2 I 0 n (t)dt [ T 2 T 2 I 0 (t)dt ] n ,

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