Abstract

Non-degenerate 2-photon excitation (ND-2PE) of a fluorophore with two laser beams of different photon energies offers an independent degree of freedom in tuning of the photon flux for each beam. This feature takes advantage of the infrared wavelengths used in degenerate 3-photon excitation (D-3PE) microscopy to achieve increased penetration depths, while preserving a relatively high 2-photon excitation cross section in comparison to that of D-3PE. Here, using spatially and temporally aligned Ti:Sapphire laser and optical parametric oscillator beams operating at near infrared (NIR) and short-wavelength infrared (SWIR) optical frequencies, we employ ND-2PE and provide a practical demonstration that a constant fluorophore emission intensity is achievable deeper into a scattering medium using ND-2PE as compared to the commonly used degenerate 2-photon excitation (D-2PE).

© 2016 Optical Society of America

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References

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2016 (1)

L. Shi, L. A. Sordillo, A. Rodríguez-Contreras, and R. Alfano, “Transmission in near-infrared optical windows for deep brain imaging,” J. Biophotonics 9(1-2), 38–43 (2016).
[Crossref] [PubMed]

2014 (6)

L.-C. Cheng, N. G. Horton, K. Wang, S.-J. Chen, and C. Xu, “Measurements of multiphoton action cross sections for multiphoton microscopy,” Biomed. Opt. Express 5(10), 3427–3433 (2014).
[Crossref] [PubMed]

L. Shi, A. Rodríguez-Contreras, and R. R. Alfano, “Gaussian beam in two-photon fluorescence imaging of rat brain microvessel,” J. Biomed. Opt. 19(12), 126006 (2014).
[Crossref] [PubMed]

G. Hong, S. Diao, J. Chang, A. L. Antaris, C. Chen, B. Zhang, S. Zhao, D. N. Atochin, P. L. Huang, K. I. Andreasson, C. J. Kuo, and H. Dai, “Through-skull fluorescence imaging of the brain in a new near-infrared window,” Nat. Photonics 8(9), 723–730 (2014).
[Crossref] [PubMed]

C. Wang, R. Liu, D. E. Milkie, W. Sun, Z. Tan, A. Kerlin, T.-W. Chen, D. S. Kim, and N. Ji, “Multiplexed aberration measurement for deep tissue imaging in vivo,” Nat. Methods 11(10), 1037–1040 (2014).
[Crossref] [PubMed]

K. Wang, D. E. Milkie, A. Saxena, P. Engerer, T. Misgeld, M. E. Bronner, J. Mumm, and E. Betzig, “Rapid adaptive optical recovery of optimal resolution over large volumes,” Nat. Methods 11(6), 625–628 (2014).
[Crossref] [PubMed]

T. Robinson, P. Valluri, G. Kennedy, A. Sardini, C. Dunsby, M. A. Neil, G. S. Baldwin, P. M. French, and A. J. de Mello, “Analysis of DNA binding and nucleotide flipping kinetics using two-color two-photon fluorescence lifetime imaging microscopy,” Anal. Chem. 86(21), 10732–10740 (2014).
[Crossref] [PubMed]

2013 (4)

T. R. Insel, S. C. Landis, F. S. Collins, and The NIH BRAIN Initiative, “Research priorities,” Science 340(6133), 687–688 (2013).
[Crossref] [PubMed]

K. Nizar, H. Uhlirova, P. Tian, P. A. Saisan, Q. Cheng, L. Reznichenko, K. L. Weldy, T. C. Steed, V. B. Sridhar, C. L. MacDonald, J. Cui, S. L. Gratiy, S. Sakadzić, D. A. Boas, T. I. Beka, G. T. Einevoll, J. Chen, E. Masliah, A. M. Dale, G. A. Silva, and A. Devor, “In vivo stimulus-induced vasodilation occurs without IP3 receptor activation and may precede astrocytic calcium increase,” J. Neurosci. 33(19), 8411–8422 (2013).
[Crossref] [PubMed]

R. Kawakami, K. Sawada, A. Sato, T. Hibi, Y. Kozawa, S. Sato, H. Yokoyama, and T. Nemoto, “Visualizing hippocampal neurons with in vivo two-photon microscopy using a 1030 nm picosecond pulse laser,” Sci. Rep. 3, 1014 (2013).
[Crossref] [PubMed]

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 (4)

P. Mahou, M. Zimmerley, K. Loulier, K. S. Matho, G. Labroille, X. Morin, W. Supatto, J. Livet, D. Débarre, and E. Beaurepaire, “Multicolor two-photon tissue imaging by wavelength mixing,” Nat. Methods 9(8), 815–818 (2012).
[Crossref] [PubMed]

L. V. Wang and S. Hu, “Photoacoustic tomography: in vivo imaging from organelles to organs,” Science 335(6075), 1458–1462 (2012).
[Crossref] [PubMed]

N. Ji, T. R. Sato, and E. Betzig, “Characterization and adaptive optical correction of aberrations during in vivo imaging in the mouse cortex,” Proc. Natl. Acad. Sci. U.S.A. 109(1), 22–27 (2012).
[Crossref] [PubMed]

E. W. Miller, J. Y. Lin, E. P. Frady, P. A. Steinbach, W. B. Kristan, and R. Y. Tsien, “Optically monitoring voltage in neurons by photo-induced electron transfer through molecular wires,” Proc. Natl. Acad. Sci. U.S.A. 109(6), 2114–2119 (2012).
[Crossref] [PubMed]

2011 (3)

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]

R. P. Barretto, T. H. Ko, J. C. Jung, T. J. Wang, G. Capps, A. C. Waters, Y. Ziv, A. Attardo, L. Recht, and M. J. Schnitzer, “Time-lapse imaging of disease progression in deep brain areas using fluorescence microendoscopy,” Nat. Med. 17(2), 223–228 (2011).
[Crossref] [PubMed]

K. A. Kasischke, E. M. Lambert, B. Panepento, A. Sun, H. A. Gelbard, R. W. Burgess, T. H. Foster, and M. Nedergaard, “Two-photon NADH imaging exposes boundaries of oxygen diffusion in cortical vascular supply regions,” J. Cereb. Blood Flow Metab. 31(1), 68–81 (2011).
[Crossref] [PubMed]

2010 (2)

S. Deng, L. Liu, G. Wang, R. Li, and Z. Xu, “Three-dimensional superresolution in two-color excitation fluorescence microscopy using theta illumination method,” Opt.-Int. J. Light Electron Opt. 121(8), 726–731 (2010).
[Crossref]

N. Ji, D. E. Milkie, and E. Betzig, “Adaptive optics via pupil segmentation for high-resolution imaging in biological tissues,” Nat. Methods 7(2), 141–147 (2010).
[Crossref] [PubMed]

2009 (2)

2008 (3)

J. N. Kerr and W. Denk, “Imaging in vivo: watching the brain in action,” Nat. Rev. Neurosci. 9(3), 195–205 (2008).
[Crossref] [PubMed]

C. Wang, L. Qiao, Z. Mao, Y. Cheng, and Z. Xu, “Reduced deep-tissue image degradation in three-dimensional multiphoton microscopy with concentric two-color two-photon fluorescence excitation,” JOSA B 25(6), 976–982 (2008).
[Crossref]

S. Quentmeier, S. Denicke, J.-E. Ehlers, R. A. Niesner, and K.-H. Gericke, “Two-color two-photon excitation using femtosecond laser pulses,” J. Phys. Chem. B 112(18), 5768–5773 (2008).
[Crossref] [PubMed]

2007 (2)

D. Fu, T. Ye, T. E. Matthews, G. Yurtsever, and W. S. Warren, “Two-color, two-photon, and excited-state absorption microscopy,” J. Biomed. Opt. 12(5), 054004 (2007).
[Crossref] [PubMed]

T. Takano, G.-F. Tian, W. Peng, N. Lou, D. Lovatt, A. J. Hansen, K. A. Kasischke, and M. Nedergaard, “Cortical spreading depression causes and coincides with tissue hypoxia,” Nat. Neurosci. 10(6), 754–762 (2007).
[Crossref] [PubMed]

2006 (2)

K. Svoboda and R. Yasuda, “Principles of two-photon excitation microscopy and its applications to neuroscience,” Neuron 50(6), 823–839 (2006).
[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]

2005 (2)

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

M. T. Caballero, P. Andrés, A. Pons, J. Lancis, and M. Martínez-Corral, “Axial resolution in two-color excitation fluorescence microscopy by phase-only binary apodization,” Opt. Commun. 246(4-6), 313–321 (2005).
[Crossref]

2004 (3)

C. Ibáñez-López, I. Escobar, G. Saavedra, and M. Martínez-Corral, “Optical-sectioning improvement in two-color excitation scanning microscopy,” Microsc. Res. Tech. 64(2), 96–102 (2004).
[Crossref] [PubMed]

J. M. Hales, D. J. Hagan, E. W. Van Stryland, K. J. Schafer, A. R. Morales, K. D. Belfield, P. Pacher, O. Kwon, E. Zojer, and J.-L. Brédas, “Resonant enhancement of two-photon absorption in substituted fluorene molecules,” J. Chem. Phys. 121(7), 3152–3160 (2004).
[Crossref] [PubMed]

A. Mizrahi, J. C. Crowley, E. Shtoyerman, and L. C. Katz, “High-resolution in vivo imaging of hippocampal dendrites and spines,” J. Neurosci. 24(13), 3147–3151 (2004).
[Crossref] [PubMed]

2003 (4)

P. Theer, M. T. Hasan, and W. Denk, “Two-photon imaging to a depth of 1000 μm in living brains by use of a Ti:Al2O3 regenerative amplifier,” Opt. Lett. 28(12), 1022–1024 (2003).
[Crossref] [PubMed]

A. Rapaport, F. Szipöcs, and M. Bass, “Dependence of two-photon-absorption-excited fluorescence on the angle between the linear polarizations of two intersecting beams,” Appl. Phys. Lett. 82(26), 4642–4644 (2003).
[Crossref]

F. Xiao, G. Wang, and Z. Xu, “Superresolution in two-color excitation fluorescence microscopy,” Opt. Commun. 228(4), 225–230 (2003).
[Crossref]

M. Lim and C. Saloma, “Primary spherical aberration in two-color (two-photon) excitation fluorescence microscopy with two confocal excitation beams,” Appl. Opt. 42(17), 3398–3406 (2003).
[Crossref] [PubMed]

2001 (1)

2000 (1)

M. O. Cambaliza and C. Saloma, “Advantages of two-color excitation fluorescence microscopy with two confocal excitation beams,” Opt. Commun. 184(1), 25–35 (2000).
[Crossref]

1997 (1)

W. Denk and K. Svoboda, “Photon upmanship: why multiphoton imaging is more than a gimmick,” Neuron 18(3), 351–357 (1997).
[Crossref] [PubMed]

1996 (4)

C. Xu, R. M. Williams, W. Zipfel, and W. W. Webb, “Multiphoton excitation cross-sections of molecular fluorophores,” Bioimaging 4(3), 198–207 (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]

J. R. Lakowicz, I. Gryczynski, H. Malak, and Z. Gryczynski, “Two-color two-photon excitation of fluorescence,” Photochem. Photobiol. 64(4), 632–635 (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,” JOSA B 13(3), 481–491 (1996).
[Crossref]

1992 (1)

S. T. Flock, S. L. Jacques, B. C. Wilson, W. M. Star, and M. J. van Gemert, “Optical properties of Intralipid: a phantom medium for light propagation studies,” Lasers Surg. Med. 12(5), 510–519 (1992).
[Crossref] [PubMed]

1981 (1)

Alfano, R.

L. Shi, L. A. Sordillo, A. Rodríguez-Contreras, and R. Alfano, “Transmission in near-infrared optical windows for deep brain imaging,” J. Biophotonics 9(1-2), 38–43 (2016).
[Crossref] [PubMed]

Alfano, R. R.

L. Shi, A. Rodríguez-Contreras, and R. R. Alfano, “Gaussian beam in two-photon fluorescence imaging of rat brain microvessel,” J. Biomed. Opt. 19(12), 126006 (2014).
[Crossref] [PubMed]

Andreasson, K. I.

G. Hong, S. Diao, J. Chang, A. L. Antaris, C. Chen, B. Zhang, S. Zhao, D. N. Atochin, P. L. Huang, K. I. Andreasson, C. J. Kuo, and H. Dai, “Through-skull fluorescence imaging of the brain in a new near-infrared window,” Nat. Photonics 8(9), 723–730 (2014).
[Crossref] [PubMed]

Andrés, P.

M. T. Caballero, P. Andrés, A. Pons, J. Lancis, and M. Martínez-Corral, “Axial resolution in two-color excitation fluorescence microscopy by phase-only binary apodization,” Opt. Commun. 246(4-6), 313–321 (2005).
[Crossref]

Antaris, A. L.

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A. Mizrahi, J. C. Crowley, E. Shtoyerman, and L. C. Katz, “High-resolution in vivo imaging of hippocampal dendrites and spines,” J. Neurosci. 24(13), 3147–3151 (2004).
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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).
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J. R. Lakowicz, I. Gryczynski, H. Malak, and Z. Gryczynski, “Two-color two-photon excitation of fluorescence,” Photochem. Photobiol. 64(4), 632–635 (1996).
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P. Mahou, M. Zimmerley, K. Loulier, K. S. Matho, G. Labroille, X. Morin, W. Supatto, J. Livet, D. Débarre, and E. Beaurepaire, “Multicolor two-photon tissue imaging by wavelength mixing,” Nat. Methods 9(8), 815–818 (2012).
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T. Takano, G.-F. Tian, W. Peng, N. Lou, D. Lovatt, A. J. Hansen, K. A. Kasischke, and M. Nedergaard, “Cortical spreading depression causes and coincides with tissue hypoxia,” Nat. Neurosci. 10(6), 754–762 (2007).
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J. R. Lakowicz, I. Gryczynski, H. Malak, and Z. Gryczynski, “Two-color two-photon excitation of fluorescence,” Photochem. Photobiol. 64(4), 632–635 (1996).
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C. Wang, L. Qiao, Z. Mao, Y. Cheng, and Z. Xu, “Reduced deep-tissue image degradation in three-dimensional multiphoton microscopy with concentric two-color two-photon fluorescence excitation,” JOSA B 25(6), 976–982 (2008).
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K. Wang, D. E. Milkie, A. Saxena, P. Engerer, T. Misgeld, M. E. Bronner, J. Mumm, and E. Betzig, “Rapid adaptive optical recovery of optimal resolution over large volumes,” Nat. Methods 11(6), 625–628 (2014).
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A. Mizrahi, J. C. Crowley, E. Shtoyerman, and L. C. Katz, “High-resolution in vivo imaging of hippocampal dendrites and spines,” J. Neurosci. 24(13), 3147–3151 (2004).
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J. M. Hales, D. J. Hagan, E. W. Van Stryland, K. J. Schafer, A. R. Morales, K. D. Belfield, P. Pacher, O. Kwon, E. Zojer, and J.-L. Brédas, “Resonant enhancement of two-photon absorption in substituted fluorene molecules,” J. Chem. Phys. 121(7), 3152–3160 (2004).
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K. Wang, D. E. Milkie, A. Saxena, P. Engerer, T. Misgeld, M. E. Bronner, J. Mumm, and E. Betzig, “Rapid adaptive optical recovery of optimal resolution over large volumes,” Nat. Methods 11(6), 625–628 (2014).
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K. A. Kasischke, E. M. Lambert, B. Panepento, A. Sun, H. A. Gelbard, R. W. Burgess, T. H. Foster, and M. Nedergaard, “Two-photon NADH imaging exposes boundaries of oxygen diffusion in cortical vascular supply regions,” J. Cereb. Blood Flow Metab. 31(1), 68–81 (2011).
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T. Robinson, P. Valluri, G. Kennedy, A. Sardini, C. Dunsby, M. A. Neil, G. S. Baldwin, P. M. French, and A. J. de Mello, “Analysis of DNA binding and nucleotide flipping kinetics using two-color two-photon fluorescence lifetime imaging microscopy,” Anal. Chem. 86(21), 10732–10740 (2014).
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R. Kawakami, K. Sawada, A. Sato, T. Hibi, Y. Kozawa, S. Sato, H. Yokoyama, and T. Nemoto, “Visualizing hippocampal neurons with in vivo two-photon microscopy using a 1030 nm picosecond pulse laser,” Sci. Rep. 3, 1014 (2013).
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J. M. Hales, D. J. Hagan, E. W. Van Stryland, K. J. Schafer, A. R. Morales, K. D. Belfield, P. Pacher, O. Kwon, E. Zojer, and J.-L. Brédas, “Resonant enhancement of two-photon absorption in substituted fluorene molecules,” J. Chem. Phys. 121(7), 3152–3160 (2004).
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K. A. Kasischke, E. M. Lambert, B. Panepento, A. Sun, H. A. Gelbard, R. W. Burgess, T. H. Foster, and M. Nedergaard, “Two-photon NADH imaging exposes boundaries of oxygen diffusion in cortical vascular supply regions,” J. Cereb. Blood Flow Metab. 31(1), 68–81 (2011).
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T. Takano, G.-F. Tian, W. Peng, N. Lou, D. Lovatt, A. J. Hansen, K. A. Kasischke, and M. Nedergaard, “Cortical spreading depression causes and coincides with tissue hypoxia,” Nat. Neurosci. 10(6), 754–762 (2007).
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C. Wang, L. Qiao, Z. Mao, Y. Cheng, and Z. Xu, “Reduced deep-tissue image degradation in three-dimensional multiphoton microscopy with concentric two-color two-photon fluorescence excitation,” JOSA B 25(6), 976–982 (2008).
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L. Shi, L. A. Sordillo, A. Rodríguez-Contreras, and R. Alfano, “Transmission in near-infrared optical windows for deep brain imaging,” J. Biophotonics 9(1-2), 38–43 (2016).
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C. Ibáñez-López, I. Escobar, G. Saavedra, and M. Martínez-Corral, “Optical-sectioning improvement in two-color excitation scanning microscopy,” Microsc. Res. Tech. 64(2), 96–102 (2004).
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K. Nizar, H. Uhlirova, P. Tian, P. A. Saisan, Q. Cheng, L. Reznichenko, K. L. Weldy, T. C. Steed, V. B. Sridhar, C. L. MacDonald, J. Cui, S. L. Gratiy, S. Sakadzić, D. A. Boas, T. I. Beka, G. T. Einevoll, J. Chen, E. Masliah, A. M. Dale, G. A. Silva, and A. Devor, “In vivo stimulus-induced vasodilation occurs without IP3 receptor activation and may precede astrocytic calcium increase,” J. Neurosci. 33(19), 8411–8422 (2013).
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K. Nizar, H. Uhlirova, P. Tian, P. A. Saisan, Q. Cheng, L. Reznichenko, K. L. Weldy, T. C. Steed, V. B. Sridhar, C. L. MacDonald, J. Cui, S. L. Gratiy, S. Sakadzić, D. A. Boas, T. I. Beka, G. T. Einevoll, J. Chen, E. Masliah, A. M. Dale, G. A. Silva, and A. Devor, “In vivo stimulus-induced vasodilation occurs without IP3 receptor activation and may precede astrocytic calcium increase,” J. Neurosci. 33(19), 8411–8422 (2013).
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Sardini, A.

T. Robinson, P. Valluri, G. Kennedy, A. Sardini, C. Dunsby, M. A. Neil, G. S. Baldwin, P. M. French, and A. J. de Mello, “Analysis of DNA binding and nucleotide flipping kinetics using two-color two-photon fluorescence lifetime imaging microscopy,” Anal. Chem. 86(21), 10732–10740 (2014).
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Sato, A.

R. Kawakami, K. Sawada, A. Sato, T. Hibi, Y. Kozawa, S. Sato, H. Yokoyama, and T. Nemoto, “Visualizing hippocampal neurons with in vivo two-photon microscopy using a 1030 nm picosecond pulse laser,” Sci. Rep. 3, 1014 (2013).
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R. Kawakami, K. Sawada, A. Sato, T. Hibi, Y. Kozawa, S. Sato, H. Yokoyama, and T. Nemoto, “Visualizing hippocampal neurons with in vivo two-photon microscopy using a 1030 nm picosecond pulse laser,” Sci. Rep. 3, 1014 (2013).
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Sato, T. R.

N. Ji, T. R. Sato, and E. Betzig, “Characterization and adaptive optical correction of aberrations during in vivo imaging in the mouse cortex,” Proc. Natl. Acad. Sci. U.S.A. 109(1), 22–27 (2012).
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R. Kawakami, K. Sawada, A. Sato, T. Hibi, Y. Kozawa, S. Sato, H. Yokoyama, and T. Nemoto, “Visualizing hippocampal neurons with in vivo two-photon microscopy using a 1030 nm picosecond pulse laser,” Sci. Rep. 3, 1014 (2013).
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K. Wang, D. E. Milkie, A. Saxena, P. Engerer, T. Misgeld, M. E. Bronner, J. Mumm, and E. Betzig, “Rapid adaptive optical recovery of optimal resolution over large volumes,” Nat. Methods 11(6), 625–628 (2014).
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Schafer, K. J.

J. M. Hales, D. J. Hagan, E. W. Van Stryland, K. J. Schafer, A. R. Morales, K. D. Belfield, P. Pacher, O. Kwon, E. Zojer, and J.-L. Brédas, “Resonant enhancement of two-photon absorption in substituted fluorene molecules,” J. Chem. Phys. 121(7), 3152–3160 (2004).
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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).
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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]

Schnitzer, M. J.

R. P. Barretto, T. H. Ko, J. C. Jung, T. J. Wang, G. Capps, A. C. Waters, Y. Ziv, A. Attardo, L. Recht, and M. J. Schnitzer, “Time-lapse imaging of disease progression in deep brain areas using fluorescence microendoscopy,” Nat. Med. 17(2), 223–228 (2011).
[Crossref] [PubMed]

Shamir, J.

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, L.

L. Shi, L. A. Sordillo, A. Rodríguez-Contreras, and R. Alfano, “Transmission in near-infrared optical windows for deep brain imaging,” J. Biophotonics 9(1-2), 38–43 (2016).
[Crossref] [PubMed]

L. Shi, A. Rodríguez-Contreras, and R. R. Alfano, “Gaussian beam in two-photon fluorescence imaging of rat brain microvessel,” J. Biomed. Opt. 19(12), 126006 (2014).
[Crossref] [PubMed]

Shtoyerman, E.

A. Mizrahi, J. C. Crowley, E. Shtoyerman, and L. C. Katz, “High-resolution in vivo imaging of hippocampal dendrites and spines,” J. Neurosci. 24(13), 3147–3151 (2004).
[Crossref] [PubMed]

Silva, G. A.

K. Nizar, H. Uhlirova, P. Tian, P. A. Saisan, Q. Cheng, L. Reznichenko, K. L. Weldy, T. C. Steed, V. B. Sridhar, C. L. MacDonald, J. Cui, S. L. Gratiy, S. Sakadzić, D. A. Boas, T. I. Beka, G. T. Einevoll, J. Chen, E. Masliah, A. M. Dale, G. A. Silva, and A. Devor, “In vivo stimulus-induced vasodilation occurs without IP3 receptor activation and may precede astrocytic calcium increase,” J. Neurosci. 33(19), 8411–8422 (2013).
[Crossref] [PubMed]

Sordillo, L. A.

L. Shi, L. A. Sordillo, A. Rodríguez-Contreras, and R. Alfano, “Transmission in near-infrared optical windows for deep brain imaging,” J. Biophotonics 9(1-2), 38–43 (2016).
[Crossref] [PubMed]

Sridhar, V. B.

K. Nizar, H. Uhlirova, P. Tian, P. A. Saisan, Q. Cheng, L. Reznichenko, K. L. Weldy, T. C. Steed, V. B. Sridhar, C. L. MacDonald, J. Cui, S. L. Gratiy, S. Sakadzić, D. A. Boas, T. I. Beka, G. T. Einevoll, J. Chen, E. Masliah, A. M. Dale, G. A. Silva, and A. Devor, “In vivo stimulus-induced vasodilation occurs without IP3 receptor activation and may precede astrocytic calcium increase,” J. Neurosci. 33(19), 8411–8422 (2013).
[Crossref] [PubMed]

Star, W. M.

S. T. Flock, S. L. Jacques, B. C. Wilson, W. M. Star, and M. J. van Gemert, “Optical properties of Intralipid: a phantom medium for light propagation studies,” Lasers Surg. Med. 12(5), 510–519 (1992).
[Crossref] [PubMed]

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K. Nizar, H. Uhlirova, P. Tian, P. A. Saisan, Q. Cheng, L. Reznichenko, K. L. Weldy, T. C. Steed, V. B. Sridhar, C. L. MacDonald, J. Cui, S. L. Gratiy, S. Sakadzić, D. A. Boas, T. I. Beka, G. T. Einevoll, J. Chen, E. Masliah, A. M. Dale, G. A. Silva, and A. Devor, “In vivo stimulus-induced vasodilation occurs without IP3 receptor activation and may precede astrocytic calcium increase,” J. Neurosci. 33(19), 8411–8422 (2013).
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E. W. Miller, J. Y. Lin, E. P. Frady, P. A. Steinbach, W. B. Kristan, and R. Y. Tsien, “Optically monitoring voltage in neurons by photo-induced electron transfer through molecular wires,” Proc. Natl. Acad. Sci. U.S.A. 109(6), 2114–2119 (2012).
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K. A. Kasischke, E. M. Lambert, B. Panepento, A. Sun, H. A. Gelbard, R. W. Burgess, T. H. Foster, and M. Nedergaard, “Two-photon NADH imaging exposes boundaries of oxygen diffusion in cortical vascular supply regions,” J. Cereb. Blood Flow Metab. 31(1), 68–81 (2011).
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C. Wang, R. Liu, D. E. Milkie, W. Sun, Z. Tan, A. Kerlin, T.-W. Chen, D. S. Kim, and N. Ji, “Multiplexed aberration measurement for deep tissue imaging in vivo,” Nat. Methods 11(10), 1037–1040 (2014).
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P. Mahou, M. Zimmerley, K. Loulier, K. S. Matho, G. Labroille, X. Morin, W. Supatto, J. Livet, D. Débarre, and E. Beaurepaire, “Multicolor two-photon tissue imaging by wavelength mixing,” Nat. Methods 9(8), 815–818 (2012).
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K. Svoboda and R. Yasuda, “Principles of two-photon excitation microscopy and its applications to neuroscience,” Neuron 50(6), 823–839 (2006).
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A. Rapaport, F. Szipöcs, and M. Bass, “Dependence of two-photon-absorption-excited fluorescence on the angle between the linear polarizations of two intersecting beams,” Appl. Phys. Lett. 82(26), 4642–4644 (2003).
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C. Wang, R. Liu, D. E. Milkie, W. Sun, Z. Tan, A. Kerlin, T.-W. Chen, D. S. Kim, and N. Ji, “Multiplexed aberration measurement for deep tissue imaging in vivo,” Nat. Methods 11(10), 1037–1040 (2014).
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Tian, G.-F.

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K. Nizar, H. Uhlirova, P. Tian, P. A. Saisan, Q. Cheng, L. Reznichenko, K. L. Weldy, T. C. Steed, V. B. Sridhar, C. L. MacDonald, J. Cui, S. L. Gratiy, S. Sakadzić, D. A. Boas, T. I. Beka, G. T. Einevoll, J. Chen, E. Masliah, A. M. Dale, G. A. Silva, and A. Devor, “In vivo stimulus-induced vasodilation occurs without IP3 receptor activation and may precede astrocytic calcium increase,” J. Neurosci. 33(19), 8411–8422 (2013).
[Crossref] [PubMed]

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E. W. Miller, J. Y. Lin, E. P. Frady, P. A. Steinbach, W. B. Kristan, and R. Y. Tsien, “Optically monitoring voltage in neurons by photo-induced electron transfer through molecular wires,” Proc. Natl. Acad. Sci. U.S.A. 109(6), 2114–2119 (2012).
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K. Nizar, H. Uhlirova, P. Tian, P. A. Saisan, Q. Cheng, L. Reznichenko, K. L. Weldy, T. C. Steed, V. B. Sridhar, C. L. MacDonald, J. Cui, S. L. Gratiy, S. Sakadzić, D. A. Boas, T. I. Beka, G. T. Einevoll, J. Chen, E. Masliah, A. M. Dale, G. A. Silva, and A. Devor, “In vivo stimulus-induced vasodilation occurs without IP3 receptor activation and may precede astrocytic calcium increase,” J. Neurosci. 33(19), 8411–8422 (2013).
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T. Robinson, P. Valluri, G. Kennedy, A. Sardini, C. Dunsby, M. A. Neil, G. S. Baldwin, P. M. French, and A. J. de Mello, “Analysis of DNA binding and nucleotide flipping kinetics using two-color two-photon fluorescence lifetime imaging microscopy,” Anal. Chem. 86(21), 10732–10740 (2014).
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S. T. Flock, S. L. Jacques, B. C. Wilson, W. M. Star, and M. J. van Gemert, “Optical properties of Intralipid: a phantom medium for light propagation studies,” Lasers Surg. Med. 12(5), 510–519 (1992).
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J. M. Hales, D. J. Hagan, E. W. Van Stryland, K. J. Schafer, A. R. Morales, K. D. Belfield, P. Pacher, O. Kwon, E. Zojer, and J.-L. Brédas, “Resonant enhancement of two-photon absorption in substituted fluorene molecules,” J. Chem. Phys. 121(7), 3152–3160 (2004).
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C. Wang, R. Liu, D. E. Milkie, W. Sun, Z. Tan, A. Kerlin, T.-W. Chen, D. S. Kim, and N. Ji, “Multiplexed aberration measurement for deep tissue imaging in vivo,” Nat. Methods 11(10), 1037–1040 (2014).
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C. Wang, L. Qiao, Z. Mao, Y. Cheng, and Z. Xu, “Reduced deep-tissue image degradation in three-dimensional multiphoton microscopy with concentric two-color two-photon fluorescence excitation,” JOSA B 25(6), 976–982 (2008).
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Wang, G.

S. Deng, L. Liu, G. Wang, R. Li, and Z. Xu, “Three-dimensional superresolution in two-color excitation fluorescence microscopy using theta illumination method,” Opt.-Int. J. Light Electron Opt. 121(8), 726–731 (2010).
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F. Xiao, G. Wang, and Z. Xu, “Superresolution in two-color excitation fluorescence microscopy,” Opt. Commun. 228(4), 225–230 (2003).
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L.-C. Cheng, N. G. Horton, K. Wang, S.-J. Chen, and C. Xu, “Measurements of multiphoton action cross sections for multiphoton microscopy,” Biomed. Opt. Express 5(10), 3427–3433 (2014).
[Crossref] [PubMed]

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).
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Wang, L. V.

L. V. Wang and S. Hu, “Photoacoustic tomography: in vivo imaging from organelles to organs,” Science 335(6075), 1458–1462 (2012).
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Wang, T. J.

R. P. Barretto, T. H. Ko, J. C. Jung, T. J. Wang, G. Capps, A. C. Waters, Y. Ziv, A. Attardo, L. Recht, and M. J. Schnitzer, “Time-lapse imaging of disease progression in deep brain areas using fluorescence microendoscopy,” Nat. Med. 17(2), 223–228 (2011).
[Crossref] [PubMed]

Warren, W. S.

D. Fu, T. Ye, T. E. Matthews, G. Yurtsever, and W. S. Warren, “Two-color, two-photon, and excited-state absorption microscopy,” J. Biomed. Opt. 12(5), 054004 (2007).
[Crossref] [PubMed]

Waters, A. C.

R. P. Barretto, T. H. Ko, J. C. Jung, T. J. Wang, G. Capps, A. C. Waters, Y. Ziv, A. Attardo, L. Recht, and M. J. Schnitzer, “Time-lapse imaging of disease progression in deep brain areas using fluorescence microendoscopy,” Nat. Med. 17(2), 223–228 (2011).
[Crossref] [PubMed]

Webb, W. W.

C. Xu, R. M. Williams, W. Zipfel, and W. W. Webb, “Multiphoton excitation cross-sections of molecular fluorophores,” Bioimaging 4(3), 198–207 (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]

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

Weldy, K. L.

K. Nizar, H. Uhlirova, P. Tian, P. A. Saisan, Q. Cheng, L. Reznichenko, K. L. Weldy, T. C. Steed, V. B. Sridhar, C. L. MacDonald, J. Cui, S. L. Gratiy, S. Sakadzić, D. A. Boas, T. I. Beka, G. T. Einevoll, J. Chen, E. Masliah, A. M. Dale, G. A. Silva, and A. Devor, “In vivo stimulus-induced vasodilation occurs without IP3 receptor activation and may precede astrocytic calcium increase,” J. Neurosci. 33(19), 8411–8422 (2013).
[Crossref] [PubMed]

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]

C. Xu, R. M. Williams, W. Zipfel, and W. W. Webb, “Multiphoton excitation cross-sections of molecular fluorophores,” Bioimaging 4(3), 198–207 (1996).
[Crossref]

Wilson, B. C.

S. T. Flock, S. L. Jacques, B. C. Wilson, W. M. Star, and M. J. van Gemert, “Optical properties of Intralipid: a phantom medium for light propagation studies,” Lasers Surg. Med. 12(5), 510–519 (1992).
[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.

Xiao, F.

F. Xiao, G. Wang, and Z. Xu, “Superresolution in two-color excitation fluorescence microscopy,” Opt. Commun. 228(4), 225–230 (2003).
[Crossref]

Xu, C.

L.-C. Cheng, N. G. Horton, K. Wang, S.-J. Chen, and C. Xu, “Measurements of multiphoton action cross sections for multiphoton microscopy,” Biomed. Opt. Express 5(10), 3427–3433 (2014).
[Crossref] [PubMed]

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

C. Xu, R. M. Williams, W. Zipfel, and W. W. Webb, “Multiphoton excitation cross-sections of molecular fluorophores,” Bioimaging 4(3), 198–207 (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]

Xu, Z.

S. Deng, L. Liu, G. Wang, R. Li, and Z. Xu, “Three-dimensional superresolution in two-color excitation fluorescence microscopy using theta illumination method,” Opt.-Int. J. Light Electron Opt. 121(8), 726–731 (2010).
[Crossref]

C. Wang, L. Qiao, Z. Mao, Y. Cheng, and Z. Xu, “Reduced deep-tissue image degradation in three-dimensional multiphoton microscopy with concentric two-color two-photon fluorescence excitation,” JOSA B 25(6), 976–982 (2008).
[Crossref]

F. Xiao, G. Wang, and Z. Xu, “Superresolution in two-color excitation fluorescence microscopy,” Opt. Commun. 228(4), 225–230 (2003).
[Crossref]

Yasuda, R.

K. Svoboda and R. Yasuda, “Principles of two-photon excitation microscopy and its applications to neuroscience,” Neuron 50(6), 823–839 (2006).
[Crossref] [PubMed]

Ye, T.

D. Fu, T. Ye, T. E. Matthews, G. Yurtsever, and W. S. Warren, “Two-color, two-photon, and excited-state absorption microscopy,” J. Biomed. Opt. 12(5), 054004 (2007).
[Crossref] [PubMed]

Yokoyama, H.

R. Kawakami, K. Sawada, A. Sato, T. Hibi, Y. Kozawa, S. Sato, H. Yokoyama, and T. Nemoto, “Visualizing hippocampal neurons with in vivo two-photon microscopy using a 1030 nm picosecond pulse laser,” Sci. Rep. 3, 1014 (2013).
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Yurtsever, G.

D. Fu, T. Ye, T. E. Matthews, G. Yurtsever, and W. S. Warren, “Two-color, two-photon, and excited-state absorption microscopy,” J. Biomed. Opt. 12(5), 054004 (2007).
[Crossref] [PubMed]

Zhang, B.

G. Hong, S. Diao, J. Chang, A. L. Antaris, C. Chen, B. Zhang, S. Zhao, D. N. Atochin, P. L. Huang, K. I. Andreasson, C. J. Kuo, and H. Dai, “Through-skull fluorescence imaging of the brain in a new near-infrared window,” Nat. Photonics 8(9), 723–730 (2014).
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Zhao, S.

G. Hong, S. Diao, J. Chang, A. L. Antaris, C. Chen, B. Zhang, S. Zhao, D. N. Atochin, P. L. Huang, K. I. Andreasson, C. J. Kuo, and H. Dai, “Through-skull fluorescence imaging of the brain in a new near-infrared window,” Nat. Photonics 8(9), 723–730 (2014).
[Crossref] [PubMed]

Zimmerley, M.

P. Mahou, M. Zimmerley, K. Loulier, K. S. Matho, G. Labroille, X. Morin, W. Supatto, J. Livet, D. Débarre, and E. Beaurepaire, “Multicolor two-photon tissue imaging by wavelength mixing,” Nat. Methods 9(8), 815–818 (2012).
[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).
[Crossref] [PubMed]

C. Xu, R. M. Williams, W. Zipfel, and W. W. Webb, “Multiphoton excitation cross-sections of molecular fluorophores,” Bioimaging 4(3), 198–207 (1996).
[Crossref]

Ziv, Y.

R. P. Barretto, T. H. Ko, J. C. Jung, T. J. Wang, G. Capps, A. C. Waters, Y. Ziv, A. Attardo, L. Recht, and M. J. Schnitzer, “Time-lapse imaging of disease progression in deep brain areas using fluorescence microendoscopy,” Nat. Med. 17(2), 223–228 (2011).
[Crossref] [PubMed]

Zojer, E.

J. M. Hales, D. J. Hagan, E. W. Van Stryland, K. J. Schafer, A. R. Morales, K. D. Belfield, P. Pacher, O. Kwon, E. Zojer, and J.-L. Brédas, “Resonant enhancement of two-photon absorption in substituted fluorene molecules,” J. Chem. Phys. 121(7), 3152–3160 (2004).
[Crossref] [PubMed]

Anal. Chem. (1)

T. Robinson, P. Valluri, G. Kennedy, A. Sardini, C. Dunsby, M. A. Neil, G. S. Baldwin, P. M. French, and A. J. de Mello, “Analysis of DNA binding and nucleotide flipping kinetics using two-color two-photon fluorescence lifetime imaging microscopy,” Anal. Chem. 86(21), 10732–10740 (2014).
[Crossref] [PubMed]

Appl. Opt. (3)

Appl. Phys. Lett. (1)

A. Rapaport, F. Szipöcs, and M. Bass, “Dependence of two-photon-absorption-excited fluorescence on the angle between the linear polarizations of two intersecting beams,” Appl. Phys. Lett. 82(26), 4642–4644 (2003).
[Crossref]

Bioimaging (1)

C. Xu, R. M. Williams, W. Zipfel, and W. W. Webb, “Multiphoton excitation cross-sections of molecular fluorophores,” Bioimaging 4(3), 198–207 (1996).
[Crossref]

Biomed. Opt. Express (1)

J. Biomed. Opt. (3)

L. Shi, A. Rodríguez-Contreras, and R. R. Alfano, “Gaussian beam in two-photon fluorescence imaging of rat brain microvessel,” J. Biomed. Opt. 19(12), 126006 (2014).
[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. Fu, T. Ye, T. E. Matthews, G. Yurtsever, and W. S. Warren, “Two-color, two-photon, and excited-state absorption microscopy,” J. Biomed. Opt. 12(5), 054004 (2007).
[Crossref] [PubMed]

J. Biophotonics (1)

L. Shi, L. A. Sordillo, A. Rodríguez-Contreras, and R. Alfano, “Transmission in near-infrared optical windows for deep brain imaging,” J. Biophotonics 9(1-2), 38–43 (2016).
[Crossref] [PubMed]

J. Cereb. Blood Flow Metab. (1)

K. A. Kasischke, E. M. Lambert, B. Panepento, A. Sun, H. A. Gelbard, R. W. Burgess, T. H. Foster, and M. Nedergaard, “Two-photon NADH imaging exposes boundaries of oxygen diffusion in cortical vascular supply regions,” J. Cereb. Blood Flow Metab. 31(1), 68–81 (2011).
[Crossref] [PubMed]

J. Chem. Phys. (1)

J. M. Hales, D. J. Hagan, E. W. Van Stryland, K. J. Schafer, A. R. Morales, K. D. Belfield, P. Pacher, O. Kwon, E. Zojer, and J.-L. Brédas, “Resonant enhancement of two-photon absorption in substituted fluorene molecules,” J. Chem. Phys. 121(7), 3152–3160 (2004).
[Crossref] [PubMed]

J. Fluoresc. (1)

S. Quentmeier, S. Denicke, and K.-H. Gericke, “Two-color two-photon fluorescence laser scanning microscopy,” J. Fluoresc. 19(6), 1037–1043 (2009).
[Crossref] [PubMed]

J. Neurosci. (2)

A. Mizrahi, J. C. Crowley, E. Shtoyerman, and L. C. Katz, “High-resolution in vivo imaging of hippocampal dendrites and spines,” J. Neurosci. 24(13), 3147–3151 (2004).
[Crossref] [PubMed]

K. Nizar, H. Uhlirova, P. Tian, P. A. Saisan, Q. Cheng, L. Reznichenko, K. L. Weldy, T. C. Steed, V. B. Sridhar, C. L. MacDonald, J. Cui, S. L. Gratiy, S. Sakadzić, D. A. Boas, T. I. Beka, G. T. Einevoll, J. Chen, E. Masliah, A. M. Dale, G. A. Silva, and A. Devor, “In vivo stimulus-induced vasodilation occurs without IP3 receptor activation and may precede astrocytic calcium increase,” J. Neurosci. 33(19), 8411–8422 (2013).
[Crossref] [PubMed]

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

J. Phys. Chem. B (1)

S. Quentmeier, S. Denicke, J.-E. Ehlers, R. A. Niesner, and K.-H. Gericke, “Two-color two-photon excitation using femtosecond laser pulses,” J. Phys. Chem. B 112(18), 5768–5773 (2008).
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JOSA B (2)

C. Wang, L. Qiao, Z. Mao, Y. Cheng, and Z. Xu, “Reduced deep-tissue image degradation in three-dimensional multiphoton microscopy with concentric two-color two-photon fluorescence excitation,” JOSA B 25(6), 976–982 (2008).
[Crossref]

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

Lasers Surg. Med. (1)

S. T. Flock, S. L. Jacques, B. C. Wilson, W. M. Star, and M. J. van Gemert, “Optical properties of Intralipid: a phantom medium for light propagation studies,” Lasers Surg. Med. 12(5), 510–519 (1992).
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Microsc. Res. Tech. (1)

C. Ibáñez-López, I. Escobar, G. Saavedra, and M. Martínez-Corral, “Optical-sectioning improvement in two-color excitation scanning microscopy,” Microsc. Res. Tech. 64(2), 96–102 (2004).
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Figures (7)

Fig. 1
Fig. 1

Schematic energy diagram demonstrating degenerate and non-degenerate multi-photon absorption of a molecule: (a) degenerate 2-photon excitation (D-2PE); (b) degenerate 3-photon excitation (3-PE); and (c) non-degenerate 2-photon excitation (ND-2PE).

Fig. 2
Fig. 2

Experimental setup for demonstration of ND-2PE. PBS, polarization beam splitter; HWP, half wave plate; DM, dichroic mirror; FS, fluorescent sample; 40XOBJ, microscope objective; BPF, band pass filter; PMT, photomultiplier. M, mirror.

Fig. 3
Fig. 3

Experimental demonstration of ND-2PE. (a) dependence of the fluorescence signal on temporal alignment; (b) fluorescence intensity dependence on NIR power (SWIR intensity is fixed at 4.16 × 1023 photons/cm2s); (c) fluorescence intensity dependence on SWIR power (NIR intensity is fixed at 1.18 × 1023 photons/cm2s).

Fig. 4
Fig. 4

Experimental setup for demonstration of penetration depth of D-2PE and ND-2PE. HWP, half wave plate; DM, dichroic mirror; FS, fluorescent sample; OBJ 1, 40X microscope objective; OBJ 2, 20X microscope objective; BPF, band pass filter; PMT, photomultiplier. The arrow indicated the direction of movement of the cuvette.

Fig. 5
Fig. 5

Relative fluorescence intensity as a function of sample depth within varying concentrations of intralipid: (a) 0.5%, (b) 0.75%, (c) 1%, (d) 1.5% and (e) 2%. (f) Attenuation length as a function of intralipid concentrations.

Fig. 6
Fig. 6

Simulation of the fluorescence intensity under D-2PE (blue) and ND-2PE (green) as a function of depth in the scattering medium.

Fig. 7
Fig. 7

Experimental demonstration that ND-2PE can excite fluorescence at greater depth.

Tables (1)

Tables Icon

Table 1 Parameters for calculating the excitation cross section ratio of D-2PE and ND-2PE

Equations (23)

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F D =K σ D I NIR_D (t,r,z) I NIR_D (t,r,z)dVdt,
F ND =K σ ND I NIR_ND (t,r,z) I SWIR_ND (t t 0 ,r r 0 ,z)dVdt,
I NIR_D ( t,r,z )= I NIR_D [ 1 ( π f τ NIR_D ) exp( t 2 τ NIR_D 2 ) ] { [ w NIR_D ( 0 ) w NIR_D ( z ) ] 2 exp[ r 2 w NIR_D 2 ( z ) ] },
I NIR_ND ( t,r,z )= I NIR_ND [ 1 ( π f τ NIR_ND ) exp( t 2 τ NIR_ND 2 ) ] { [ w NIR_ND ( 0 ) w NIR_ND ( z ) ] 2 exp[ r 2 w NIR_ND 2 ( z ) ] },
I SWIR_ND ( t t 0 ,r r 0 ,z )= I SWIR_ND { 1 ( π f τ SWIR_ND ) exp[ ( t t 0 ) 2 τ SWIR_ND 2 ] } { [ w SWIR_ND ( 0 ) w SWIR_ND ( z ) ] 2 exp[ ( r r 0 ) 2 w SWIR_ND 2 ( z ) ] },
I NIR_D = P NIR_D ω NIR_D 1 π w NIR_D 2 (0) ,
I NIR_ND = P NIR_ND ω NIR_ND 1 π w NIR_ND 2 (0) ,
I SWIR_ND = P SWIR_ND ω SWIR_ND 1 π w SWIR_ND 2 (0) .
w NIR_D ( z )= w NIR_D ( 0 ) 1+ [ z λ NIR_D π w NIR_D 2 ( 0 ) ] 2 ,
w NIR_ND ( z )= w NIR_ND ( 0 ) 1+ [ z λ NIR_ND π w NIR_ND 2 ( 0 ) ] 2 ,
w SWIR_ND ( z )= w SWIR_ND ( 0 ) 1+ [ z λ SWIR_ND π w SWIR_ND 2 ( 0 ) ] 2 ,
F D (z)=K σ D I NIR_D I NIR_D T D S D ,
F ND (z)=K σ ND I NIR_ND I SWIR_ND T ND S ND ,
T D = [ 1 ( π f τ NIR_D ) exp( t 2 τ NIR_D 2 ) ][ 1 ( π f τ NIR_D ) exp( t 2 τ NIR_D 2 ) ]dt = 1 π ( f τ NIR_D ) 2 exp( 2 t 2 τ NIR_D 2 ) dt,
S D = { [ w NIR_D (0) w NIR_D (z) ] 2 exp[ r 2 w NIR_D 2 (z) ] } { [ w NIR_D (0) w NIR_D (z) ] 2 exp[ r 2 w NIR_D 2 (z) ] }dV = [ w NIR_D (0) w NIR_D (z) ] 4 exp[ 2 r 2 w NIR_D 2 (z) ] dV,
T ND = [ 1 ( π f τ NIR_ND ) exp( t 2 τ NIR_ND 2 ) ][ 1 ( π f τ SWIR_ND ) exp( t 2 τ SWIR_ND 2 ) ]dt = 1 ( π f τ NIR_ND ) 1 ( π f τ SWIR_ND ) exp[ t 2 ( 1 τ NIR_ND 2 + 1 τ SWIR_ND 2 ) ] dt,
S ND = { [ w NIR_ND (0) w NIR_ND (z) ] 2 exp[ r 2 w NIR_ND 2 (z) ] } { [ w SWIR_ND (0) w SWIR_ND (z) ] 2 exp[ r 2 w SWIR_ND 2 (z) ] }dV = [ w NIR_ND (0) w NIR_ND (z) ] 2 [ w SWIR_ND (0) w SWIR_ND (z) ] 2 exp{ r 2 [ 1 w NIR_ND 2 (z) + 1 w SWIR_ND 2 (z) ] } dV.
F ND F D = σ ND I NIR_ND I SWIR_ND ξ σ D I NIR_D I NIR_D ,
F ND F D = σ ND I SWIR ξ σ D I NIR .
I NIR (z)= I NIR (0)exp( z α NIR ),
I SWIR (z)= I SWIR (0)exp( z α SWIR ),
I D_2PE (z)=A I NIR 2 (0)exp( 2z α NIR ),
I ND_2PE (z)=A I NIR (0) I SWIR (0)exp[ z( 1 α NIR + 1 α SWIR ) ],

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