Abstract

We demonstrate that high-quality images of the deep regions of a thick sample can be obtained from its surface by multi-focal multiphoton microscopy (MMM). The MMM system incorporates a spatial light modulator to separate the excitation beam into a multi-focal excitation beam and modulate the pre-distortion wavefront to correct spherical aberration (SA) caused by a refractive index mismatch between the immersion medium and the biological sample. When fluorescent beads in transparent epoxy resin were observed using four SA-corrected focal beams, the fluorescence signal of the obtained images was ~52 times higher than that obtained without SA correction until a depth of ~1100 μm, similar to the result for single-focal multiphoton microscopy (SMM). The MMM scanning time was four times less than that for SMM, and MMM showed an improved fluorescence intensity and depth resolution for an image of blood vessels in the brain of a mouse stained with a fluorescent dye.

© 2017 Optical Society of America

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2017 (1)

2016 (1)

W. Yang, J. Kang, M. L. Carrillo-Reid, E. Pnevmatikakis, L. Paninski, R. Yuste, and D. S. Peterka, “Simultaneous multi-plane imaging of neural circuits,” Neron 89(2) 269–284 (2016).
[Crossref]

2015 (3)

L. Kong, J. Tang, J. P. Little, Y. Yu, T. Lammermann, C. P. Lin, R. N. Germain, and M. Cui, “Continuous volumetric imaging via an optical phase-locked ultrasound lens,” Nat. Methods 12, 759–762 (2015).
[Crossref] [PubMed]

N. Matsumoto, T. Inoue, A. Matsumoto, and S. Okazaki, “Correction of depth-induced spherical aberration for deep observation using two-photon excitation fluorescence microscopy with spatial light modulator,” Bio. Opt. Express 6(7), 2575–2587 (2015).
[Crossref]

S. P. Poland, N. Krstajić, J. Monypenny, S. Coelho, D. Tyndall, R. J. Walker, V. Devauges, J. Richardson, N. Dutton, P. Barber, D. D. Li, K. Suhling, T. Ng, R. K. Henderson, and S. M. Ameer-Beg, “A high speed multifocal multiphoton fluorescence lifetime imaging microscope for live-cell FRET imaging,” Bio. Opt. Express 6(2), 277–296 (2015).
[Crossref]

2014 (5)

2013 (4)

C. O. Yanez, A. R. Morales, X. Yue, T. Urakami, M. Komatsu, T. A. H. Jarvinen, and K. D. Belfield, “Deep vascular imaging in wounds by two-photon fluorescence microscopy,” PLos One 8(7), e67559 (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]

S. Coelho, S. Poland, N. Krstajic, D. Li, J. Moneypenny, R. Walker, D. Tyndall, T. Ng, R. Henderson, and S. Ameer-Beg, “multi-focal multiphoton microscopy with adaptive optical correction,” Proc. SPIE 8588, 858817 (2013).
[Crossref]

M. T. Ke, S. Fujimoto, and T. Imai, “SeeDB: A simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction,” Nat. Neurosci. 16, 1154–1161 (2013).
[Crossref] [PubMed]

2012 (1)

Y. Shao, W. Qin, H. Lin, J. Qu, X. Peng, H. Niu, and B. Z. Gao, “multi-focal multiphoton microscopy based on a spatial light modulator,” Appl. Phys. B 107, 653–657 (2012).
[Crossref]

2011 (1)

T. A. Planchon, L. Gao, D. E. Milkie, M. W. Davidson, J. A. Galbraith, C. G. Galbraith, and E. Betzig, “Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination,” Nat. Methods 8, 417–423 (2011).
[Crossref] [PubMed]

2010 (1)

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

2008 (3)

Y. Li, Y. Song, L. Zhao, G. Gaidosh, AM Laties, and R. Wen, “Direct labeling and visualization of blood vessels with lipophilic carbocyanine dye DiI”, Nat. Protocols 3, 1703–1708 (2008).
[Crossref]

N. Matsumoto, T. Ando, T. Inoue, Y. Ohtake, N. Fukuchi, and T. Hara, “Generation of high-quality higher-order Laguerre Gaussian beams using liquid-crystal-on-silicon spatial light modulators,” J. Opt. Soc. Am. A 25(7), 1642–1650 (2008).
[Crossref]

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

2007 (3)

2004 (1)

E. Theofanidou, L. Wilson, W. J. Hassack, and J. Arlt, “Spherical aberration correction for optical tweezers,” Opt. Commun. 236(1–3), 145–150 (2004).
[Crossref]

2003 (2)

2001 (1)

A. Egner, J. Bewersdorf, and S. W. Hell, “Comparison of the axial resolution of practical Nipkow-disk confocal fluorescence microscopy with that of multifocal multiphoton microscopy: theory and experiment,” J. Microscopy 206, 24–32 (2001).
[Crossref]

2000 (2)

T. Nielsen, M. Fricke, D. Hellweg, and P. Andersen, “High efficiency beam splitter for multi-focal mutiphoton microscopy,” J. Microscopy 201, 368–376 (2000).
[Crossref]

M. A. A. Neil, R. Juškaitis, M. J. Booth, T. Wilson, T. Tanaka, and S. Kawata, “Adaptive aberration correction in a two-photon microscope,” J. Microscopy 200(2), 105–108 (2000).
[Crossref]

1999 (3)

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

K. H. Kim, C. Buehler, and P. T. C. So, “High-speed, two-photon scanning microscope,” Appl. Opt. 38, 6004–6009 (1999).
[Crossref]

J. Bewersdorf, R. Pick, and S. W. Hell, “Multifocal multiphoton microscopy,” Opt. Lett. 23, 655–657 (1999).
[Crossref]

1995 (1)

1992 (1)

Alexeev, I.

Ameer-Beg, S.

S. Coelho, S. Poland, N. Krstajic, D. Li, J. Moneypenny, R. Walker, D. Tyndall, T. Ng, R. Henderson, and S. Ameer-Beg, “multi-focal multiphoton microscopy with adaptive optical correction,” Proc. SPIE 8588, 858817 (2013).
[Crossref]

Ameer-Beg, S. M.

S. P. Poland, N. Krstajić, J. Monypenny, S. Coelho, D. Tyndall, R. J. Walker, V. Devauges, J. Richardson, N. Dutton, P. Barber, D. D. Li, K. Suhling, T. Ng, R. K. Henderson, and S. M. Ameer-Beg, “A high speed multifocal multiphoton fluorescence lifetime imaging microscope for live-cell FRET imaging,” Bio. Opt. Express 6(2), 277–296 (2015).
[Crossref]

Andersen, P.

T. Nielsen, M. Fricke, D. Hellweg, and P. Andersen, “High efficiency beam splitter for multi-focal mutiphoton microscopy,” J. Microscopy 201, 368–376 (2000).
[Crossref]

Ando, T.

Antolini, R.

Arlt, J.

E. Theofanidou, L. Wilson, W. J. Hassack, and J. Arlt, “Spherical aberration correction for optical tweezers,” Opt. Commun. 236(1–3), 145–150 (2004).
[Crossref]

Bahlmann, K.

Barber, P.

S. P. Poland, N. Krstajić, J. Monypenny, S. Coelho, D. Tyndall, R. J. Walker, V. Devauges, J. Richardson, N. Dutton, P. Barber, D. D. Li, K. Suhling, T. Ng, R. K. Henderson, and S. M. Ameer-Beg, “A high speed multifocal multiphoton fluorescence lifetime imaging microscope for live-cell FRET imaging,” Bio. Opt. Express 6(2), 277–296 (2015).
[Crossref]

Baum, M.

Belfield, K. D.

C. O. Yanez, A. R. Morales, X. Yue, T. Urakami, M. Komatsu, T. A. H. Jarvinen, and K. D. Belfield, “Deep vascular imaging in wounds by two-photon fluorescence microscopy,” PLos One 8(7), e67559 (2013).
[Crossref] [PubMed]

Bellve, K.

Betzig, E.

T. A. Planchon, L. Gao, D. E. Milkie, M. W. Davidson, J. A. Galbraith, C. G. Galbraith, and E. Betzig, “Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination,” Nat. Methods 8, 417–423 (2011).
[Crossref] [PubMed]

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

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

Bewersdorf, J.

A. Egner, J. Bewersdorf, and S. W. Hell, “Comparison of the axial resolution of practical Nipkow-disk confocal fluorescence microscopy with that of multifocal multiphoton microscopy: theory and experiment,” J. Microscopy 206, 24–32 (2001).
[Crossref]

J. Bewersdorf, R. Pick, and S. W. Hell, “Multifocal multiphoton microscopy,” Opt. Lett. 23, 655–657 (1999).
[Crossref]

Booker, G. R.

Booth, M. J.

P. S. Salter, M. Baum, I. Alexeev, M. Schmidt, and M. J. Booth, “Exploring the depth range for three-dimensional laser machining with aberration correction,” Opt. Express 22(15), 17644–17656 (2014).
[Crossref] [PubMed]

M. J. Booth, “Adaptive optics in microscopy,” Phil. Trans R. Soc. A 365, 2829–2843 (2007).
[Crossref] [PubMed]

M. A. A. Neil, R. Juškaitis, M. J. Booth, T. Wilson, T. Tanaka, and S. Kawata, “Adaptive aberration correction in a two-photon microscope,” J. Microscopy 200(2), 105–108 (2000).
[Crossref]

Buehler, C.

Carrillo-Reid, M. L.

W. Yang, J. Kang, M. L. Carrillo-Reid, E. Pnevmatikakis, L. Paninski, R. Yuste, and D. S. Peterka, “Simultaneous multi-plane imaging of neural circuits,” Neron 89(2) 269–284 (2016).
[Crossref]

Cha, J. W.

J. W. Cha, V. R. Singh, K. H. Kim, J. Subramanian, Q. Peng, H. Yu, E. Nedivi, and P. T. C. So, “Reassignment of scattering emission photons in multi-focal multiphoton microscopy,” Sci. Rep. 4, 5153 (2014).
[Crossref]

Choudhury, A.

Coelho, S.

S. P. Poland, N. Krstajić, J. Monypenny, S. Coelho, D. Tyndall, R. J. Walker, V. Devauges, J. Richardson, N. Dutton, P. Barber, D. D. Li, K. Suhling, T. Ng, R. K. Henderson, and S. M. Ameer-Beg, “A high speed multifocal multiphoton fluorescence lifetime imaging microscope for live-cell FRET imaging,” Bio. Opt. Express 6(2), 277–296 (2015).
[Crossref]

S. Coelho, S. Poland, N. Krstajic, D. Li, J. Moneypenny, R. Walker, D. Tyndall, T. Ng, R. Henderson, and S. Ameer-Beg, “multi-focal multiphoton microscopy with adaptive optical correction,” Proc. SPIE 8588, 858817 (2013).
[Crossref]

Cui, M.

L. Kong, J. Tang, J. P. Little, Y. Yu, T. Lammermann, C. P. Lin, R. N. Germain, and M. Cui, “Continuous volumetric imaging via an optical phase-locked ultrasound lens,” Nat. Methods 12, 759–762 (2015).
[Crossref] [PubMed]

Dandliker, R.

Davidson, M. W.

T. A. Planchon, L. Gao, D. E. Milkie, M. W. Davidson, J. A. Galbraith, C. G. Galbraith, and E. Betzig, “Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination,” Nat. Methods 8, 417–423 (2011).
[Crossref] [PubMed]

Denk, W.

Devauges, V.

S. P. Poland, N. Krstajić, J. Monypenny, S. Coelho, D. Tyndall, R. J. Walker, V. Devauges, J. Richardson, N. Dutton, P. Barber, D. D. Li, K. Suhling, T. Ng, R. K. Henderson, and S. M. Ameer-Beg, “A high speed multifocal multiphoton fluorescence lifetime imaging microscope for live-cell FRET imaging,” Bio. Opt. Express 6(2), 277–296 (2015).
[Crossref]

Duan, X.

Dutton, N.

S. P. Poland, N. Krstajić, J. Monypenny, S. Coelho, D. Tyndall, R. J. Walker, V. Devauges, J. Richardson, N. Dutton, P. Barber, D. D. Li, K. Suhling, T. Ng, R. K. Henderson, and S. M. Ameer-Beg, “A high speed multifocal multiphoton fluorescence lifetime imaging microscope for live-cell FRET imaging,” Bio. Opt. Express 6(2), 277–296 (2015).
[Crossref]

Egner, A.

A. Egner, J. Bewersdorf, and S. W. Hell, “Comparison of the axial resolution of practical Nipkow-disk confocal fluorescence microscopy with that of multifocal multiphoton microscopy: theory and experiment,” J. Microscopy 206, 24–32 (2001).
[Crossref]

Ellisman, M. H.

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

Fan, G. Y.

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

Fantini, S.

Fricke, M.

T. Nielsen, M. Fricke, D. Hellweg, and P. Andersen, “High efficiency beam splitter for multi-focal mutiphoton microscopy,” J. Microscopy 201, 368–376 (2000).
[Crossref]

Froner, E.

Fujimoto, S.

M. T. Ke, S. Fujimoto, and T. Imai, “SeeDB: A simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction,” Nat. Neurosci. 16, 1154–1161 (2013).
[Crossref] [PubMed]

Fujisaki, H.

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

Fukuchi, N.

Fukushi, Y.

Gaidosh, G.

Y. Li, Y. Song, L. Zhao, G. Gaidosh, AM Laties, and R. Wen, “Direct labeling and visualization of blood vessels with lipophilic carbocyanine dye DiI”, Nat. Protocols 3, 1703–1708 (2008).
[Crossref]

Galbraith, C. G.

T. A. Planchon, L. Gao, D. E. Milkie, M. W. Davidson, J. A. Galbraith, C. G. Galbraith, and E. Betzig, “Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination,” Nat. Methods 8, 417–423 (2011).
[Crossref] [PubMed]

Galbraith, J. A.

T. A. Planchon, L. Gao, D. E. Milkie, M. W. Davidson, J. A. Galbraith, C. G. Galbraith, and E. Betzig, “Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination,” Nat. Methods 8, 417–423 (2011).
[Crossref] [PubMed]

Gale, M. T.

Gao, B. Z.

Y. Shao, W. Qin, H. Lin, J. Qu, X. Peng, H. Niu, and B. Z. Gao, “multi-focal multiphoton microscopy based on a spatial light modulator,” Appl. Phys. B 107, 653–657 (2012).
[Crossref]

Gao, L.

T. A. Planchon, L. Gao, D. E. Milkie, M. W. Davidson, J. A. Galbraith, C. G. Galbraith, and E. Betzig, “Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination,” Nat. Methods 8, 417–423 (2011).
[Crossref] [PubMed]

Germain, R. N.

L. Kong, J. Tang, J. P. Little, Y. Yu, T. Lammermann, C. P. Lin, R. N. Germain, and M. Cui, “Continuous volumetric imaging via an optical phase-locked ultrasound lens,” Nat. Methods 12, 759–762 (2015).
[Crossref] [PubMed]

Gobe, G. C.

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S. Coelho, S. Poland, N. Krstajic, D. Li, J. Moneypenny, R. Walker, D. Tyndall, T. Ng, R. Henderson, and S. Ameer-Beg, “multi-focal multiphoton microscopy with adaptive optical correction,” Proc. SPIE 8588, 858817 (2013).
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S. P. Poland, N. Krstajić, J. Monypenny, S. Coelho, D. Tyndall, R. J. Walker, V. Devauges, J. Richardson, N. Dutton, P. Barber, D. D. Li, K. Suhling, T. Ng, R. K. Henderson, and S. M. Ameer-Beg, “A high speed multifocal multiphoton fluorescence lifetime imaging microscope for live-cell FRET imaging,” Bio. Opt. Express 6(2), 277–296 (2015).
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Hibi, T.

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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D. M. Small, W. Y. Sanchez, S. Roy, M. J. Hickey, and G. C. Gobe, “Multiphoton fluorescence microscopy of the live kidney in health and disease,” J. Biomed. Opt. 19(2), 020901 (2014).
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N. Ji, J. C Magee, and E. Betzig, “Adaptive optics via pupil segmentation for high-resolution imaging in biological tissues,” Nat. Methods 7, 141–147 (2010).
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N. Ji, J. C Magee, and E. Betzig, “High-speed, low-photodamage nonlinear imaging using passive pulse splitters,” Nat. Methods 5, 197–202 (2008).
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M. A. A. Neil, R. Juškaitis, M. J. Booth, T. Wilson, T. Tanaka, and S. Kawata, “Adaptive aberration correction in a two-photon microscope,” J. Microscopy 200(2), 105–108 (2000).
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W. Yang, J. Kang, M. L. Carrillo-Reid, E. Pnevmatikakis, L. Paninski, R. Yuste, and D. S. Peterka, “Simultaneous multi-plane imaging of neural circuits,” Neron 89(2) 269–284 (2016).
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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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M. T. Ke, S. Fujimoto, and T. Imai, “SeeDB: A simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction,” Nat. Neurosci. 16, 1154–1161 (2013).
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Kirber, M.

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C. O. Yanez, A. R. Morales, X. Yue, T. Urakami, M. Komatsu, T. A. H. Jarvinen, and K. D. Belfield, “Deep vascular imaging in wounds by two-photon fluorescence microscopy,” PLos One 8(7), e67559 (2013).
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Kozawa, Y.

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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S. P. Poland, N. Krstajić, J. Monypenny, S. Coelho, D. Tyndall, R. J. Walker, V. Devauges, J. Richardson, N. Dutton, P. Barber, D. D. Li, K. Suhling, T. Ng, R. K. Henderson, and S. M. Ameer-Beg, “A high speed multifocal multiphoton fluorescence lifetime imaging microscope for live-cell FRET imaging,” Bio. Opt. Express 6(2), 277–296 (2015).
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S. Coelho, S. Poland, N. Krstajic, D. Li, J. Moneypenny, R. Walker, D. Tyndall, T. Ng, R. Henderson, and S. Ameer-Beg, “multi-focal multiphoton microscopy with adaptive optical correction,” Proc. SPIE 8588, 858817 (2013).
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L. Kong, J. Tang, J. P. Little, Y. Yu, T. Lammermann, C. P. Lin, R. N. Germain, and M. Cui, “Continuous volumetric imaging via an optical phase-locked ultrasound lens,” Nat. Methods 12, 759–762 (2015).
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Y. Li, Y. Song, L. Zhao, G. Gaidosh, AM Laties, and R. Wen, “Direct labeling and visualization of blood vessels with lipophilic carbocyanine dye DiI”, Nat. Protocols 3, 1703–1708 (2008).
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Li, D.

S. Coelho, S. Poland, N. Krstajic, D. Li, J. Moneypenny, R. Walker, D. Tyndall, T. Ng, R. Henderson, and S. Ameer-Beg, “multi-focal multiphoton microscopy with adaptive optical correction,” Proc. SPIE 8588, 858817 (2013).
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Li, D. D.

S. P. Poland, N. Krstajić, J. Monypenny, S. Coelho, D. Tyndall, R. J. Walker, V. Devauges, J. Richardson, N. Dutton, P. Barber, D. D. Li, K. Suhling, T. Ng, R. K. Henderson, and S. M. Ameer-Beg, “A high speed multifocal multiphoton fluorescence lifetime imaging microscope for live-cell FRET imaging,” Bio. Opt. Express 6(2), 277–296 (2015).
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Li, H.

Li, X.

Li, Y.

Y. Li, Y. Song, L. Zhao, G. Gaidosh, AM Laties, and R. Wen, “Direct labeling and visualization of blood vessels with lipophilic carbocyanine dye DiI”, Nat. Protocols 3, 1703–1708 (2008).
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Lin, C. P.

L. Kong, J. Tang, J. P. Little, Y. Yu, T. Lammermann, C. P. Lin, R. N. Germain, and M. Cui, “Continuous volumetric imaging via an optical phase-locked ultrasound lens,” Nat. Methods 12, 759–762 (2015).
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Lin, H.

Y. Shao, W. Qin, H. Lin, J. Qu, X. Peng, H. Niu, and B. Z. Gao, “multi-focal multiphoton microscopy based on a spatial light modulator,” Appl. Phys. B 107, 653–657 (2012).
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L. Kong, J. Tang, J. P. Little, Y. Yu, T. Lammermann, C. P. Lin, R. N. Germain, and M. Cui, “Continuous volumetric imaging via an optical phase-locked ultrasound lens,” Nat. Methods 12, 759–762 (2015).
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Magee, J. C

N. Ji, J. C Magee, and E. Betzig, “Adaptive optics via pupil segmentation for high-resolution imaging in biological tissues,” Nat. Methods 7, 141–147 (2010).
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N. Ji, J. C Magee, and E. Betzig, “High-speed, low-photodamage nonlinear imaging using passive pulse splitters,” Nat. Methods 5, 197–202 (2008).
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Matsumoto, A.

N. Matsumoto, T. Inoue, A. Matsumoto, and S. Okazaki, “Correction of depth-induced spherical aberration for deep observation using two-photon excitation fluorescence microscopy with spatial light modulator,” Bio. Opt. Express 6(7), 2575–2587 (2015).
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McGonagle, W.

Milkie, D. E.

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Miyawaki, A.

G. Y. Fan, H. Fujisaki, A. Miyawaki, R. -K. Tsay, R. Y. Tsien, and M. H. Ellisman, “Video-Rate Scanning two-photon excitation fluorescence microscopy and ratio imaging with cameleons,” Biophys. J. 76, 2412–2420 (1999).
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Moneypenny, J.

S. Coelho, S. Poland, N. Krstajic, D. Li, J. Moneypenny, R. Walker, D. Tyndall, T. Ng, R. Henderson, and S. Ameer-Beg, “multi-focal multiphoton microscopy with adaptive optical correction,” Proc. SPIE 8588, 858817 (2013).
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S. P. Poland, N. Krstajić, J. Monypenny, S. Coelho, D. Tyndall, R. J. Walker, V. Devauges, J. Richardson, N. Dutton, P. Barber, D. D. Li, K. Suhling, T. Ng, R. K. Henderson, and S. M. Ameer-Beg, “A high speed multifocal multiphoton fluorescence lifetime imaging microscope for live-cell FRET imaging,” Bio. Opt. Express 6(2), 277–296 (2015).
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Morales, A. R.

C. O. Yanez, A. R. Morales, X. Yue, T. Urakami, M. Komatsu, T. A. H. Jarvinen, and K. D. Belfield, “Deep vascular imaging in wounds by two-photon fluorescence microscopy,” PLos One 8(7), e67559 (2013).
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J. W. Cha, V. R. Singh, K. H. Kim, J. Subramanian, Q. Peng, H. Yu, E. Nedivi, and P. T. C. So, “Reassignment of scattering emission photons in multi-focal multiphoton microscopy,” Sci. Rep. 4, 5153 (2014).
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M. A. A. Neil, R. Juškaitis, M. J. Booth, T. Wilson, T. Tanaka, and S. Kawata, “Adaptive aberration correction in a two-photon microscope,” J. Microscopy 200(2), 105–108 (2000).
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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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S. P. Poland, N. Krstajić, J. Monypenny, S. Coelho, D. Tyndall, R. J. Walker, V. Devauges, J. Richardson, N. Dutton, P. Barber, D. D. Li, K. Suhling, T. Ng, R. K. Henderson, and S. M. Ameer-Beg, “A high speed multifocal multiphoton fluorescence lifetime imaging microscope for live-cell FRET imaging,” Bio. Opt. Express 6(2), 277–296 (2015).
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S. Coelho, S. Poland, N. Krstajic, D. Li, J. Moneypenny, R. Walker, D. Tyndall, T. Ng, R. Henderson, and S. Ameer-Beg, “multi-focal multiphoton microscopy with adaptive optical correction,” Proc. SPIE 8588, 858817 (2013).
[Crossref]

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T. Nielsen, M. Fricke, D. Hellweg, and P. Andersen, “High efficiency beam splitter for multi-focal mutiphoton microscopy,” J. Microscopy 201, 368–376 (2000).
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Y. Shao, W. Qin, H. Lin, J. Qu, X. Peng, H. Niu, and B. Z. Gao, “multi-focal multiphoton microscopy based on a spatial light modulator,” Appl. Phys. B 107, 653–657 (2012).
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Okazaki, S.

N. Matsumoto, T. Inoue, A. Matsumoto, and S. Okazaki, “Correction of depth-induced spherical aberration for deep observation using two-photon excitation fluorescence microscopy with spatial light modulator,” Bio. Opt. Express 6(7), 2575–2587 (2015).
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Otsu, T.

Paninski, L.

W. Yang, J. Kang, M. L. Carrillo-Reid, E. Pnevmatikakis, L. Paninski, R. Yuste, and D. S. Peterka, “Simultaneous multi-plane imaging of neural circuits,” Neron 89(2) 269–284 (2016).
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Peng, Q.

J. W. Cha, V. R. Singh, K. H. Kim, J. Subramanian, Q. Peng, H. Yu, E. Nedivi, and P. T. C. So, “Reassignment of scattering emission photons in multi-focal multiphoton microscopy,” Sci. Rep. 4, 5153 (2014).
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Peng, X.

Y. Shao, W. Qin, H. Lin, J. Qu, X. Peng, H. Niu, and B. Z. Gao, “multi-focal multiphoton microscopy based on a spatial light modulator,” Appl. Phys. B 107, 653–657 (2012).
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W. Yang, J. Kang, M. L. Carrillo-Reid, E. Pnevmatikakis, L. Paninski, R. Yuste, and D. S. Peterka, “Simultaneous multi-plane imaging of neural circuits,” Neron 89(2) 269–284 (2016).
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Planchon, T. A.

T. A. Planchon, L. Gao, D. E. Milkie, M. W. Davidson, J. A. Galbraith, C. G. Galbraith, and E. Betzig, “Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination,” Nat. Methods 8, 417–423 (2011).
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W. Yang, J. Kang, M. L. Carrillo-Reid, E. Pnevmatikakis, L. Paninski, R. Yuste, and D. S. Peterka, “Simultaneous multi-plane imaging of neural circuits,” Neron 89(2) 269–284 (2016).
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S. Coelho, S. Poland, N. Krstajic, D. Li, J. Moneypenny, R. Walker, D. Tyndall, T. Ng, R. Henderson, and S. Ameer-Beg, “multi-focal multiphoton microscopy with adaptive optical correction,” Proc. SPIE 8588, 858817 (2013).
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S. P. Poland, N. Krstajić, J. Monypenny, S. Coelho, D. Tyndall, R. J. Walker, V. Devauges, J. Richardson, N. Dutton, P. Barber, D. D. Li, K. Suhling, T. Ng, R. K. Henderson, and S. M. Ameer-Beg, “A high speed multifocal multiphoton fluorescence lifetime imaging microscope for live-cell FRET imaging,” Bio. Opt. Express 6(2), 277–296 (2015).
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Qin, W.

Y. Shao, W. Qin, H. Lin, J. Qu, X. Peng, H. Niu, and B. Z. Gao, “multi-focal multiphoton microscopy based on a spatial light modulator,” Appl. Phys. B 107, 653–657 (2012).
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Y. Shao, W. Qin, H. Lin, J. Qu, X. Peng, H. Niu, and B. Z. Gao, “multi-focal multiphoton microscopy based on a spatial light modulator,” Appl. Phys. B 107, 653–657 (2012).
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Reich, R.

Richardson, J.

S. P. Poland, N. Krstajić, J. Monypenny, S. Coelho, D. Tyndall, R. J. Walker, V. Devauges, J. Richardson, N. Dutton, P. Barber, D. D. Li, K. Suhling, T. Ng, R. K. Henderson, and S. M. Ameer-Beg, “A high speed multifocal multiphoton fluorescence lifetime imaging microscope for live-cell FRET imaging,” Bio. Opt. Express 6(2), 277–296 (2015).
[Crossref]

Roy, S.

D. M. Small, W. Y. Sanchez, S. Roy, M. J. Hickey, and G. C. Gobe, “Multiphoton fluorescence microscopy of the live kidney in health and disease,” J. Biomed. Opt. 19(2), 020901 (2014).
[Crossref] [PubMed]

Sacconi, L.

Salter, P. S.

Sanchez, W. Y.

D. M. Small, W. Y. Sanchez, S. Roy, M. J. Hickey, and G. C. Gobe, “Multiphoton fluorescence microscopy of the live kidney in health and disease,” J. Biomed. Opt. 19(2), 020901 (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).
[Crossref] [PubMed]

Sato, S.

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]

Sawada, K.

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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Shao, Y.

Y. Shao, W. Qin, H. Lin, J. Qu, X. Peng, H. Niu, and B. Z. Gao, “multi-focal multiphoton microscopy based on a spatial light modulator,” Appl. Phys. B 107, 653–657 (2012).
[Crossref]

Singh, V. R.

J. W. Cha, V. R. Singh, K. H. Kim, J. Subramanian, Q. Peng, H. Yu, E. Nedivi, and P. T. C. So, “Reassignment of scattering emission photons in multi-focal multiphoton microscopy,” Sci. Rep. 4, 5153 (2014).
[Crossref]

Small, D. M.

D. M. Small, W. Y. Sanchez, S. Roy, M. J. Hickey, and G. C. Gobe, “Multiphoton fluorescence microscopy of the live kidney in health and disease,” J. Biomed. Opt. 19(2), 020901 (2014).
[Crossref] [PubMed]

So, P. T. C.

Song, Y.

Y. Li, Y. Song, L. Zhao, G. Gaidosh, AM Laties, and R. Wen, “Direct labeling and visualization of blood vessels with lipophilic carbocyanine dye DiI”, Nat. Protocols 3, 1703–1708 (2008).
[Crossref]

Subramanian, J.

J. W. Cha, V. R. Singh, K. H. Kim, J. Subramanian, Q. Peng, H. Yu, E. Nedivi, and P. T. C. So, “Reassignment of scattering emission photons in multi-focal multiphoton microscopy,” Sci. Rep. 4, 5153 (2014).
[Crossref]

Suhling, K.

S. P. Poland, N. Krstajić, J. Monypenny, S. Coelho, D. Tyndall, R. J. Walker, V. Devauges, J. Richardson, N. Dutton, P. Barber, D. D. Li, K. Suhling, T. Ng, R. K. Henderson, and S. M. Ameer-Beg, “A high speed multifocal multiphoton fluorescence lifetime imaging microscope for live-cell FRET imaging,” Bio. Opt. Express 6(2), 277–296 (2015).
[Crossref]

Taghizadeh, M. R.

Takamoto, H.

Tanaka, T.

M. A. A. Neil, R. Juškaitis, M. J. Booth, T. Wilson, T. Tanaka, and S. Kawata, “Adaptive aberration correction in a two-photon microscope,” J. Microscopy 200(2), 105–108 (2000).
[Crossref]

Tang, J.

L. Kong, J. Tang, J. P. Little, Y. Yu, T. Lammermann, C. P. Lin, R. N. Germain, and M. Cui, “Continuous volumetric imaging via an optical phase-locked ultrasound lens,” Nat. Methods 12, 759–762 (2015).
[Crossref] [PubMed]

Terakawa, S.

Theer, P.

Theofanidou, E.

E. Theofanidou, L. Wilson, W. J. Hassack, and J. Arlt, “Spherical aberration correction for optical tweezers,” Opt. Commun. 236(1–3), 145–150 (2004).
[Crossref]

Török, P.

Toyoda, H.

Tsay, R. -K.

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

Tsien, R. Y.

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

Tyndall, D.

S. P. Poland, N. Krstajić, J. Monypenny, S. Coelho, D. Tyndall, R. J. Walker, V. Devauges, J. Richardson, N. Dutton, P. Barber, D. D. Li, K. Suhling, T. Ng, R. K. Henderson, and S. M. Ameer-Beg, “A high speed multifocal multiphoton fluorescence lifetime imaging microscope for live-cell FRET imaging,” Bio. Opt. Express 6(2), 277–296 (2015).
[Crossref]

S. Coelho, S. Poland, N. Krstajic, D. Li, J. Moneypenny, R. Walker, D. Tyndall, T. Ng, R. Henderson, and S. Ameer-Beg, “multi-focal multiphoton microscopy with adaptive optical correction,” Proc. SPIE 8588, 858817 (2013).
[Crossref]

Urakami, T.

C. O. Yanez, A. R. Morales, X. Yue, T. Urakami, M. Komatsu, T. A. H. Jarvinen, and K. D. Belfield, “Deep vascular imaging in wounds by two-photon fluorescence microscopy,” PLos One 8(7), e67559 (2013).
[Crossref] [PubMed]

Varga, P.

Walker, R.

S. Coelho, S. Poland, N. Krstajic, D. Li, J. Moneypenny, R. Walker, D. Tyndall, T. Ng, R. Henderson, and S. Ameer-Beg, “multi-focal multiphoton microscopy with adaptive optical correction,” Proc. SPIE 8588, 858817 (2013).
[Crossref]

Walker, R. J.

S. P. Poland, N. Krstajić, J. Monypenny, S. Coelho, D. Tyndall, R. J. Walker, V. Devauges, J. Richardson, N. Dutton, P. Barber, D. D. Li, K. Suhling, T. Ng, R. K. Henderson, and S. M. Ameer-Beg, “A high speed multifocal multiphoton fluorescence lifetime imaging microscope for live-cell FRET imaging,” Bio. Opt. Express 6(2), 277–296 (2015).
[Crossref]

Wang, T. D.

Wen, R.

Y. Li, Y. Song, L. Zhao, G. Gaidosh, AM Laties, and R. Wen, “Direct labeling and visualization of blood vessels with lipophilic carbocyanine dye DiI”, Nat. Protocols 3, 1703–1708 (2008).
[Crossref]

Wilson, L.

E. Theofanidou, L. Wilson, W. J. Hassack, and J. Arlt, “Spherical aberration correction for optical tweezers,” Opt. Commun. 236(1–3), 145–150 (2004).
[Crossref]

Wilson, T.

M. A. A. Neil, R. Juškaitis, M. J. Booth, T. Wilson, T. Tanaka, and S. Kawata, “Adaptive aberration correction in a two-photon microscope,” J. Microscopy 200(2), 105–108 (2000).
[Crossref]

Yanez, C. O.

C. O. Yanez, A. R. Morales, X. Yue, T. Urakami, M. Komatsu, T. A. H. Jarvinen, and K. D. Belfield, “Deep vascular imaging in wounds by two-photon fluorescence microscopy,” PLos One 8(7), e67559 (2013).
[Crossref] [PubMed]

Yang, W.

W. Yang, J. Kang, M. L. Carrillo-Reid, E. Pnevmatikakis, L. Paninski, R. Yuste, and D. S. Peterka, “Simultaneous multi-plane imaging of neural circuits,” Neron 89(2) 269–284 (2016).
[Crossref]

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).
[Crossref] [PubMed]

Yu, H.

J. W. Cha, V. R. Singh, K. H. Kim, J. Subramanian, Q. Peng, H. Yu, E. Nedivi, and P. T. C. So, “Reassignment of scattering emission photons in multi-focal multiphoton microscopy,” Sci. Rep. 4, 5153 (2014).
[Crossref]

Yu, Y.

L. Kong, J. Tang, J. P. Little, Y. Yu, T. Lammermann, C. P. Lin, R. N. Germain, and M. Cui, “Continuous volumetric imaging via an optical phase-locked ultrasound lens,” Nat. Methods 12, 759–762 (2015).
[Crossref] [PubMed]

Yue, X.

C. O. Yanez, A. R. Morales, X. Yue, T. Urakami, M. Komatsu, T. A. H. Jarvinen, and K. D. Belfield, “Deep vascular imaging in wounds by two-photon fluorescence microscopy,” PLos One 8(7), e67559 (2013).
[Crossref] [PubMed]

Yuste, R.

W. Yang, J. Kang, M. L. Carrillo-Reid, E. Pnevmatikakis, L. Paninski, R. Yuste, and D. S. Peterka, “Simultaneous multi-plane imaging of neural circuits,” Neron 89(2) 269–284 (2016).
[Crossref]

Zhao, L.

Y. Li, Y. Song, L. Zhao, G. Gaidosh, AM Laties, and R. Wen, “Direct labeling and visualization of blood vessels with lipophilic carbocyanine dye DiI”, Nat. Protocols 3, 1703–1708 (2008).
[Crossref]

Appl. Opt. (2)

Appl. Phys. B (1)

Y. Shao, W. Qin, H. Lin, J. Qu, X. Peng, H. Niu, and B. Z. Gao, “multi-focal multiphoton microscopy based on a spatial light modulator,” Appl. Phys. B 107, 653–657 (2012).
[Crossref]

Bio. Opt. Express (2)

N. Matsumoto, T. Inoue, A. Matsumoto, and S. Okazaki, “Correction of depth-induced spherical aberration for deep observation using two-photon excitation fluorescence microscopy with spatial light modulator,” Bio. Opt. Express 6(7), 2575–2587 (2015).
[Crossref]

S. P. Poland, N. Krstajić, J. Monypenny, S. Coelho, D. Tyndall, R. J. Walker, V. Devauges, J. Richardson, N. Dutton, P. Barber, D. D. Li, K. Suhling, T. Ng, R. K. Henderson, and S. M. Ameer-Beg, “A high speed multifocal multiphoton fluorescence lifetime imaging microscope for live-cell FRET imaging,” Bio. Opt. Express 6(2), 277–296 (2015).
[Crossref]

Biophys. J. (1)

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

J. Biomed. Opt. (1)

D. M. Small, W. Y. Sanchez, S. Roy, M. J. Hickey, and G. C. Gobe, “Multiphoton fluorescence microscopy of the live kidney in health and disease,” J. Biomed. Opt. 19(2), 020901 (2014).
[Crossref] [PubMed]

J. Microscopy (3)

A. Egner, J. Bewersdorf, and S. W. Hell, “Comparison of the axial resolution of practical Nipkow-disk confocal fluorescence microscopy with that of multifocal multiphoton microscopy: theory and experiment,” J. Microscopy 206, 24–32 (2001).
[Crossref]

T. Nielsen, M. Fricke, D. Hellweg, and P. Andersen, “High efficiency beam splitter for multi-focal mutiphoton microscopy,” J. Microscopy 201, 368–376 (2000).
[Crossref]

M. A. A. Neil, R. Juškaitis, M. J. Booth, T. Wilson, T. Tanaka, and S. Kawata, “Adaptive aberration correction in a two-photon microscope,” J. Microscopy 200(2), 105–108 (2000).
[Crossref]

J. Opt. Soc. Am. A (2)

Nat. Methods (4)

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

L. Kong, J. Tang, J. P. Little, Y. Yu, T. Lammermann, C. P. Lin, R. N. Germain, and M. Cui, “Continuous volumetric imaging via an optical phase-locked ultrasound lens,” Nat. Methods 12, 759–762 (2015).
[Crossref] [PubMed]

T. A. Planchon, L. Gao, D. E. Milkie, M. W. Davidson, J. A. Galbraith, C. G. Galbraith, and E. Betzig, “Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination,” Nat. Methods 8, 417–423 (2011).
[Crossref] [PubMed]

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

Nat. Neurosci. (1)

M. T. Ke, S. Fujimoto, and T. Imai, “SeeDB: A simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction,” Nat. Neurosci. 16, 1154–1161 (2013).
[Crossref] [PubMed]

Nat. Protocols (1)

Y. Li, Y. Song, L. Zhao, G. Gaidosh, AM Laties, and R. Wen, “Direct labeling and visualization of blood vessels with lipophilic carbocyanine dye DiI”, Nat. Protocols 3, 1703–1708 (2008).
[Crossref]

Neron (1)

W. Yang, J. Kang, M. L. Carrillo-Reid, E. Pnevmatikakis, L. Paninski, R. Yuste, and D. S. Peterka, “Simultaneous multi-plane imaging of neural circuits,” Neron 89(2) 269–284 (2016).
[Crossref]

Opt. Commun. (1)

E. Theofanidou, L. Wilson, W. J. Hassack, and J. Arlt, “Spherical aberration correction for optical tweezers,” Opt. Commun. 236(1–3), 145–150 (2004).
[Crossref]

Opt. Express (6)

Opt. Lett. (3)

Phil. Trans R. Soc. A (1)

M. J. Booth, “Adaptive optics in microscopy,” Phil. Trans R. Soc. A 365, 2829–2843 (2007).
[Crossref] [PubMed]

PLos One (1)

C. O. Yanez, A. R. Morales, X. Yue, T. Urakami, M. Komatsu, T. A. H. Jarvinen, and K. D. Belfield, “Deep vascular imaging in wounds by two-photon fluorescence microscopy,” PLos One 8(7), e67559 (2013).
[Crossref] [PubMed]

Proc. SPIE (1)

S. Coelho, S. Poland, N. Krstajic, D. Li, J. Moneypenny, R. Walker, D. Tyndall, T. Ng, R. Henderson, and S. Ameer-Beg, “multi-focal multiphoton microscopy with adaptive optical correction,” Proc. SPIE 8588, 858817 (2013).
[Crossref]

Sci. Rep. (2)

J. W. Cha, V. R. Singh, K. H. Kim, J. Subramanian, Q. Peng, H. Yu, E. Nedivi, and P. T. C. So, “Reassignment of scattering emission photons in multi-focal multiphoton microscopy,” Sci. Rep. 4, 5153 (2014).
[Crossref]

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]

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Figures (8)

Fig. 1
Fig. 1

Schematic of the experimental MLSM system using an SLM. The solid line (red) and dashed lines (green) represent the excitation beams and fluorescence, respectively. By changing the CGH applied to the SLM, the system can perform four types of scans: MMM with SA correction, MMM without SA correction, SMM with SA correction, and SMM without SA correction. To reduce the influence of the spread of the fluorescence, the multiple fluorescence are detected by every two anodes of the mPMT. The blue double-head arrow indicates the polarization direction of the excitation beam.

Fig. 2
Fig. 2

The scanner performed a raster scan with: (a) four excitation beams and (b) a single excitation beams. The system performed MMM when four excitation beams are scanned, and SMM when a single excitation beam is scanned. The resulting x-y images observed using (c) MMM and (d) SMM. The red arrows indicate the boundaries of the scanning areas. The scale bar indicates 20 μm.

Fig. 3
Fig. 3

Intensity of the excitation beam for MMM and SMM. To clarify the effect of the SA correction in the deeper regions, the intensity of the excitation beam was changed depending on the observation depth. The intensity of the excitation beam was measured under the objective lens.

Fig. 4
Fig. 4

Results of observations of the fluorescent polystyrene beads in transparent epoxy resin with a dry objective lens (NA = 0.7). (a), (b) Y Z projected images for an optical depth of −35 μm to 1100 μm from MMM scans performed with and without SA correction, respectively. (c), (d) Y Z projected images from SMM scans performed with and without SA correction, respectively. (e)–(p) Magnified Y Z projected images for optical depths of (e)–(h) −35 μm to 100 μm, (i)–(l) 450 μm to 550 μm, and (m)–(p) 950 μm to 1050 μm. The red arrows indicate the boundary of the scanning area. The scale bar indicates 20 μm.

Fig. 5
Fig. 5

(a) Ratio of the fluorescence intensity with SA correction to that without SA correction. (b) Ratio of the observed beads length (FWHM) with SA correction to that without SA correction.

Fig. 6
Fig. 6

Y Z projected images of blood vessels from the cerebrum of a mouse stained with DiI. A dry objective lens (NA = 0.7) was used, and the objective lens was moved in increments of 2.0 μm. (a), (b) Y Z projected images for an optical depth of 0 μm to 754 μm for MMM scans performed with and without SA correction, respectively. (c), (d) Y Z projected images for SMM scans performed with and without SA correction, respectively. (e)–(h) Magnified Y Z projected images for an optical depth of 242 μm to 424 μm (areas surrounded by yellow dashed squares in Figs. 6(a) to 6(d)). The scale bar indicates 100 μm.

Fig. 7
Fig. 7

Observed x-y images of blood vessels from the cerebrum of a mouse stained with DiI. (a), (b) x-y images at an optical depth of 345 μm for MMM scans performed with and without SA correction, respectively. (c), (d) x-y images for SMM scans performed with and without SA correction, respectively. The scale bar indicates 20 μm. The magnified view of the vessel in the orange dashed box is shown on the upper left of Figs. 7 (a) to (d). (e) the fluorescence intensity of the vessel along the green dashed line in the orange dashed box.

Fig. 8
Fig. 8

Intensity of the excitation beam for MMM and SMM. To clarify the effect of the SA correction in the deeper region, the intensity of the excitation beam was changed depending on the observation depth. The intensity of the excitation beam was measured under the objective lens. Compared to SMM, the intensity of the excitation beam for MMM was 4.6 times higher in the shallow depth region. The power of the laser reached its limit when the MMM scan was performed at an optical depth of over 412 μm.

Equations (3)

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ϕ 2 ( ρ ) = 2 π d λ ( ( 1 + η ) n 2 2 ( N A ¯ ρ ) 2 n 1 2 ( N A ¯ ρ ) 2 ) ,
Ratio experiment ( α , β , γ ) = Q with SA correction ( α , β , γ ) Q without SA correction ( α , β , γ ) ,
Ratio simulation ( γ ) = ( max | ( A exp ( ϕ f l a t ) ) | * DE SLM ( γ ) max | ( A exp ( ϕ a b e r r a t i o n ( γ ) ) ) | ) 4 ,

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