Abstract

Fast, volumetric imaging over large scales has been a long-standing challenge in biological microscopy. To address this challenge, we report an augmented variant of confocal microscopy that uses a series of reflecting pinholes axially distributed in the detection space, such that each pinhole probes a different depth within the sample. We thus obtain simultaneous multiplane imaging without the need for axial scanning. Our microscope technique is versatile and configured here to provide two-color fluorescence imaging with a field of view larger than a millimeter at video rate. Its general applicability is demonstrated with neuronal imaging of both Caenorhabditis elegans and mouse brains in vivo.

© 2019 Optical Society of America under the terms of the OSA Open Access Publishing Agreement

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2018 (4)

S. Xiao, H.-A. Tseng, H. Gritton, X. Han, and J. Mertz, “Video-rate volumetric neuronal imaging using 3D targeted illumination,” Sci. Rep. 8, 7921 (2018).
[Crossref]

W. J. Shain, N. A. Vickers, J. Li, X. Han, T. Bifano, and J. Mertz, “Axial localization with modulated-illumination extended-depth-of-field microscopy,” Biomed. Opt. Express 9, 1771–1782 (2018).
[Crossref]

Q. Hu, P. Li, Y. Xiong, Y. Wang, X. Lv, and S. Zeng, “Simultaneous two-plane, two-photon imaging based on spatial multiplexing,” Opt. Lett. 43, 4598–4601 (2018).
[Crossref]

K. Burnett, E. Edsinger, and D. R. Albrecht, “Rapid and gentle hydrogel encapsulation of living organisms enables long-term microscopy over multiple hours,” Commun. Biol. 1, 73 (2018).
[Crossref]

2017 (6)

R. Lu, W. Sun, Y. Liang, A. Kerlin, J. Bierfeld, J. D. Seelig, D. E. Wilson, B. Scholl, B. Mohar, M. Tanimoto, and M. Koyama, “Video-rate volumetric functional imaging of the brain at synaptic resolution,” Nat. Neurosci. 20, 620–628 (2017).
[Crossref]

A. Song, A. S. Charles, S. A. Koay, J. L. Gauthier, S. Y. Thiberge, J. W. Pillow, and D. W. Tank, “Volumetric two-photon imaging of neurons using stereoscopy (vtwins),” Nat. Methods 14, 420–426 (2017).
[Crossref]

C. Roider, R. Piestun, and A. Jesacher, “3d image scanning microscopy with engineered excitation and detection,” Optica 4, 1373–1381 (2017).
[Crossref]

W. Yang and R. Yuste, “In vivo imaging of neural activity,” Nat. Methods 14, 349–359 (2017).
[Crossref]

T.-H. Chen, J. Ault, H. Stone, and C. Arnold, “High-speed axial-scanning wide-field microscopy for volumetric particle tracking velocimetry,” Exp. Fluids 58, 41 (2017).
[Crossref]

W. J. Shain, N. A. Vickers, B. B. Goldberg, T. Bifano, and J. Mertz, “Extended depth-of-field microscopy with a high-speed deformable mirror,” Opt. Lett. 42, 995–998 (2017).
[Crossref]

2016 (7)

N. Ji, J. Freeman, and S. L. Smith, “Technologies for imaging neural activity in large volumes,” Nat. Neurosci. 19, 1154–1164 (2016).
[Crossref]

N. C. Pégard, H.-Y. Liu, N. Antipa, M. Gerlock, H. Adesnik, and L. Waller, “Compressive light-field microscopy for 3D neural activity recording,” Optica 3, 517–524 (2016).
[Crossref]

K. N. S. Nadella, H. Roš, C. Baragli, V. A. Griffiths, G. Konstantinou, T. Koimtzis, G. J. Evans, P. A. Kirkby, and R. A. Silver, “Random-access scanning microscopy for 3D imaging in awake behaving animals,” Nat. Methods 13, 1001–1004 (2016).
[Crossref]

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

A. I. Mohammed, H. J. Gritton, H.-A. Tseng, M. E. Bucklin, Z. Yao, and X. Han, “An integrative approach for analyzing hundreds of neurons in task performing mice using wide-field calcium imaging,” Sci. Rep. 6, 20986 (2016).
[Crossref]

A. Dubbs, J. Guevara, and R. Yuste, “moco: Fast motion correction for calcium imaging,” Front. Neuroinf. 10, 6 (2016).
[Crossref]

E. A. Pnevmatikakis, D. Soudry, Y. Gao, T. A. Machado, J. Merel, D. Pfau, T. Reardon, Y. Mu, C. Lacefield, W. Yang, and M. Ahrens, “Simultaneous denoising, deconvolution, and demixing of calcium imaging data,” Neuron 89, 285–299 (2016).
[Crossref]

2015 (2)

L. Kong, J. Tang, J. P. Little, Y. Yu, T. Lämmermann, 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]

M. B. Bouchard, V. Voleti, C. S. Mendes, C. Lacefield, W. B. Grueber, R. S. Mann, R. M. Bruno, and E. M. Hillman, “Swept confocally-aligned planar excitation (scape) microscopy for high-speed volumetric imaging of behaving organisms,” Nat. Photonics 9, 113–119 (2015).
[Crossref]

2014 (2)

R. Prevedel, Y.-G. Yoon, M. Hoffmann, N. Pak, G. Wetzstein, S. Kato, T. Schrödel, R. Raskar, M. Zimmer, E. S. Boyden, and A. Vaziri, “Simultaneous whole-animal 3D imaging of neuronal activity using light-field microscopy,” Nat. Methods 11, 727–730 (2014).
[Crossref]

M. Duocastella, G. Vicidomini, and A. Diaspro, “Simultaneous multiplane confocal microscopy using acoustic tunable lenses,” Opt. Express 22, 19293–19301 (2014).
[Crossref]

2013 (2)

T. Schrödel, R. Prevedel, K. Aumayr, M. Zimmer, and A. Vaziri, “Brain-wide 3D imaging of neuronal activity in Caenorhabditis elegans with sculpted light,” Nat. Methods 10, 1013–1020 (2013).
[Crossref]

S. Quirin, D. S. Peterka, and R. Yuste, “Instantaneous three-dimensional sensing using spatial light modulator illumination with extended depth of field imaging,” Opt. Express 21, 16007–16021 (2013).
[Crossref]

2012 (2)

S. Abrahamsson, J. Chen, B. Hajj, S. Stallinga, A. Y. Katsov, J. Wisniewski, G. Mizuguchi, P. Soule, F. Mueller, C. D. Darzacq, and X. Darzacq, “Fast multicolor 3D imaging using aberration-corrected multifocus microscopy,” Nat. Methods 10, 60–63 (2012).
[Crossref]

C. Yang, K. Shi, M. Zhou, S. Zheng, S. Yin, and Z. Liu, “Z-microscopy for parallel axial imaging with micro mirror array,” Appl. Phys. Lett. 101, 231111 (2012).
[Crossref]

2011 (2)

B. F. Grewe, F. F. Voigt, M. van ’t Hoff, and F. Helmchen, “Fast two-layer two-photon imaging of neuronal cell populations using an electrically tunable lens,” Biomed. Opt. Express 2, 2035–2046 (2011).
[Crossref]

A. Cheng, J. T. Goncalves, P. Golshani, K. Arisaka, and C. Portera-Cailliau, “Simultaneous two-photon calcium imaging at different depths with spatiotemporal multiplexing,” Nat. Methods 8, 139–142 (2011).
[Crossref]

2010 (1)

2009 (1)

2008 (1)

Y. Otsu, V. Bormuth, J. Wong, B. Mathieu, G. P. Dugué, A. Feltz, and S. Dieudonné, “Optical monitoring of neuronal activity at high frame rate with a digital random-access multiphoton (ramp) microscope,” J. Neurosci. Methods 173, 259–270 (2008).
[Crossref]

2007 (2)

2004 (1)

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science 305, 1007–1009 (2004).
[Crossref]

2003 (1)

T. A. Pologruto, B. L. Sabatini, and K. Svoboda, “Scanimage: flexible software for operating laser scanning microscopes,” Biomed. Eng. online 2, 13 (2003).
[Crossref]

1999 (1)

1995 (1)

Abrahamsson, S.

S. Abrahamsson, J. Chen, B. Hajj, S. Stallinga, A. Y. Katsov, J. Wisniewski, G. Mizuguchi, P. Soule, F. Mueller, C. D. Darzacq, and X. Darzacq, “Fast multicolor 3D imaging using aberration-corrected multifocus microscopy,” Nat. Methods 10, 60–63 (2012).
[Crossref]

Adesnik, H.

Ahrens, M.

E. A. Pnevmatikakis, D. Soudry, Y. Gao, T. A. Machado, J. Merel, D. Pfau, T. Reardon, Y. Mu, C. Lacefield, W. Yang, and M. Ahrens, “Simultaneous denoising, deconvolution, and demixing of calcium imaging data,” Neuron 89, 285–299 (2016).
[Crossref]

Albrecht, D. R.

K. Burnett, E. Edsinger, and D. R. Albrecht, “Rapid and gentle hydrogel encapsulation of living organisms enables long-term microscopy over multiple hours,” Commun. Biol. 1, 73 (2018).
[Crossref]

Amir, W.

Antipa, N.

Arisaka, K.

A. Cheng, J. T. Goncalves, P. Golshani, K. Arisaka, and C. Portera-Cailliau, “Simultaneous two-photon calcium imaging at different depths with spatiotemporal multiplexing,” Nat. Methods 8, 139–142 (2011).
[Crossref]

Arnold, C.

T.-H. Chen, J. Ault, H. Stone, and C. Arnold, “High-speed axial-scanning wide-field microscopy for volumetric particle tracking velocimetry,” Exp. Fluids 58, 41 (2017).
[Crossref]

Arnold, C. B.

Ault, J.

T.-H. Chen, J. Ault, H. Stone, and C. Arnold, “High-speed axial-scanning wide-field microscopy for volumetric particle tracking velocimetry,” Exp. Fluids 58, 41 (2017).
[Crossref]

Aumayr, K.

T. Schrödel, R. Prevedel, K. Aumayr, M. Zimmer, and A. Vaziri, “Brain-wide 3D imaging of neuronal activity in Caenorhabditis elegans with sculpted light,” Nat. Methods 10, 1013–1020 (2013).
[Crossref]

Baragli, C.

K. N. S. Nadella, H. Roš, C. Baragli, V. A. Griffiths, G. Konstantinou, T. Koimtzis, G. J. Evans, P. A. Kirkby, and R. A. Silver, “Random-access scanning microscopy for 3D imaging in awake behaving animals,” Nat. Methods 13, 1001–1004 (2016).
[Crossref]

Beaurepaire, E.

Bierfeld, J.

R. Lu, W. Sun, Y. Liang, A. Kerlin, J. Bierfeld, J. D. Seelig, D. E. Wilson, B. Scholl, B. Mohar, M. Tanimoto, and M. Koyama, “Video-rate volumetric functional imaging of the brain at synaptic resolution,” Nat. Neurosci. 20, 620–628 (2017).
[Crossref]

Bifano, T.

Blanchard, P. M.

Booth, M. J.

Bormuth, V.

Y. Otsu, V. Bormuth, J. Wong, B. Mathieu, G. P. Dugué, A. Feltz, and S. Dieudonné, “Optical monitoring of neuronal activity at high frame rate with a digital random-access multiphoton (ramp) microscope,” J. Neurosci. Methods 173, 259–270 (2008).
[Crossref]

Botcherby, E. J.

Bouchard, M. B.

M. B. Bouchard, V. Voleti, C. S. Mendes, C. Lacefield, W. B. Grueber, R. S. Mann, R. M. Bruno, and E. M. Hillman, “Swept confocally-aligned planar excitation (scape) microscopy for high-speed volumetric imaging of behaving organisms,” Nat. Photonics 9, 113–119 (2015).
[Crossref]

Boyden, E. S.

R. Prevedel, Y.-G. Yoon, M. Hoffmann, N. Pak, G. Wetzstein, S. Kato, T. Schrödel, R. Raskar, M. Zimmer, E. S. Boyden, and A. Vaziri, “Simultaneous whole-animal 3D imaging of neuronal activity using light-field microscopy,” Nat. Methods 11, 727–730 (2014).
[Crossref]

Bruno, R. M.

M. B. Bouchard, V. Voleti, C. S. Mendes, C. Lacefield, W. B. Grueber, R. S. Mann, R. M. Bruno, and E. M. Hillman, “Swept confocally-aligned planar excitation (scape) microscopy for high-speed volumetric imaging of behaving organisms,” Nat. Photonics 9, 113–119 (2015).
[Crossref]

Bucklin, M. E.

A. I. Mohammed, H. J. Gritton, H.-A. Tseng, M. E. Bucklin, Z. Yao, and X. Han, “An integrative approach for analyzing hundreds of neurons in task performing mice using wide-field calcium imaging,” Sci. Rep. 6, 20986 (2016).
[Crossref]

Burnett, K.

K. Burnett, E. Edsinger, and D. R. Albrecht, “Rapid and gentle hydrogel encapsulation of living organisms enables long-term microscopy over multiple hours,” Commun. Biol. 1, 73 (2018).
[Crossref]

Carriles, R.

Carrillo-Reid, L.

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

Cathey, W. T.

Charles, A. S.

A. Song, A. S. Charles, S. A. Koay, J. L. Gauthier, S. Y. Thiberge, J. W. Pillow, and D. W. Tank, “Volumetric two-photon imaging of neurons using stereoscopy (vtwins),” Nat. Methods 14, 420–426 (2017).
[Crossref]

Chen, J.

S. Abrahamsson, J. Chen, B. Hajj, S. Stallinga, A. Y. Katsov, J. Wisniewski, G. Mizuguchi, P. Soule, F. Mueller, C. D. Darzacq, and X. Darzacq, “Fast multicolor 3D imaging using aberration-corrected multifocus microscopy,” Nat. Methods 10, 60–63 (2012).
[Crossref]

Chen, T.-H.

T.-H. Chen, J. Ault, H. Stone, and C. Arnold, “High-speed axial-scanning wide-field microscopy for volumetric particle tracking velocimetry,” Exp. Fluids 58, 41 (2017).
[Crossref]

Cheng, A.

A. Cheng, J. T. Goncalves, P. Golshani, K. Arisaka, and C. Portera-Cailliau, “Simultaneous two-photon calcium imaging at different depths with spatiotemporal multiplexing,” Nat. Methods 8, 139–142 (2011).
[Crossref]

Cui, M.

L. Kong, J. Tang, J. P. Little, Y. Yu, T. Lämmermann, 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]

Dalgarno, H. I.

Dalgarno, P. A.

Darzacq, C. D.

S. Abrahamsson, J. Chen, B. Hajj, S. Stallinga, A. Y. Katsov, J. Wisniewski, G. Mizuguchi, P. Soule, F. Mueller, C. D. Darzacq, and X. Darzacq, “Fast multicolor 3D imaging using aberration-corrected multifocus microscopy,” Nat. Methods 10, 60–63 (2012).
[Crossref]

Darzacq, X.

S. Abrahamsson, J. Chen, B. Hajj, S. Stallinga, A. Y. Katsov, J. Wisniewski, G. Mizuguchi, P. Soule, F. Mueller, C. D. Darzacq, and X. Darzacq, “Fast multicolor 3D imaging using aberration-corrected multifocus microscopy,” Nat. Methods 10, 60–63 (2012).
[Crossref]

Del Bene, F.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science 305, 1007–1009 (2004).
[Crossref]

Diaspro, A.

Dieudonné, S.

Y. Otsu, V. Bormuth, J. Wong, B. Mathieu, G. P. Dugué, A. Feltz, and S. Dieudonné, “Optical monitoring of neuronal activity at high frame rate with a digital random-access multiphoton (ramp) microscope,” J. Neurosci. Methods 173, 259–270 (2008).
[Crossref]

Dowski, E. R.

Dubbs, A.

A. Dubbs, J. Guevara, and R. Yuste, “moco: Fast motion correction for calcium imaging,” Front. Neuroinf. 10, 6 (2016).
[Crossref]

Dugué, G. P.

Y. Otsu, V. Bormuth, J. Wong, B. Mathieu, G. P. Dugué, A. Feltz, and S. Dieudonné, “Optical monitoring of neuronal activity at high frame rate with a digital random-access multiphoton (ramp) microscope,” J. Neurosci. Methods 173, 259–270 (2008).
[Crossref]

Duocastella, M.

Durfee, C. G.

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Pillow, J. W.

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Pnevmatikakis, E.

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E. A. Pnevmatikakis, D. Soudry, Y. Gao, T. A. Machado, J. Merel, D. Pfau, T. Reardon, Y. Mu, C. Lacefield, W. Yang, and M. Ahrens, “Simultaneous denoising, deconvolution, and demixing of calcium imaging data,” Neuron 89, 285–299 (2016).
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Quirin, S.

Raskar, R.

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E. A. Pnevmatikakis, D. Soudry, Y. Gao, T. A. Machado, J. Merel, D. Pfau, T. Reardon, Y. Mu, C. Lacefield, W. Yang, and M. Ahrens, “Simultaneous denoising, deconvolution, and demixing of calcium imaging data,” Neuron 89, 285–299 (2016).
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Roš, H.

K. N. S. Nadella, H. Roš, C. Baragli, V. A. Griffiths, G. Konstantinou, T. Koimtzis, G. J. Evans, P. A. Kirkby, and R. A. Silver, “Random-access scanning microscopy for 3D imaging in awake behaving animals,” Nat. Methods 13, 1001–1004 (2016).
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R. Lu, W. Sun, Y. Liang, A. Kerlin, J. Bierfeld, J. D. Seelig, D. E. Wilson, B. Scholl, B. Mohar, M. Tanimoto, and M. Koyama, “Video-rate volumetric functional imaging of the brain at synaptic resolution,” Nat. Neurosci. 20, 620–628 (2017).
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R. Prevedel, Y.-G. Yoon, M. Hoffmann, N. Pak, G. Wetzstein, S. Kato, T. Schrödel, R. Raskar, M. Zimmer, E. S. Boyden, and A. Vaziri, “Simultaneous whole-animal 3D imaging of neuronal activity using light-field microscopy,” Nat. Methods 11, 727–730 (2014).
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T. Schrödel, R. Prevedel, K. Aumayr, M. Zimmer, and A. Vaziri, “Brain-wide 3D imaging of neuronal activity in Caenorhabditis elegans with sculpted light,” Nat. Methods 10, 1013–1020 (2013).
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R. Lu, W. Sun, Y. Liang, A. Kerlin, J. Bierfeld, J. D. Seelig, D. E. Wilson, B. Scholl, B. Mohar, M. Tanimoto, and M. Koyama, “Video-rate volumetric functional imaging of the brain at synaptic resolution,” Nat. Neurosci. 20, 620–628 (2017).
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Shi, K.

C. Yang, K. Shi, M. Zhou, S. Zheng, S. Yin, and Z. Liu, “Z-microscopy for parallel axial imaging with micro mirror array,” Appl. Phys. Lett. 101, 231111 (2012).
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Silver, R. A.

K. N. S. Nadella, H. Roš, C. Baragli, V. A. Griffiths, G. Konstantinou, T. Koimtzis, G. J. Evans, P. A. Kirkby, and R. A. Silver, “Random-access scanning microscopy for 3D imaging in awake behaving animals,” Nat. Methods 13, 1001–1004 (2016).
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N. Ji, J. Freeman, and S. L. Smith, “Technologies for imaging neural activity in large volumes,” Nat. Neurosci. 19, 1154–1164 (2016).
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A. Song, A. S. Charles, S. A. Koay, J. L. Gauthier, S. Y. Thiberge, J. W. Pillow, and D. W. Tank, “Volumetric two-photon imaging of neurons using stereoscopy (vtwins),” Nat. Methods 14, 420–426 (2017).
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E. A. Pnevmatikakis, D. Soudry, Y. Gao, T. A. Machado, J. Merel, D. Pfau, T. Reardon, Y. Mu, C. Lacefield, W. Yang, and M. Ahrens, “Simultaneous denoising, deconvolution, and demixing of calcium imaging data,” Neuron 89, 285–299 (2016).
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S. Abrahamsson, J. Chen, B. Hajj, S. Stallinga, A. Y. Katsov, J. Wisniewski, G. Mizuguchi, P. Soule, F. Mueller, C. D. Darzacq, and X. Darzacq, “Fast multicolor 3D imaging using aberration-corrected multifocus microscopy,” Nat. Methods 10, 60–63 (2012).
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Stallinga, S.

S. Abrahamsson, J. Chen, B. Hajj, S. Stallinga, A. Y. Katsov, J. Wisniewski, G. Mizuguchi, P. Soule, F. Mueller, C. D. Darzacq, and X. Darzacq, “Fast multicolor 3D imaging using aberration-corrected multifocus microscopy,” Nat. Methods 10, 60–63 (2012).
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J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science 305, 1007–1009 (2004).
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J. Neurosci. Methods (1)

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[Crossref]

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[Crossref]

S. Abrahamsson, J. Chen, B. Hajj, S. Stallinga, A. Y. Katsov, J. Wisniewski, G. Mizuguchi, P. Soule, F. Mueller, C. D. Darzacq, and X. Darzacq, “Fast multicolor 3D imaging using aberration-corrected multifocus microscopy,” Nat. Methods 10, 60–63 (2012).
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Nat. Photonics (1)

M. B. Bouchard, V. Voleti, C. S. Mendes, C. Lacefield, W. B. Grueber, R. S. Mann, R. M. Bruno, and E. M. Hillman, “Swept confocally-aligned planar excitation (scape) microscopy for high-speed volumetric imaging of behaving organisms,” Nat. Photonics 9, 113–119 (2015).
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Neuron (2)

E. A. Pnevmatikakis, D. Soudry, Y. Gao, T. A. Machado, J. Merel, D. Pfau, T. Reardon, Y. Mu, C. Lacefield, W. Yang, and M. Ahrens, “Simultaneous denoising, deconvolution, and demixing of calcium imaging data,” Neuron 89, 285–299 (2016).
[Crossref]

W. Yang, J.-E. K. Miller, L. Carrillo-Reid, E. Pnevmatikakis, L. Paninski, R. Yuste, and D. S. Peterka, “Simultaneous multi-plane imaging of neural circuits,” Neuron 89, 269–284 (2016).
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Opt. Express (3)

Opt. Lett. (5)

Optica (2)

Sci. Rep. (2)

S. Xiao, H.-A. Tseng, H. Gritton, X. Han, and J. Mertz, “Video-rate volumetric neuronal imaging using 3D targeted illumination,” Sci. Rep. 8, 7921 (2018).
[Crossref]

A. I. Mohammed, H. J. Gritton, H.-A. Tseng, M. E. Bucklin, Z. Yao, and X. Han, “An integrative approach for analyzing hundreds of neurons in task performing mice using wide-field calcium imaging,” Sci. Rep. 6, 20986 (2016).
[Crossref]

Science (1)

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science 305, 1007–1009 (2004).
[Crossref]

Other (1)

S. Weisenburger, R. Prevedel, and A. Vaziri, “Quantitative evaluation of two-photon calcium imaging modalities for high-speed volumetric calcium imaging in scattering brain tissue,” bioRxiv (2017), p. 115659.

Supplementary Material (5)

NameDescription
» Supplement 1       Supplementary Information
» Visualization 1       In vivo volumetric calcium imaging of a whole moving C. elegans. This video was cropped from a large field of view acquired a 7.5 Hz. Each channel is displayed in a different color.
» Visualization 2       In vivo volumetric calcium imaging of the head ganglia of a C.elegans acquired at 30 Hz. Each channel is displayed in a different color.
» Visualization 3       30 Hz large scale in vivo calcium imaging of a mouse hippocampus. Extended depth of field images are displayed.
» Visualization 4       30 Hz in vivo calcium imaging of a mouse hippocampus. Extended depth of field images are displayed.

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Figures (4)

Fig. 1.
Fig. 1. Multi-Z confocal microscopy. (a) Simplified schematic of the experimental setup. The multiplane detection unit comprises a series of axially distributed reflecting pinholes, each probing a different depth within the sample. (b) Axially extended illumination is obtained by underfilling the back aperture of the MO. The full NA of the MO is used for detection. (c) Transverse (xy) and axial (xz) PSF measured with a subdiffraction size bead, and associated x and z profiles. Horizontal scale bar, 5 μm. Vertical scale bar, 10 μm. (d) Bead signal recorded by each detection channel at different z positions of the stage. Continuous lines correspond to Lorentzian fits. (e) Different imaging planes simultaneously acquired of Aspergillus conidiophores. Scale bar, 200 μm.
Fig. 2.
Fig. 2. In vivo volumetric imaging of C. elegans. (a) Extended depth of field image showing the nuclei marked with NLSmCherry of four different worms in the FOV. 1024×1024 pixels; scale bar, 200 μm. (b) Simultaneous imaging of a single worm [green rectangle in (a)] at different depths and the resulting volumetric rendering. Scale bar, 200 μm. (c, d) Close up view [blue rectangle in(a)], showing the nuclei (in red) and the neurons displaying activity (in green). (e) Activity of the 42 neurons identified in (d). (f) Standard deviation of the calcium activity recorded in the head ganglion of a worm in each imaging plane. Each channel is displayed with a different color and in log scale. (see Visualization 2 for a video of intensity variations). 512×512 pixels; scale bar, 50 μm. (g) Activity of the 32 neurons identified in (f) and recorded over 1000 s. Each row corresponds to a time-series of an individual neuron, as shown in the insets.
Fig. 3.
Fig. 3. Video-rate volumetric Ca2+ imaging in mouse brain expressing GCaMP6f (note that viral injection here led to a labeled area somewhat smaller than our FOV). (a) Extended depth of field image recorded in the hippocampus at 30 frames per second and averaged over 20 s (see Visualization 3 for the video). 512×512 pixels; scale bar, 100 μm. Inset with 3× zoom illustrates cellular resolution. (b) Identification of neurons in each image plane using constrained non-negative matrix factorization (overlay with Panel a is shown in Supplement 1). (c) Activity of the 826 neurons identified in (a). (d) Magnified view of the neuronal activity traces for the region indicated by the red rectangle in (c).
Fig. 4.
Fig. 4. Augmented volumetric imaging using an electrically tunable lens controlled by a square-wave voltage VETL. (a) Multi-Z images obtained for VETL=0 and VETL=5V corresponding to a focal shift of 150 μm. (b) Resulting volumetric acquisition of fixed mouse brain vasculature with structures color-coded by depth. 512×512 pixels; scale bar 300 μm.