Abstract

The combination of optical clearing with light microscopy has a number of applications in the whole-brain imaging of mice. However, the initial processing time of optical clearing is time consuming, and the protocol is complicated. We propose a novel method based on on-line optical clearing. Agarose-embedded mouse brain was immersed in the optical clearing reagent, and clearing of the brain was achieved ~100 μm beneath the sample surface. After imaging, the cleared layer was removed, thereby allowing layer-by-layer clearing and imaging. No pre-immersion was required, and we demonstrated that on-line optical clearing can reduce the whole-brain imaging time by half.

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

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2017 (2)

K. Seiriki, A. Kasai, T. Hashimoto, W. Schulze, M. Niu, S. Yamaguchi, T. Nakazawa, K. I. Inoue, S. Uezono, M. Takada, Y. Naka, H. Igarashi, M. Tanuma, J. A. Waschek, Y. Ago, K. F. Tanaka, A. Hayata-Takano, K. Nagayasu, N. Shintani, R. Hashimoto, Y. Kunii, M. Hino, J. Matsumoto, H. Yabe, T. Nagai, K. Fujita, T. Matsuda, K. Takuma, A. Baba, and H. Hashimoto, “High-speed and scalable whole-brain imaging in rodents and primates,” Neuron 94(6), 1085–1100 (2017).
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[Crossref] [PubMed]

2016 (4)

M. N. Economo, N. G. Clack, L. D. Lavis, C. R. Gerfen, K. Svoboda, E. W. Myers, and J. Chandrashekar, “A platform for brain-wide imaging and reconstruction of individual neurons,” eLife 5, e10566 (2016).
[Crossref] [PubMed]

H. Gong, D. Xu, J. Yuan, X. Li, C. Guo, J. Peng, Y. Li, L. A. Schwarz, A. Li, B. Hu, B. Xiong, Q. Sun, Y. Zhang, J. Liu, Q. Zhong, T. Xu, S. Zeng, and Q. Luo, “High-throughput dual-colour precision imaging for brain-wide connectome with cytoarchitectonic landmarks at the cellular level,” Nat. Commun. 7(1), 12142 (2016).
[Crossref] [PubMed]

M. T. Ke, Y. Nakai, S. Fujimoto, R. Takayama, S. Yoshida, T. S. Kitajima, M. Sato, and T. Imai, “Super-resolution mapping of neuronal circuitry with an index-optimized clearing agent,” Cell Reports 14(11), 2718–2732 (2016).
[Crossref] [PubMed]

C. Pan, R. Cai, F. P. Quacquarelli, A. Ghasemigharagoz, A. Lourbopoulos, P. Matryba, N. Plesnila, M. Dichgans, F. Hellal, and A. Ertürk, “Shrinkage-mediated imaging of entire organs and organisms using uDISCO,” Nat. Methods 13(10), 859–867 (2016).
[Crossref] [PubMed]

2015 (5)

H. Hama, H. Hioki, K. Namiki, T. Hoshida, H. Kurokawa, F. Ishidate, T. Kaneko, T. Akagi, T. Saito, T. Saido, and A. Miyawaki, “ScaleS: An optical clearing palette for biological imaging,” Nat. Neurosci. 18(10), 1518–1529 (2015).
[Crossref] [PubMed]

B. Hou, D. Zhang, S. Zhao, M. Wei, Z. Yang, S. Wang, J. Wang, X. Zhang, B. Liu, L. Fan, Y. Li, Z. Qiu, C. Zhang, and T. Jiang, “Scalable and DiI-compatible optical clearance of the mammalian brain,” Front. Neuroanat. 9, 19 (2015).
[Crossref] [PubMed]

J. Yuan, H. Gong, A. Li, X. Li, S. Chen, S. Zeng, and Q. Luo, “Visible rodent brain-wide networks at single-neuron resolution,” Front. Neuroanat. 9, 70 (2015).
[Crossref] [PubMed]

E. Murray, J. H. Cho, D. Goodwin, T. Ku, J. Swaney, S. Y. Kim, H. Choi, Y. G. Park, J. Y. Park, A. Hubbert, M. McCue, S. Vassallo, N. Bakh, M. P. Frosch, V. J. Wedeen, H. S. Seung, and K. Chung, “Simple, scalable proteomic imaging for high-dimensional profiling of intact systems,” Cell 163(6), 1500–1514 (2015).
[Crossref] [PubMed]

S. Y. Kim, J. H. Cho, E. Murray, N. Bakh, H. Choi, K. Ohn, L. Ruelas, A. Hubbert, M. McCue, S. L. Vassallo, P. J. Keller, and K. Chung, “Stochastic electrotransport selectively enhances the transport of highly electromobile molecules,” Proc. Natl. Acad. Sci. U.S.A. 112(46), E6274–E6283 (2015).
[Crossref] [PubMed]

2014 (4)

Y. Sun, A. Q. Nguyen, J. P. Nguyen, L. Le, D. Saur, J. Choi, E. M. Callaway, and X. Xu, “Cell-type-specific circuit connectivity of hippocampal CA1 revealed through Cre-dependent rabies tracing,” Cell Reports 7(1), 269–280 (2014).
[Crossref] [PubMed]

B. Zingg, H. Hintiryan, L. Gou, M. Y. Song, M. Bay, M. S. Bienkowski, N. N. Foster, S. Yamashita, I. Bowman, A. W. Toga, and H. W. Dong, “Neural networks of the mouse neocortex,” Cell 156(5), 1096–1111 (2014).
[Crossref] [PubMed]

E. A. Susaki, K. Tainaka, D. Perrin, F. Kishino, T. Tawara, T. M. Watanabe, C. Yokoyama, H. Onoe, M. Eguchi, S. Yamaguchi, T. Abe, H. Kiyonari, Y. Shimizu, A. Miyawaki, H. Yokota, and H. R. Ueda, “Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis,” Cell 157(3), 726–739 (2014).
[Crossref] [PubMed]

N. Renier, Z. Wu, D. J. Simon, J. Yang, P. Ariel, and M. Tessier-Lavigne, “iDISCO: A simple, rapid method to immunolabel large tissue samples for volume imaging,” Cell 159(4), 896–910 (2014).
[Crossref] [PubMed]

2013 (6)

M. T. Ke, S. Fujimoto, and T. Imai, “SeeDB: A simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction,” Nat. Neurosci. 16(8), 1154–1161 (2013).
[Crossref] [PubMed]

Z. J. Huang and H. Zeng, “Genetic approaches to neural circuits in the mouse,” Annu. Rev. Neurosci. 36(1), 183–215 (2013).
[Crossref] [PubMed]

P. Osten and T. W. Margrie, “Mapping brain circuitry with a light microscope,” Nat. Methods 10(6), 515–523 (2013).
[Crossref] [PubMed]

T. Kuwajima, A. A. Sitko, P. Bhansali, C. Jurgens, W. Guido, and C. Mason, “ClearT: A detergent- and solvent-free clearing method for neuronal and non-neuronal tissue,” Development 140(6), 1364–1368 (2013).
[Crossref] [PubMed]

Z. Yang, B. Hu, Y. Zhang, Q. Luo, and H. Gong, “Development of a plastic embedding method for large-volume and fluorescent-protein-expressing tissues,” PLoS One 8(4), e60877 (2013).
[Crossref] [PubMed]

K. Chung and K. Deisseroth, “CLARITY for mapping the nervous system,” Nat. Methods 10(6), 508–513 (2013).
[Crossref] [PubMed]

2012 (2)

A. Ertürk, K. Becker, N. Jährling, C. P. Mauch, C. D. Hojer, J. G. Egen, F. Hellal, F. Bradke, M. Sheng, and H. U. Dodt, “Three-dimensional imaging of solvent-cleared organs using 3DISCO,” Nat. Protoc. 7(11), 1983–1995 (2012).
[Crossref] [PubMed]

J. Wang, Y. Zhang, T. H. Xu, Q. M. Luo, and D. Zhu, “An innovative transparent cranial window based on skull optical clearing,” Laser Phys. Lett. 9(6), 469–473 (2012).
[Crossref]

2011 (1)

H. Hama, H. Kurokawa, H. Kawano, R. Ando, T. Shimogori, H. Noda, K. Fukami, A. Sakaue-Sawano, and A. Miyawaki, “Scale: A chemical approach for fluorescence imaging and reconstruction of transparent mouse brain,” Nat. Neurosci. 14(11), 1481–1488 (2011).
[Crossref] [PubMed]

2010 (2)

A. Li, H. Gong, B. Zhang, Q. Wang, C. Yan, J. Wu, Q. Liu, S. Zeng, and Q. Luo, “Micro-optical sectioning tomography to obtain a high-resolution atlas of the mouse brain,” Science 330(6009), 1404–1408 (2010).
[Crossref] [PubMed]

L. Madisen, T. A. Zwingman, S. M. Sunkin, S. W. Oh, H. A. Zariwala, H. Gu, L. L. Ng, R. D. Palmiter, M. J. Hawrylycz, A. R. Jones, E. S. Lein, and H. Zeng, “A robust and high-throughput Cre reporting and characterization system for the whole mouse brain,” Nat. Neurosci. 13(1), 133–140 (2010).
[Crossref] [PubMed]

2008 (1)

L. Luo, E. M. Callaway, and K. Svoboda, “Genetic dissection of neural circuits,” Neuron 57(5), 634–660 (2008).
[Crossref] [PubMed]

2007 (2)

K. D. Micheva and S. J. Smith, “Array tomography: a new tool for imaging the molecular architecture and ultrastructure of neural circuits,” Neuron 55(1), 25–36 (2007).
[Crossref] [PubMed]

H. U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: Three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods 4(4), 331–336 (2007).
[Crossref] [PubMed]

2004 (1)

H. Markram, M. Toledo-Rodriguez, Y. Wang, A. Gupta, G. Silberberg, and C. Wu, “Interneurons of the neocortical inhibitory system,” Nat. Rev. Neurosci. 5(10), 793–807 (2004).
[Crossref] [PubMed]

2002 (1)

R. K. Wang, “Signal degradation by multiple scattering in optical coherence tomography of dense tissue: a Monte Carlo study towards optical clearing of biotissues,” Phys. Med. Biol. 47(13), 2281–2299 (2002).
[Crossref] [PubMed]

Abe, T.

E. A. Susaki, K. Tainaka, D. Perrin, F. Kishino, T. Tawara, T. M. Watanabe, C. Yokoyama, H. Onoe, M. Eguchi, S. Yamaguchi, T. Abe, H. Kiyonari, Y. Shimizu, A. Miyawaki, H. Yokota, and H. R. Ueda, “Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis,” Cell 157(3), 726–739 (2014).
[Crossref] [PubMed]

Ago, Y.

K. Seiriki, A. Kasai, T. Hashimoto, W. Schulze, M. Niu, S. Yamaguchi, T. Nakazawa, K. I. Inoue, S. Uezono, M. Takada, Y. Naka, H. Igarashi, M. Tanuma, J. A. Waschek, Y. Ago, K. F. Tanaka, A. Hayata-Takano, K. Nagayasu, N. Shintani, R. Hashimoto, Y. Kunii, M. Hino, J. Matsumoto, H. Yabe, T. Nagai, K. Fujita, T. Matsuda, K. Takuma, A. Baba, and H. Hashimoto, “High-speed and scalable whole-brain imaging in rodents and primates,” Neuron 94(6), 1085–1100 (2017).
[Crossref] [PubMed]

Akagi, T.

H. Hama, H. Hioki, K. Namiki, T. Hoshida, H. Kurokawa, F. Ishidate, T. Kaneko, T. Akagi, T. Saito, T. Saido, and A. Miyawaki, “ScaleS: An optical clearing palette for biological imaging,” Nat. Neurosci. 18(10), 1518–1529 (2015).
[Crossref] [PubMed]

Albeanu, D. F.

A. Narasimhan, K. Umadevi Venkataraju, J. Mizrachi, D. F. Albeanu, and P. Osten, “Oblique light-sheet tomography: fast and high resolution volumetric imaging of mouse brains,” bioRxiv (2017).

Ando, R.

H. Hama, H. Kurokawa, H. Kawano, R. Ando, T. Shimogori, H. Noda, K. Fukami, A. Sakaue-Sawano, and A. Miyawaki, “Scale: A chemical approach for fluorescence imaging and reconstruction of transparent mouse brain,” Nat. Neurosci. 14(11), 1481–1488 (2011).
[Crossref] [PubMed]

Ariel, P.

N. Renier, Z. Wu, D. J. Simon, J. Yang, P. Ariel, and M. Tessier-Lavigne, “iDISCO: A simple, rapid method to immunolabel large tissue samples for volume imaging,” Cell 159(4), 896–910 (2014).
[Crossref] [PubMed]

Baba, A.

K. Seiriki, A. Kasai, T. Hashimoto, W. Schulze, M. Niu, S. Yamaguchi, T. Nakazawa, K. I. Inoue, S. Uezono, M. Takada, Y. Naka, H. Igarashi, M. Tanuma, J. A. Waschek, Y. Ago, K. F. Tanaka, A. Hayata-Takano, K. Nagayasu, N. Shintani, R. Hashimoto, Y. Kunii, M. Hino, J. Matsumoto, H. Yabe, T. Nagai, K. Fujita, T. Matsuda, K. Takuma, A. Baba, and H. Hashimoto, “High-speed and scalable whole-brain imaging in rodents and primates,” Neuron 94(6), 1085–1100 (2017).
[Crossref] [PubMed]

Bakh, N.

E. Murray, J. H. Cho, D. Goodwin, T. Ku, J. Swaney, S. Y. Kim, H. Choi, Y. G. Park, J. Y. Park, A. Hubbert, M. McCue, S. Vassallo, N. Bakh, M. P. Frosch, V. J. Wedeen, H. S. Seung, and K. Chung, “Simple, scalable proteomic imaging for high-dimensional profiling of intact systems,” Cell 163(6), 1500–1514 (2015).
[Crossref] [PubMed]

S. Y. Kim, J. H. Cho, E. Murray, N. Bakh, H. Choi, K. Ohn, L. Ruelas, A. Hubbert, M. McCue, S. L. Vassallo, P. J. Keller, and K. Chung, “Stochastic electrotransport selectively enhances the transport of highly electromobile molecules,” Proc. Natl. Acad. Sci. U.S.A. 112(46), E6274–E6283 (2015).
[Crossref] [PubMed]

Bay, M.

B. Zingg, H. Hintiryan, L. Gou, M. Y. Song, M. Bay, M. S. Bienkowski, N. N. Foster, S. Yamashita, I. Bowman, A. W. Toga, and H. W. Dong, “Neural networks of the mouse neocortex,” Cell 156(5), 1096–1111 (2014).
[Crossref] [PubMed]

Becker, K.

A. Ertürk, K. Becker, N. Jährling, C. P. Mauch, C. D. Hojer, J. G. Egen, F. Hellal, F. Bradke, M. Sheng, and H. U. Dodt, “Three-dimensional imaging of solvent-cleared organs using 3DISCO,” Nat. Protoc. 7(11), 1983–1995 (2012).
[Crossref] [PubMed]

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T. Kuwajima, A. A. Sitko, P. Bhansali, C. Jurgens, W. Guido, and C. Mason, “ClearT: A detergent- and solvent-free clearing method for neuronal and non-neuronal tissue,” Development 140(6), 1364–1368 (2013).
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B. Zingg, H. Hintiryan, L. Gou, M. Y. Song, M. Bay, M. S. Bienkowski, N. N. Foster, S. Yamashita, I. Bowman, A. W. Toga, and H. W. Dong, “Neural networks of the mouse neocortex,” Cell 156(5), 1096–1111 (2014).
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H. U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: Three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods 4(4), 331–336 (2007).
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T. Jiang, B. Long, H. Gong, T. Xu, X. Li, Z. Duan, A. Li, L. Deng, Q. Zhong, X. Peng, and J. Yuan, “A platform for efficient identification of molecular phenotypes of brain-wide neural circuits,” Sci. Rep. 7(1), 13891 (2017).
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A. Ertürk, K. Becker, N. Jährling, C. P. Mauch, C. D. Hojer, J. G. Egen, F. Hellal, F. Bradke, M. Sheng, and H. U. Dodt, “Three-dimensional imaging of solvent-cleared organs using 3DISCO,” Nat. Protoc. 7(11), 1983–1995 (2012).
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A. Ertürk, K. Becker, N. Jährling, C. P. Mauch, C. D. Hojer, J. G. Egen, F. Hellal, F. Bradke, M. Sheng, and H. U. Dodt, “Three-dimensional imaging of solvent-cleared organs using 3DISCO,” Nat. Protoc. 7(11), 1983–1995 (2012).
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E. Murray, J. H. Cho, D. Goodwin, T. Ku, J. Swaney, S. Y. Kim, H. Choi, Y. G. Park, J. Y. Park, A. Hubbert, M. McCue, S. Vassallo, N. Bakh, M. P. Frosch, V. J. Wedeen, H. S. Seung, and K. Chung, “Simple, scalable proteomic imaging for high-dimensional profiling of intact systems,” Cell 163(6), 1500–1514 (2015).
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C. Pan, R. Cai, F. P. Quacquarelli, A. Ghasemigharagoz, A. Lourbopoulos, P. Matryba, N. Plesnila, M. Dichgans, F. Hellal, and A. Ertürk, “Shrinkage-mediated imaging of entire organs and organisms using uDISCO,” Nat. Methods 13(10), 859–867 (2016).
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T. Jiang, B. Long, H. Gong, T. Xu, X. Li, Z. Duan, A. Li, L. Deng, Q. Zhong, X. Peng, and J. Yuan, “A platform for efficient identification of molecular phenotypes of brain-wide neural circuits,” Sci. Rep. 7(1), 13891 (2017).
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B. Zingg, H. Hintiryan, L. Gou, M. Y. Song, M. Bay, M. S. Bienkowski, N. N. Foster, S. Yamashita, I. Bowman, A. W. Toga, and H. W. Dong, “Neural networks of the mouse neocortex,” Cell 156(5), 1096–1111 (2014).
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L. Madisen, T. A. Zwingman, S. M. Sunkin, S. W. Oh, H. A. Zariwala, H. Gu, L. L. Ng, R. D. Palmiter, M. J. Hawrylycz, A. R. Jones, E. S. Lein, and H. Zeng, “A robust and high-throughput Cre reporting and characterization system for the whole mouse brain,” Nat. Neurosci. 13(1), 133–140 (2010).
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T. Kuwajima, A. A. Sitko, P. Bhansali, C. Jurgens, W. Guido, and C. Mason, “ClearT: A detergent- and solvent-free clearing method for neuronal and non-neuronal tissue,” Development 140(6), 1364–1368 (2013).
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H. Gong, D. Xu, J. Yuan, X. Li, C. Guo, J. Peng, Y. Li, L. A. Schwarz, A. Li, B. Hu, B. Xiong, Q. Sun, Y. Zhang, J. Liu, Q. Zhong, T. Xu, S. Zeng, and Q. Luo, “High-throughput dual-colour precision imaging for brain-wide connectome with cytoarchitectonic landmarks at the cellular level,” Nat. Commun. 7(1), 12142 (2016).
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K. Seiriki, A. Kasai, T. Hashimoto, W. Schulze, M. Niu, S. Yamaguchi, T. Nakazawa, K. I. Inoue, S. Uezono, M. Takada, Y. Naka, H. Igarashi, M. Tanuma, J. A. Waschek, Y. Ago, K. F. Tanaka, A. Hayata-Takano, K. Nagayasu, N. Shintani, R. Hashimoto, Y. Kunii, M. Hino, J. Matsumoto, H. Yabe, T. Nagai, K. Fujita, T. Matsuda, K. Takuma, A. Baba, and H. Hashimoto, “High-speed and scalable whole-brain imaging in rodents and primates,” Neuron 94(6), 1085–1100 (2017).
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K. Seiriki, A. Kasai, T. Hashimoto, W. Schulze, M. Niu, S. Yamaguchi, T. Nakazawa, K. I. Inoue, S. Uezono, M. Takada, Y. Naka, H. Igarashi, M. Tanuma, J. A. Waschek, Y. Ago, K. F. Tanaka, A. Hayata-Takano, K. Nagayasu, N. Shintani, R. Hashimoto, Y. Kunii, M. Hino, J. Matsumoto, H. Yabe, T. Nagai, K. Fujita, T. Matsuda, K. Takuma, A. Baba, and H. Hashimoto, “High-speed and scalable whole-brain imaging in rodents and primates,” Neuron 94(6), 1085–1100 (2017).
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K. Seiriki, A. Kasai, T. Hashimoto, W. Schulze, M. Niu, S. Yamaguchi, T. Nakazawa, K. I. Inoue, S. Uezono, M. Takada, Y. Naka, H. Igarashi, M. Tanuma, J. A. Waschek, Y. Ago, K. F. Tanaka, A. Hayata-Takano, K. Nagayasu, N. Shintani, R. Hashimoto, Y. Kunii, M. Hino, J. Matsumoto, H. Yabe, T. Nagai, K. Fujita, T. Matsuda, K. Takuma, A. Baba, and H. Hashimoto, “High-speed and scalable whole-brain imaging in rodents and primates,” Neuron 94(6), 1085–1100 (2017).
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L. Madisen, T. A. Zwingman, S. M. Sunkin, S. W. Oh, H. A. Zariwala, H. Gu, L. L. Ng, R. D. Palmiter, M. J. Hawrylycz, A. R. Jones, E. S. Lein, and H. Zeng, “A robust and high-throughput Cre reporting and characterization system for the whole mouse brain,” Nat. Neurosci. 13(1), 133–140 (2010).
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K. Seiriki, A. Kasai, T. Hashimoto, W. Schulze, M. Niu, S. Yamaguchi, T. Nakazawa, K. I. Inoue, S. Uezono, M. Takada, Y. Naka, H. Igarashi, M. Tanuma, J. A. Waschek, Y. Ago, K. F. Tanaka, A. Hayata-Takano, K. Nagayasu, N. Shintani, R. Hashimoto, Y. Kunii, M. Hino, J. Matsumoto, H. Yabe, T. Nagai, K. Fujita, T. Matsuda, K. Takuma, A. Baba, and H. Hashimoto, “High-speed and scalable whole-brain imaging in rodents and primates,” Neuron 94(6), 1085–1100 (2017).
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C. Pan, R. Cai, F. P. Quacquarelli, A. Ghasemigharagoz, A. Lourbopoulos, P. Matryba, N. Plesnila, M. Dichgans, F. Hellal, and A. Ertürk, “Shrinkage-mediated imaging of entire organs and organisms using uDISCO,” Nat. Methods 13(10), 859–867 (2016).
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K. Seiriki, A. Kasai, T. Hashimoto, W. Schulze, M. Niu, S. Yamaguchi, T. Nakazawa, K. I. Inoue, S. Uezono, M. Takada, Y. Naka, H. Igarashi, M. Tanuma, J. A. Waschek, Y. Ago, K. F. Tanaka, A. Hayata-Takano, K. Nagayasu, N. Shintani, R. Hashimoto, Y. Kunii, M. Hino, J. Matsumoto, H. Yabe, T. Nagai, K. Fujita, T. Matsuda, K. Takuma, A. Baba, and H. Hashimoto, “High-speed and scalable whole-brain imaging in rodents and primates,” Neuron 94(6), 1085–1100 (2017).
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B. Zingg, H. Hintiryan, L. Gou, M. Y. Song, M. Bay, M. S. Bienkowski, N. N. Foster, S. Yamashita, I. Bowman, A. W. Toga, and H. W. Dong, “Neural networks of the mouse neocortex,” Cell 156(5), 1096–1111 (2014).
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H. Hama, H. Hioki, K. Namiki, T. Hoshida, H. Kurokawa, F. Ishidate, T. Kaneko, T. Akagi, T. Saito, T. Saido, and A. Miyawaki, “ScaleS: An optical clearing palette for biological imaging,” Nat. Neurosci. 18(10), 1518–1529 (2015).
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A. Ertürk, K. Becker, N. Jährling, C. P. Mauch, C. D. Hojer, J. G. Egen, F. Hellal, F. Bradke, M. Sheng, and H. U. Dodt, “Three-dimensional imaging of solvent-cleared organs using 3DISCO,” Nat. Protoc. 7(11), 1983–1995 (2012).
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K. Seiriki, A. Kasai, T. Hashimoto, W. Schulze, M. Niu, S. Yamaguchi, T. Nakazawa, K. I. Inoue, S. Uezono, M. Takada, Y. Naka, H. Igarashi, M. Tanuma, J. A. Waschek, Y. Ago, K. F. Tanaka, A. Hayata-Takano, K. Nagayasu, N. Shintani, R. Hashimoto, Y. Kunii, M. Hino, J. Matsumoto, H. Yabe, T. Nagai, K. Fujita, T. Matsuda, K. Takuma, A. Baba, and H. Hashimoto, “High-speed and scalable whole-brain imaging in rodents and primates,” Neuron 94(6), 1085–1100 (2017).
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L. Madisen, T. A. Zwingman, S. M. Sunkin, S. W. Oh, H. A. Zariwala, H. Gu, L. L. Ng, R. D. Palmiter, M. J. Hawrylycz, A. R. Jones, E. S. Lein, and H. Zeng, “A robust and high-throughput Cre reporting and characterization system for the whole mouse brain,” Nat. Neurosci. 13(1), 133–140 (2010).
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L. Madisen, T. A. Zwingman, S. M. Sunkin, S. W. Oh, H. A. Zariwala, H. Gu, L. L. Ng, R. D. Palmiter, M. J. Hawrylycz, A. R. Jones, E. S. Lein, and H. Zeng, “A robust and high-throughput Cre reporting and characterization system for the whole mouse brain,” Nat. Neurosci. 13(1), 133–140 (2010).
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T. Jiang, B. Long, H. Gong, T. Xu, X. Li, Z. Duan, A. Li, L. Deng, Q. Zhong, X. Peng, and J. Yuan, “A platform for efficient identification of molecular phenotypes of brain-wide neural circuits,” Sci. Rep. 7(1), 13891 (2017).
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H. Gong, D. Xu, J. Yuan, X. Li, C. Guo, J. Peng, Y. Li, L. A. Schwarz, A. Li, B. Hu, B. Xiong, Q. Sun, Y. Zhang, J. Liu, Q. Zhong, T. Xu, S. Zeng, and Q. Luo, “High-throughput dual-colour precision imaging for brain-wide connectome with cytoarchitectonic landmarks at the cellular level,” Nat. Commun. 7(1), 12142 (2016).
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Figures (6)

Fig. 1
Fig. 1 (a) Schematic of the proposed on-line optical clearing method used for light microscopic imaging of the whole brain. In the on-line optical clearing method, the sample is immersed in the optical clearing solution, and clearing of the sample surface during sectioning. (b) Schematic diagram of serpentine scanning to obtain a complete coronal view and imaging of a thicker layer in the depth direction.
Fig. 2
Fig. 2 Capabilities of the water-soluble reagents for rapid sample clearing. (a) Pure glycerol, saturated sorbitol, sucrose, and fructose solutions were employed to clear 500-μm brain slices on the grid (1 mm). (b) The refractive indices of the fructose solution before and after urea addition. (c) The viscosity of the fructose solution before and after urea addition. (d) The relative transmission rise of a 100-μm brain slice before and after optical clearing for 1 min and 2 min.
Fig. 3
Fig. 3 (a)–(f) and (g)–(l) Images of the brain sample obtained using our previously described WVT system before and after clearing at intervals of 20 μm. The root mean square values of the images were used to define the image quality. (m) The curve for root mean square attenuation with depth by Gaussian fitting.
Fig. 4
Fig. 4 (a) and (b) Intensities of the fluorescence signals from GFP-labeled brain slices before and after clearing for 1 min, respectively. (c) Average cell brightness values in the areas marked with white dotted circles, and normalized by the sample brightness before clearing.
Fig. 5
Fig. 5 Registration maps of the upper and subsequent layers after cutting and clearing. (a) Schematic of the imaging and cutting thickness. (b) A coronal view of the redundant area before cutting. (c) The corrected coronal view of the corresponding area after cutting. (d) A merged map of the foreground color of (b) set to red and the foreground color of (c) set to green. (e), (f), and (g) Enlarged views of the red areas in (b), (c), and (d), respectively. (h), (i), and (j) Medial-lateral, dorsal-ventral, and anterior-posterior axis displacement from the upper layer to the next layer before and after cutting and clearing, respectively.
Fig. 6
Fig. 6 A complete brain data set for an adult Thy1-GFP mouse. (a) Outline of the adult (8-week-old) Thy1-GFP mouse brain. (b)–(h) 10 μm coronal slices of the mouse brain at the corresponding positions (b)–(h), respectively. The coronal slices are displayed at interval of 2 mm.

Tables (1)

Tables Icon

Table 1 Times required both with and without clearing

Metrics