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Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain

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Optical methods for viewing neuronal populations and projections in the intact mammalian brain are needed, but light scattering prevents imaging deep into brain structures. We imaged fixed brain tissue using Scale, an aqueous reagent that renders biological samples optically transparent but completely preserves fluorescent signals in the clarified structures. In Scale-treated mouse brain, neurons labeled with genetically encoded fluorescent proteins were visualized at an unprecedented depth in millimeter-scale networks and at subcellular resolution. The improved depth and scale of imaging permitted comprehensive three-dimensional reconstructions of cortical, callosal and hippocampal projections whose extent was limited only by the working distance of the objective lenses. In the intact neurogenic niche of the dentate gyrus, Scale allowed the quantitation of distances of neural stem cells to blood vessels. Our findings suggest that the Scale method will be useful for light microscopy–based connectomics of cellular networks in brain and other tissues.

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Figure 1: Tissue clearing performance of ScaleA2.
Figure 2: Comparison of ScaleA2 with BABB.
Figure 3: Three-dimensional reconstructions of YFP-expressing neurons in ScaleA2-treated brain samples of YFP-H mice.
Figure 4: Visualization of labeled callosal connections in the intact mouse brain.
Figure 5: Quantitation of the distances between proliferating neural stem cell (PNSC) nuclei and blood vessels in the subgranular zone (SGZ) of adult mice.
Figure 6: Three-dimensional reconstructions of Fucci transgenic mouse embryos treated with ScaleU2.
Figure 7: Immunohistochemistry on sections restored from ScaleA2.

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  • 13 October 2011

    In the HTML version of this article initially published online, Greek μ characters were misformatted as the letter m and a prime sign was omitted. The errors have been corrected in the HTML version of this article.


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We thank H. Sakurai, H. Otsuka and M. Hirano for general assistance, F. Ishidate, B. Zimmermann, R. Wolleschensky, Y. Watanabe, E. Nakasho, H. Kimura, T. Tajima and S. Horie for help with acquiring and analyzing images, RIKEN BSI-Olympus Collaboration Center for technical support, Y. Yoshihara (RIKEN), M. Yamaguchi and K. Mori (The University of Tokyo) for the Nestin promoter–GFP transgenic mice, J.R. Sanes (Harvard) for the YFP-H and GFP-M lines, E. Takahashi (RIKEN) for helpful advice on transgenic mice, S. J. Smith (Stanford) and J.W. Lichtman (Harvard) for helpful advice on tissue clearing, and D. Mou (Harvard), A. Govindarajan, K. Rockland and S. Tonegawa (Massachusetts Institute of Technology), A. Moore and C. Yokoyama (RIKEN) for critical comments. This work was partly supported by grants from Japan Ministry of Education, Culture, Sports, Science and Technology Grant-in-Aid for Scientific Research on Priority Areas and the Human Frontier Science Program.

Author information

Authors and Affiliations



H.H. and A.M. conceived and designed the study. H.H. performed all the experiments and analyzed the data. H. Kurokawa devised the algorithms and analyzed the data. H. Kawano constructed the TPEFM system. R.A. performed in vitro experiments using fluorescent proteins. T.S. designed and performed the experiments that imaged callosal connections. H.N. refined the algorithms. K.F. contributed to data analysis. A.S.-S. performed the experiments using Fucci transgenic mouse embryos. A.M. supervised the project and wrote the manuscript with the help of H.H.

Corresponding author

Correspondence to Atsushi Miyawaki.

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Competing interests

H. Hama, H. Kurokawa, H. Kawano, R. Ando and A. Miyawaki hold the patent for the Scale technique.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–9 and Table 1 (PDF 2935 kb)

Supplementary Video 1

Visualizing the 3D architecture of neuronal networks comprised of YFP-expressing neurons in a long quadratic prism (2 mm). A series of X − Y images through the 3D reconstruction data (500 × 500 × 2,000 μm volume) from the cerebral surface to the hippocampus of the YFP-H mouse (13 weeks old). TPEFM with a non-descanned detector and a 20× objective (NA 1.0, WD 2.0 mm) was used. (MPG 5166 kb)

Supplementary Video 2

Visualizing the 3D architecure of neuronal networks comprised of YFP-expressing neurons in a very long quadratic prism (4 mm). A series of X−Y images through the 3D reconstruction data (500 × 500 × 4,000 μm volume) from the cerebral surface to the dentate gyrus of the YFP-H mouse (13 weeks old). TPEFM with a non-descanned detector and a custom designed 25× objective lens (NA 1.0, WD 4.0 mm) was used. (MPG 11434 kb)

Supplementary Video 3

YFP-labeled pyramidal neurons in layers II and III in the right hemisphere and their callosal axons travelling into the left hemisphere. A series of X−Y images through the 3D reconstruction data (10 × 10 × 0.75 mm volume) from anterior to posterior of a brain (10 days old) containing the corpus callosum. A population of layer II/III pyramidal neurons on the right side is highlighted with EYFP fluorescence. A macro zoom confocal microscopy system was used. (MPG 2234 kb)

Supplementary Video 4

Nuclei of proliferating neural stem cells exclusively localized in the subgranular zone in association with a network of blood vessels. Animation (zooming in) of 3D image data (500 × 500 × 1,400 μm volume) in the hippocampal dentate gyrus of a #504 adult (7 weeks old) mouse extensively labeled with Texas Red-labeled lectin. Red, blood vessels; Green, nuclei of proliferating neural stem cells (PNSC) emitting mAG-hGem(1/110) fluorescence. TPEFM with two non-descanned detectors was used. (MPG 2794 kb)

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Hama, H., Kurokawa, H., Kawano, H. et al. Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain. Nat Neurosci 14, 1481–1488 (2011).

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