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

Nature Neuroscience volume 14, pages 14811488 (2011) | Download Citation

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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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  • 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.


  1. 1.

    et al. Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature 450, 56–62 (2007).

  2. 2.

    & Array tomography: a new tool for imaging the molecular architecture and ultrastructure of neural circuits. Neuron 55, 25–36 (2007).

  3. 3.

    & Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure. PLoS Biol. 2, e329 (2004).

  4. 4.

    , & 3D structural imaging of the brain with photons and electrons. Curr. Opin. Neurobiol. 18, 633–641 (2008).

  5. 5.

    et al. Anatomical and functional imaging of neurons using 2-photon laser scanning microscopy. J. Neurosci. Methods 54, 151–162 (1994).

  6. 6.

    & Photon upmanship: why multiphoton imaging is more than a gimmick. Neuron 18, 351–357 (1997).

  7. 7.

    , & Nonlinear magic: multiphoton microscopy in the biosciences. Nat. Biotechnol. 21, 1369–1377 (2003).

  8. 8.

    & Deep tissue two-photon microscopy. Nat. Methods 2, 932–940 (2005).

  9. 9.

    & On the fundamental imaging-depth limit in two-photon microscopy. J. Opt. Soc. Am. A Opt. Image Sci. Vis. 23, 3139–3149 (2006).

  10. 10.

    et al. Insect NMDA receptors mediate juvenile hormone biosynthesis. Proc. Natl. Acad. Sci. USA 99, 37–42 (2002).

  11. 11.

    & High-resolution confocal imaging and three-dimensional rendering. Methods 30, 86–93 (2003).

  12. 12.

    , , , & A map of olfactory representation in the Drosophila mushroom body. Cell 128, 1205–1217 (2007).

  13. 13.

    et al. Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain. Nat. Methods 4, 331–336 (2007).

  14. 14.

    , & 3D-reconstruction of blood vessels by ultramicroscopy. Organogenesis 5, 145–148 (2009).

  15. 15.

    , , & Multiphoton microscopy of cleared mouse organs. J. Biomed. Opt. 15, 036017 (2010).

  16. 16.

    et al. Correlations of neuronal and microvascular densities in murine cortex revealed by direct counting and colocaliztion of nuclei and vessels. J. Neurosci. 29, 14553–14570 (2009).

  17. 17.

    et al. Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Neuron 28, 41–51 (2000).

  18. 18.

    , , & Clascá, F. Mapping of fluorescent protein–expressing neurons and axon pathways in adult and developing Thy1-eYFP-H transgenic mice. Brain Res. 1345, 59–72 (2010).

  19. 19.

    , & Effect of pH on thermal- and chemical-induced denaturation of GFP. Appl. Biochem. Biotechnol. 126, 149–156 (2005).

  20. 20.

    , & Two-photon molecular excitation in laser scanning microscopy. in The Handbook of Confocal Microscopy (ed. J. Pawley) 445–458 (Plenum Press, New York, 1995).

  21. 21.

    , & Commissure formation in the mammalian forebrain. Curr. Opin. Neurobiol. 17, 3–14 (2007).

  22. 22.

    et al. Activity-dependent development of callosal projections in the somatosensory cortex. J. Neurosci. 27, 11334–11342 (2007).

  23. 23.

    & Gene application with in utero electroporation in mouse embryonic brain. Dev. Growth Differ. 50, 499–506 (2008).

  24. 24.

    et al. A specialized vascular niche for adult neural stem cells. Cell Stem Cell 3, 279–288 (2008).

  25. 25.

    et al. Adult SVZ stem cells lie in a vascular niche: a quantitative analysis of niche cell-cell interactions. Cell Stem Cell 3, 289–300 (2008).

  26. 26.

    , , & Visualization of neurogenesis in the central nervous system using nestin promoter–GFP transgenic mice. Neuroreport 11, 1991–1996 (2000).

  27. 27.

    et al. Visualizing spatiotemporal dynamics of multicellular cell-cycle progression. Cell 132, 487–498 (2008).

  28. 28.

    et al. Generation of transgenic non-human primates with germline transmission. Nature 459, 523–527 (2009).

  29. 29.

    , & Mechanisms regulating the development of the corpus callosam and its agenesis in mouse and human. Clin. Genet. 66, 276–289 (2004).

  30. 30.

    , , & Sulforhodamine 101 as a specific marker of astroglia in the neocortex in vivo. Nat. Methods 1, 31–37 (2004).

  31. 31.

    , , , & Diffusion and imaging properties of three new lipophilic tracers, NeuroVue Maroon, NeuroVue Red and NeuroVue Green and their use for double and triple labeling of neuronal profile. Brain Res. Bull. 66, 249–258 (2005).

  32. 32.

    , & Alteration of neuronal firing properties after in vivo experience in a FosGFP transgenic mouse. J. Neurosci. 24, 6466–6475 (2004).

  33. 33.

    et al. In vivo two-photon imaging reveals a role of Arc in enhancing orientation specificity in visual cortex. Cell 126, 389–402 (2006).

  34. 34.

    , & Orthogonal-plane fluorescence optical sectioning: three-dimensional imaging of macroscopic biological specimens. J. Microsc. 170, 229–236 (1993).

  35. 35.

    , , , & Optical sectioning deep inside live embryos by selective plane illumination microscopy. Science 305, 1007–1009 (2004).

  36. 36.

    et al. Volumetric tomography of fluorescent proteins through small animals in vivo. Proc. Natl. Acad. Sci. USA 102, 18252–18257 (2005).

  37. 37.

    et al. Optical projection tomography as a tool for 3D microscopy and gene expression studies. Science 296, 541–545 (2002).

  38. 38.

    Optical projection tomography as a new tool for studying embryo anatomy. J. Anat. 202, 175–181 (2003).

  39. 39.

    & Ome sweet ome: what can the genome tell us about the connectome? Curr. Opin. Neurobiol. 18, 346–353 (2008).

  40. 40.

    & Seeing circuits assemble. Neuron 60, 441–448 (2008).

  41. 41.

    Reading the book of memory: sparse sampling versus dense mapping of connectomes. Neuron 62, 17–29 (2009).

  42. 42.

    Contrast enhancement in light microscopy. Curr. Protoc. Cytom. 2.1.1–2.1.11. (2001).

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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


  1. Brain Science Institute, RIKEN, Wako-city, Saitama, Japan.

    • Hiroshi Hama
    • , Hiroshi Kurokawa
    • , Hiroyuki Kawano
    • , Ryoko Ando
    • , Tomomi Shimogori
    • , Hisayori Noda
    • , Asako Sakaue-Sawano
    •  & Atsushi Miyawaki
  2. School of Life Science, Tokyo University of Pharmacy and Life Science, Hachioji, Tokyo, Japan.

    • Hiroshi Kurokawa
    •  & Kiyoko Fukami
  3. Life Function and Dynamics, Exploratory Research for Advanced Technology, Japan Science and Technology Agency, Wako-city, Saitama, Japan.

    • Hiroyuki Kawano
    • , Asako Sakaue-Sawano
    •  & Atsushi Miyawaki
  4. Tokyo Institute of Technology, Meguro-ku, Tokyo, Japan.

    • Hisayori Noda


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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.

Competing interests

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

Corresponding author

Correspondence to Atsushi Miyawaki.

Supplementary information

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  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–9 and Table 1


  1. 1.

    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.

  2. 2.

    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.

  3. 3.

    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.

  4. 4.

    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.

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