Technical Report | Published:

ScaleS: an optical clearing palette for biological imaging

Nature Neuroscience volume 18, pages 15181529 (2015) | Download Citation


Optical clearing methods facilitate deep biological imaging by mitigating light scattering in situ. Multi-scale high-resolution imaging requires preservation of tissue integrity for accurate signal reconstruction. However, existing clearing reagents contain chemical components that could compromise tissue structure, preventing reproducible anatomical and fluorescence signal stability. We developed ScaleS, a sorbitol-based optical clearing method that provides stable tissue preservation for immunochemical labeling and three-dimensional (3D) signal rendering. ScaleS permitted optical reconstructions of aged and diseased brain in Alzheimer's disease models, including mapping of 3D networks of amyloid plaques, neurons and microglia, and multi-scale tracking of single plaques by successive fluorescence and electron microscopy. Human clinical samples from Alzheimer's disease patients analyzed via reversible optical re-sectioning illuminated plaque pathogenesis in the z axis. Comparative benchmarking of contemporary clearing agents showed superior signal and structure preservation by ScaleS. These findings suggest that ScaleS is a simple and reproducible method for accurate visualization of biological tissue.

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We thank H. Sakurai for general assistance, RIKEN BSI-Olympus Collaboration Center for technical support, K. Higuchi, K. Ishihara, D. Nishiwaki, Y. Ue, K. Okazaki, A. Yaguchi, Y. Sato, H. Sakuma, K. Koga and B. Zimmermann for help with acquiring and analyzing LM images, K. Okamoto-Furuta and H. Kohda (Division of Electron Microscopic Study, Center for Anatomical Studies, Graduate School of Medicine, Kyoto University) for their technical assistance in EM, G. Augustine (Duke University) for the ChR2-YFP Tg mice, J.R. Sanes (Harvard University) for the YFP-H line, R. Takahashi, Dr. K. Sohya and T. Tsumoto for advice on Tg mice, and C. Yokoyama and A. Terashima for critical reading and editing of the manuscript. This work was supported in part by grants from the Japan Ministry of Education, Culture, Sports, Science and Technology Grant-in-Aid for Scientific Research on Priority Areas, the Human Frontier Science Program, and the Brain Mapping by Integrated Neurotechnologies for Disease Studies (Brain/MINDS) from Japan Agency for Medical Research and development, AMED.

Author information


  1. Laboratory for Cell Function Dynamics, Brain Science Institute, RIKEN, Wako-city, Saitama, Japan.

    • Hiroshi Hama
    • , Kana Namiki
    • , Hiroshi Kurokawa
    • , Fumiyoshi Ishidate
    •  & Atsushi Miyawaki
  2. Department of Morphological Brain Science, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-Cho, Sakyo-ku, Kyoto, Japan.

    • Hiroyuki Hioki
    •  & Takeshi Kaneko
  3. Biotechnological Optics Research Team, Center for Advanced Photonics, RIKEN, Wako-city, Saitama, Japan.

    • Tetsushi Hoshida
    •  & Atsushi Miyawaki
  4. Support Unit for Animal Resources Development, Brain Science Institute, RIKEN, Wako-city, Saitama, Japan.

    • Takumi Akagi
  5. Laboratory for Proteolytic Neuroscience, Brain Science Institute, RIKEN, Wako-city, Saitama, Japan.

    • Takashi Saito
    •  & Takaomi Saido


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A.M., H. Hama, H. Hioki and T.H. conceived the study. A.M., H. Hama, K.N., H. Hioki, T.H. and H.K. planned and executed the LM experiments and analyzed the data. H. Hioki, T.K. and T.A. planned and executed the EM experiments. H.K. devised the algorithms for data analysis. F.I. contributed to image data acquisition. T. Saido and T. Saito supervised the experiments that used AD mouse model and patient brains. A.M. wrote the manuscript and supervised the project.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Atsushi Miyawaki.

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    Supplementary Text and Figures

    Supplementary Figures 1–12 and Supplementary Tables 1–3

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    Supplementary Methods Checklist


  1. 1.

    3D visualization of the spatial association between plaques and blood vessels.

    Animation (zooming in and out) of 3D image data (580 × 580 × 860 mm volume) in the cereberal cortex of a 20-month-old AppNL-F/NL-F mouse. The entire vasculature was labelled with Texas Red-labeled lectin. Aβ plaques in the left hemisphere were immunolabelled with AbScale using Alexa488-6E10. Red, blood vessels; Green, Aβ plaques. Images were acquired using SPIM. Backward and forward perspective images were created at different depths and from different angles. See Fig. 4d.

  2. 2.

    Rapid clarification of brain slices by ScaleSQ.

    Time-lapse imaging of coronal slices (1 mm thick) prepared from an 8-week-old YFP-H mice during the incubation in ScaleSQ(0) and ScaleSQ(5) at 37 °C. Transmitted-light bright-field images were acquired with a digital single lens reflex camera (Nikon). See Fig. 7.

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