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Whole-brain block-face serial microscopy tomography at subcellular resolution using FAST

Nature Protocols volume 14, pages 1509–1529 (2019)Cite this article

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Abstract

Here, we describe an optimized and detailed protocol for block-face serial microscopy tomography (FAST). FAST enables high-speed serial section fluorescence imaging of fixed brains at an axonal spatial resolution and subsequent image data processing. It renders brain-wide anatomical and functional analyses, including structural profiling of nuclear-stained brain at the single-cell level, cell-type-specific mapping with reporter animal brains and neuronal tracing with anterograde/retrograde labeling. Light-sheet fluorescence microscopy of cleared brains is advantageous in regard to imaging speed, but its spatial resolution is generally limited, whereas the opposite is true for conventional confocal microscopy. FAST offers a solution to overcome these technical limitations. This protocol describes detailed procedures for assembling the FAST hardware, sample preparation, imaging and image processing. A single imaging session takes as little as 2.4 h per mouse brain, and sample preparation requires 1 to several days, depending on pretreatments; however, multiple samples can be prepared simultaneously. We anticipate that FAST will contribute to unbiased and hypothesis-free approaches for a better understanding of brain systems.

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Fig. 1: Overview of FAST imaging analyses.
Fig. 2: Assembling FAST hardware.
Fig. 3: Design of the sample chamber.
Fig. 4: FAST imaging procedures.
Fig. 5: Whole-brain imaging of nuclear-stained brain at a voxel resolution of 2.5 µm3.
Fig. 6: Chemical staining of a whole mouse brain and multicolor imaging.
Fig. 7: Whole-brain imaging of a fluorescent-reporter animal brain: Arc-dVenus mouse.
Fig. 8: Whole-brain imaging of axonal projections from the anterior cingulate cortex.
Fig. 9: Multicolor whole-brain imaging with two different fluorescent proteins whose expression is driven by the cell-type-specific promoter and enhancer.

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Acknowledgements

We are grateful to S. Yamaguchi (Gifu University) for providing Arc-dVenus-reporter mice; to K.-I. Inoue (Kyoto University) and M. Takada (Kyoto University) for providing and supporting the AAV-CMV-tdTomato vectors and the plasmid vector to express the AAV-PHP.eB capsid; to A. Yamanaka (Nagoya University) for providing the AAV plasmid vector, including the mouse alpha-CaMKII promoter; to G. Fishell (Harvard Medical School) for providing the pAAV-mDlx-GFP-Fishell-1 plasmid vector (AAV-mDLX-EGFP in Fig. 9) (Addgene, plasmid no. 83900)31; to K. Fujita (Osaka University) and T. Nagai (Osaka University) for helpful suggestions on the setup of the FAST apparatus; and to T. Hashimoto (Shizuoka University) for valuable support in image data processing. We also thank T. Funato (Nikon Instech), M. Sato (COMS), H. Tanaka (Yokogawa Electric), O. Kunitaki (Andor Technology) and S. Kameishi (Dosaka EM) for their valuable suggestions and support. This work was supported in part by JSPS KAKENHI, grant nos. JP17H06842 (K.S.), JP18K19498 (K.S.), JP17H05054 (A.K.) and JP17H03989 (H.H.); the JSPS Research Fellowships for Young Scientists, grant no. JP18J10350 (M.N.); MEXT KAKENHI, grant nos. JP18H05416 (H.H.) and JP18H05132 (A.K.); AMED, grant nos. JP18dm0107122h (H.H.), JP18dm0207061h (H.H.) and JP18am0101084; and grants from the Takeda Science Foundation, Japan (A.K.).

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Authors and Affiliations

  1. Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan

    Kaoru Seiriki, Atsushi Kasai, Takanobu Nakazawa, Misaki Niu, Yuichiro Naka, Masato Tanuma, Hisato Igarashi, Kosei Yamaura, Atsuko Hayata-Takano, Yukio Ago & Hitoshi Hashimoto

  2. Interdisciplinary Program for Biomedical Sciences, Institute for Transdisciplinary Graduate Degree Programs, Osaka University, Osaka, Japan

    Kaoru Seiriki

  3. Department of Pharmacology, Graduate School of Dentistry, Osaka University, Osaka, Japan

    Takanobu Nakazawa

  4. Molecular Research Center for Children’s Mental Development, United Graduate School of Child Development, Osaka University, Kanazawa University, Hamamatsu University School of Medicine, Chiba University and University of Fukui, Osaka, Japan

    Atsuko Hayata-Takano & Hitoshi Hashimoto

  5. Laboratory of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan

    Yukio Ago

  6. Division of Bioscience, Institute for Datability Science, Osaka University, Osaka, Japan

    Hitoshi Hashimoto

  7. Transdimensional Life Imaging Division, Institute for Open and Transdisciplinary Research Initiatives, Osaka University, Osaka, Japan

    Hitoshi Hashimoto

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  1. Kaoru Seiriki
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Contributions

Conceptualization, K.S., A.K. and H.H.; methodology, K.S., A.K., T.N. and H.H.; sample preparation and imaging, K.S., A.K., M.N., Y.N., M.T., H.I. and K.Y.; investigation, K.S., A.K., M.N., A.H.-T., Y.A. and H.H.; writing, K.S., A.K., T.N. and H.H.; funding acquisition, K.S., A.K., M.N. and H.H.

Corresponding authors

Correspondence to Atsushi Kasai or Hitoshi Hashimoto.

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The authors declare no competing interests.

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Journal peer review information: Nature Protocols thanks Pavel Osten and other anonymous reviewer(s) for their contribution to the peer review of this work.

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Key reference using this protocol

Seiriki, K. et al. Neuron 94, 1085–1100.e6 (2017): https://doi.org/10.1016/j.neuron.2017.05.017

Integrated supplementary information

Supplementary Figure 1 Example of imaging software settings for whole-brain imaging.

An example of the imaging protocol in Andor iQ software. Trigger out signal and reloading protocol must be included for the subsequent sectioning procedure and automatic repetition using the CP-700 program. In addition to this protocol setup, the external start mode is required to receive the start signal from the CP-700 program after sectioning. The mechanical laser shutter must be kept open for high-speed imaging; ‘561 ShutterKeepOpen’ is a user-designated protocol to keep the shutter of the 561-nm laser open.

Supplementary information

Supplementary Text and Figures

Supplementary Figure 1, Supplementary Tables 1 and 2

Reporting Summary

Supplementary Data

FASTitcher scripts, along with a user guide and a small sample dataset.

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Seiriki, K., Kasai, A., Nakazawa, T. et al. Whole-brain block-face serial microscopy tomography at subcellular resolution using FAST. Nat Protoc 14, 1509–1529 (2019). https://doi.org/10.1038/s41596-019-0148-4

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