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Rapid specimen preparation to improve the throughput of electron microscopic volume imaging for three-dimensional analyses of subcellular ultrastructures with serial block-face scanning electron microscopy

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Abstract

Serial block-face imaging using scanning electron microscopy enables rapid observations of three-dimensional ultrastructures in a large volume of biological specimens. However, such imaging usually requires days for sample preparation to reduce charging and increase image contrast. In this study, we report a rapid procedure to acquire serial electron microscopic images within 1 day for three-dimensional analyses of subcellular ultrastructures. This procedure is based on serial block-face with two major modifications, including a new sample treatment device and direct polymerization on the rivets, to reduce the time and workload needed. The modified procedure without uranyl acetate can produce tens of embedded samples observable under serial block-face scanning electron microscopy within 1 day. The serial images obtained are similar to the block-face images acquired by common procedures, and are applicable to three-dimensional reconstructions at a subcellular resolution. Using this approach, regional immune deposits and the double contour or heterogeneous thinning of basement membranes were observed in the glomerular capillary loops of an autoimmune nephropathy model. These modifications provide options to improve the throughput of three-dimensional electron microscopic examinations, and will ultimately be beneficial for the wider application of volume imaging in life science and clinical medicine.

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Acknowledgments

This work was partly supported by JSPS KAKENHI Grant Number 25870281 (to SS) and 26860129 (to NO), Comprehensive Brain Science Network (CBSN) (to NO) and The Cooperative Study Programs of National Institute for Physiological Sciences (to NO and SS).

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Authors and Affiliations

  1. Department of Anatomy and Molecular Histology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 1110 Shimokato, Chuo-City, Yamanashi, 409-3898, Japan

    Truc Quynh Thai, Huy Bang Nguyen, Sei Saitoh, Bao Wu, Yurika Saitoh & Nobuhiko Ohno

  2. Department of Occupational Therapy, Health Science University, 7187 Kodachi, Fujikawaguchiko, Yamanashi, 401-0380, Japan

    Satoshi Shimo

  3. Department of Histology and Cytology, Faculty of Veterinary Medicine, Zagazig University, Zagazig, 44519, Egypt

    Yaser Hosny Ali Elewa

  4. Laboratory of Anatomy, Department of Biomedical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo City, Hokkaido, 060-0818, Japan

    Yaser Hosny Ali Elewa, Osamu Ichii & Yasuhiro Kon

  5. Department of Pathology, Tohoku University, Sendai, Miyagi, 980-8575, Japan

    Takashi Takaki & Kensuke Joh

  6. Center for Multidisciplinary Brain Research, National Institute for Physiological Sciences, Okazaki, Aichi, 444-8787, Japan

    Nobuhiko Ohno

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  1. Truc Quynh Thai
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  2. Huy Bang Nguyen
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  8. Osamu Ichii
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  9. Yasuhiro Kon
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  10. Takashi Takaki
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  11. Kensuke Joh
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  12. Nobuhiko Ohno
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Corresponding author

Correspondence to Nobuhiko Ohno.

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Thai, T.Q., Nguyen, H.B., Saitoh, S. et al. Rapid specimen preparation to improve the throughput of electron microscopic volume imaging for three-dimensional analyses of subcellular ultrastructures with serial block-face scanning electron microscopy. Med Mol Morphol 49, 154–162 (2016). https://doi.org/10.1007/s00795-016-0134-7

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  • DOI: https://doi.org/10.1007/s00795-016-0134-7

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