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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

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).

Author information

Correspondence to Nobuhiko Ohno.

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Supplementary material 3 (MPG 7984 kb)

Supplementary material 4 (MPG 5560 kb)

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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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Keywords

  • Serial block-face scanning electron microscopy
  • Tissue preparation
  • High throughput
  • Uranyl acetate