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Freeze Stabilization and Cryopreparation Technique for Visualizing the Water Distribution in Woody Tissues by X-Ray Imaging and Cryo-scanning Electron Microscopy

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Electron Microscopy

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1117))

Abstract

The protocol of freeze stabilization and cryopreparation techniques to examine the distribution of water in living tree stems by X-ray imaging and cryo-scanning electron microscopy have been developed and described. The brief procedures are as follows. Firstly, a portion of transpiring stem is frozen in the standing state with liquid nitrogen to stabilize the water that is present in the conducting tissue. After filling with liquid nitrogen, discs are then collected from the frozen portion of the stem and stored in liquid nitrogen. In a low-temperature room, the samples for X-ray imaging are sectioned with a fine handsaw, and trimmed sample blokes for cryo-scanning electron microscopy are cleanly planed using a sliding microtome. Finally, the frozen sections are irradiated in a soft X-ray apparatus, and the small blocks are examined in cryo-scanning electron microscope after freeze-etching and metal coating.

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Acknowledgments

We wish to thank Dr. Seizo Fujikawa for explanations of the basics of cryo-SEM, Dr. Ryo Funada and the late Dr. Jun Ohtani for their helpful comments, Dr. Takayuki Shiraiwa, Dr. Keita Arakawa, Dr. Daisuke Takezawa, and the late Dr. Takuya Fukuzawa for use of the low-temperature room at the Institute of Low Temperature Science of Hokkaido University, and Mr. Toshiaki Itoh, Mr. Kunio Shinbori, and Mr. Shigeo Kon’no for their technical support.

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Authors and Affiliations

  1. Kyushu Research Forest, Graduate School of Agriculture, Kyushu University, Miyazaki, Kyushu, Japan

    Yasuhiro Utsumi

  2. Laboratory of Wood Biology, Graduate School of Agriculture, Hokkaido University, Sapporo, Japan

    Yuzou Sano

Authors
  1. Yasuhiro Utsumi
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  2. Yuzou Sano
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Editor information

Editors and Affiliations

  1. University of Western Australia, Crawley, Australia

    John Kuo

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Utsumi, Y., Sano, Y. (2014). Freeze Stabilization and Cryopreparation Technique for Visualizing the Water Distribution in Woody Tissues by X-Ray Imaging and Cryo-scanning Electron Microscopy. In: Kuo, J. (eds) Electron Microscopy. Methods in Molecular Biology, vol 1117. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-776-1_30

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  • DOI: https://doi.org/10.1007/978-1-62703-776-1_30

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-775-4

  • Online ISBN: 978-1-62703-776-1

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