Good Ultrastructural Preservation of Human Tissues and Cultured Cells by Glutaraldehyde Fixation, Sandwich Freezing, and Freeze-Substitution

Published on Mar 25, 2020in Cytologia0.795
· DOI :10.1508/CYTOLOGIA.85.15
Masashi Yamaguchi20
Estimated H-index: 20
(Chiba University),
Seiichiro Wakabayashi2
Estimated H-index: 2
(Chiba University)
+ 8 AuthorsHiroji Chibana24
Estimated H-index: 24
(Chiba University)
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Abstract
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Observing cells and cell components in three dimensions at high magnification in transmission electron microscopy requires preparing serial ultrathin sections of the specimen. Although preparing serial ultrathin sections is considered to be very difficult, it is rather easy if the proper method is used. In this paper, we show a step-by-step procedure for safely obtaining serial ultrathin sections of microorganisms. The key points of this method are: 1) to use the large part of the specimen and a...
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// Shigeki Aoki 1, * , Michie Morita 1, * , Takuya Hirao 1 , Masashi Yamaguchi 2 , Reika Shiratori 1 , Megumi Kikuya 1 , Hiroji Chibana 2 and Kousei Ito 1 1 Laboratory of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba-city, Chiba 260-8675, Japan 2 Medical Mycology Research Center, Chiba University, Chiba-city, Chiba 260-8673, Japan * These authors have contributed equally to this work Correspondence to: Shigeki Aoki, email: aokishigeki@chiba-u.jp Keywords: ...
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: Structome analysis, the quantitative three-dimensional structural analysis of whole cells at the electron microscopic level, of Exophiala dermatitidis (black yeast), Saccharomyces cerevisiae, Mycobacterium tuberculosis (MTB) and Myojin spiral bacteria (MSB) have already been reported. Here, the results of the structome analysis of Escherichia coli cells based on transmission electron microscope observation of serial ultrathin sections was reported, and compared with the data obtained from phas...
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: Structome analysis is a useful tool for identification of unknown microorganisms that cannot be cultured. In 2012, we discovered a unique deep-sea microorganism with a cell structure intermediate between those of prokaryotes and eukaryotes and described its features using freeze-substitution electron microscopy and structome analysis (quantitative and three-dimensional structural analysis of a whole cell at the electron microscopic level). We named it Myojin parakaryote Here we describe, using...
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We have been using sandwich freezing of living yeast and bacteria followed by freeze-substitution for observing close-to-native ultrastructure of cells. Recently, sandwich freezing of glutaraldehyde-fixed cultured cells and human tissues have been found to give excellent preservation of ultrastructure of cells and tissues. These studies, however, have been conducted using a handmade sandwich freezing device and have been limited in a few laboratories. To spread the use of this method to other la...
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