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Chromosome Interior Observation by Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM) Using Ionic Liquid Technique

Published online by Cambridge University Press:  10 July 2014

Tohru Hamano
Affiliation:
Laboratory of Dynamic Cell Biology, Department of Biotechnology, Graduate School of Engineering, Osaka University, Yamadaoka, Suita, Osaka 565-0871, Japan
Astari Dwiranti
Affiliation:
Laboratory of Dynamic Cell Biology, Department of Biotechnology, Graduate School of Engineering, Osaka University, Yamadaoka, Suita, Osaka 565-0871, Japan
Kohei Kaneyoshi
Affiliation:
Laboratory of Dynamic Cell Biology, Department of Biotechnology, Graduate School of Engineering, Osaka University, Yamadaoka, Suita, Osaka 565-0871, Japan
Shota Fukuda
Affiliation:
Laboratory of Dynamic Cell Biology, Department of Biotechnology, Graduate School of Engineering, Osaka University, Yamadaoka, Suita, Osaka 565-0871, Japan
Reo Kometani
Affiliation:
Laboratory of Nano Mechanics, Department of Mechanical Engineering, Graduate School of Engineering, The University of Tokyo, Hongo, Bunkyo, Tokyo 113-8685, Japan
Masayuki Nakao
Affiliation:
Department of Engineering Synthesis, Graduate School of Engineering, The University of Tokyo, Hongo, Bunkyo, Tokyo 113-8685, Japan
Hideaki Takata
Affiliation:
Laboratory of Dynamic Cell Biology, Department of Biotechnology, Graduate School of Engineering, Osaka University, Yamadaoka, Suita, Osaka 565-0871, Japan Frontier Research Base for Global Young Researchers, Graduate School of Engineering, Osaka University, Yamadaoka, Suita,Osaka 565-0871, Japan
Susumu Uchiyama
Affiliation:
Laboratory of Dynamic Cell Biology, Department of Biotechnology, Graduate School of Engineering, Osaka University, Yamadaoka, Suita, Osaka 565-0871, Japan
Nobuko Ohmido
Affiliation:
Department of Human Environmental Science, Division of Living Environment, Graduate School of Human Development and Environment, Kobe University, Tsurukabuto, Nada, Kobe 657-8501, Japan.
Kiichi Fukui*
Affiliation:
Laboratory of Dynamic Cell Biology, Department of Biotechnology, Graduate School of Engineering, Osaka University, Yamadaoka, Suita, Osaka 565-0871, Japan
*
*Corresponding author. kfukui@bio.eng.osaka-u.ac.jp

Abstract

Attempts to elucidate chromosome structure have long remained elusive. Electron microscopy is useful for chromosome structure research because of its high resolution and magnification. However, biological samples such as chromosomes need to be subjected to various preparation steps, including dehydration, drying, and metal/carbon coating, which may induce shrinkage and artifacts. The ionic liquid technique has recently been developed and it enables sample preparation without dehydration, drying, or coating, providing a sample that is closer to the native condition. Concurrently, focused ion beam/scanning electron microscopy (FIB/SEM) has been developed, allowing the investigation and direct analysis of chromosome interiors. In this study, we investigated chromosome interiors by FIB/SEM using plant and human chromosomes prepared by the ionic liquid technique. As a result, two types of chromosomes, with and without cavities, were visualized, both for barley and human chromosomes prepared by critical point drying. However, chromosome interiors were revealed only as a solid structure, lacking cavities, when prepared by the ionic liquid technique. Our results suggest that the existence and size of cavities depend on the preparation procedures. We conclude that combination of the ionic liquid technique and FIB/SEM is a powerful tool for chromosome study.

Type
Biological Applications
Copyright
© Microscopy Society of America 2014 

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