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A Novel Technique for Observing the Internal Ultrastructure of Human Chromosomes with Known Karyotype

Published online by Cambridge University Press:  04 July 2008

Mohammad Ghazizadeh ,
Yoshihiro Sasaki ,
Tatsuo Oguro ,
Shigeru Sato ,
Seiko Egawa ,
Kyoko Inoue ,
Akiko Adachi ,
Hajime Shimizu  and
Oichi Kawanami
Mohammad Ghazizadeh*
Affiliation:
Department of Molecular Pathology, Institute of Gerontology, Nippon Medical School, Kawasaki, Japan Central Institute for Electron Microscopic Researches, Nippon Medical School, Tokyo, Japan
Yoshihiro Sasaki
Affiliation:
Central Institute for Electron Microscopic Researches, Nippon Medical School, Tokyo, Japan
Tatsuo Oguro
Affiliation:
Division of Surgical Pathology, Chiba Hokusoh Hospital, Nippon Medical School, Chiba, Japan
Shigeru Sato
Affiliation:
Central Institute for Electron Microscopic Researches, Nippon Medical School, Tokyo, Japan
Seiko Egawa
Affiliation:
Department of Molecular Pathology, Institute of Gerontology, Nippon Medical School, Kawasaki, Japan
Kyoko Inoue
Affiliation:
Central Institute for Electron Microscopic Researches, Nippon Medical School, Tokyo, Japan
Akiko Adachi
Affiliation:
Central Institute for Electron Microscopic Researches, Nippon Medical School, Tokyo, Japan
Hajime Shimizu
Affiliation:
Department of Molecular Pathology, Institute of Gerontology, Nippon Medical School, Kawasaki, Japan
Oichi Kawanami
Affiliation:
Department of Molecular Pathology, Institute of Gerontology, Nippon Medical School, Kawasaki, Japan
*
Corresponding author. E-mail: ciem@nms.ac.jp
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Abstract

Observation of the internal ultrastructure of human chromosomes by transmission electron microscopy (TEM) has frequently been attempted in spite of the difficulties in detaching metaphase chromosome spreads from the glass slide for further processing. In this study we have used a method in which metaphase chromosome spreads were prepared on a flexible thermoplastic membrane (ACLAR) film. To assess chromosome identity, a diamidino-phenylindole staining and karyotying was first done using a conventional cytogenetic system. The chromosome spreads were then fixed with 1% osmium tetroxide, stained with freshly prepared 2% tannic acid, dehydrated, and flat-embedded in epoxy resin. The resin sheet was easily detachable and carried whole chromosome spreads. By this method, TEM observation of chromosomes from normal human lymphocytes allowed a thorough examination of the ultrastructure of centromeres, telomeres, fragile sites, and other chromosomal regions. Various ultrastructural patterns including thick electron dense boundaries, less dense internal regions, and extended chromatin loops at the periphery of the chromosomes were discernible. Application of the present method to chromosome research is expected to provide comprehensive information on the internal ultrastructure of different chromosomal regions in relation to function.

Keywords

human chromosomesinternal ultrastructurekaryotypingflat embeddingACLAR filmtransmission electron microscopy
Type
Biological Applications
Information
Microscopy and Microanalysis , Volume 14 , Issue 4 , August 2008 , pp. 357 - 361
Copyright
Copyright © Microscopy Society of America 2008

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References

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