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A Method for Observing Silver-Stained Osteocytes In Situ in 3-μm Sections Using Ultra-High Voltage Electron Microscopy Tomography

Published online by Cambridge University Press:  27 August 2009

Hiroshi Kamioka ,
Sakhr A. Murshid ,
Yoshihito Ishihara ,
Naoko Kajimura ,
Toshiaki Hasegawa ,
Ryoko Ando ,
Yasuyo Sugawara ,
Takashi Yamashiro ,
Akio Takaoka  and
Teruko Takano-Yamamoto
Hiroshi Kamioka*
Affiliation:
Department of Orthodontics, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan
Sakhr A. Murshid
Affiliation:
Division of Orthodontics and Dentofacial Orthopedics, Tohoku University Graduate School of Dentistry, Sendai, Japan
Yoshihito Ishihara
Affiliation:
Department of Orthodontics, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan
Naoko Kajimura
Affiliation:
Research Center for Ultra-High Voltage Electron Microscopy, Osaka University, Osaka, Japan
Toshiaki Hasegawa
Affiliation:
Research Center for Ultra-High Voltage Electron Microscopy, Osaka University, Osaka, Japan
Ryoko Ando
Affiliation:
Department of Orthodontics, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan
Yasuyo Sugawara
Affiliation:
Department of Orthodontics, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan
Takashi Yamashiro
Affiliation:
Department of Orthodontics, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan
Akio Takaoka
Affiliation:
Research Center for Ultra-High Voltage Electron Microscopy, Osaka University, Osaka, Japan
Teruko Takano-Yamamoto
Affiliation:
Division of Orthodontics and Dentofacial Orthopedics, Tohoku University Graduate School of Dentistry, Sendai, Japan
*
Corresponding author. E-mail: kamioka@md.okayama-u.ac.jp
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Abstract

Osteocytes are surrounded by hard bone matrix, and it has not been possible previously to directly observe the in situ architecture of osteocyte morphology in bone. Electron microscope tomography, however, is a technique that has the unique potential to provide three-dimensional (3D) visualization of cellular ultrastructure. This approach is based on reconstruction of 3D volumes from a tilt series of electron micrographs of cells, and resolution at the nanometer level has been achieved. We applied electron microscope tomography to thick sections of silver-stained osteocytes in bone using a Hitachi H-3000 ultra-high voltage electron microscope equipped with a 360° tilt specimen holder, at an accelerating voltage of 2 MeV. Osteocytes with numerous processes and branches were clearly seen in the serial tilt series acquired from 3-μm-thick sections. Reconstruction of young osteocytes showed the 3D topographic morphology of the cell body and processes at high resolution. This morphological data on osteocytes should provide useful information to those who study osteocyte physiology and the several models used to explain their mechanosensory properties.

Keywords

osteocyteelectron tomographysilver stainingthree-dimensional reconstructionultra-high voltage electron microscopy
Type
Tomography Applications
Information
Microscopy and Microanalysis , Volume 15 , Issue 5 , October 2009 , pp. 377 - 383
Copyright
Copyright © Microscopy Society of America 2009

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