Histochemistry and Cell Biology

, Volume 149, Issue 4, pp 423–432 | Cite as

Three-dimensional ultrastructure of osteocytes assessed by focused ion beam-scanning electron microscopy (FIB-SEM)

  • Tomoka HasegawaEmail author
  • Tomomaya Yamamoto
  • Hiromi Hongo
  • Zixuan Qiu
  • Miki Abe
  • Takuma Kanesaki
  • Kawori Tanaka
  • Takashi Endo
  • Paulo Henrique Luiz de Freitas
  • Minqi Li
  • Norio Amizuka
Original Paper


The aim of this study is to demonstrate the application of focused ion beam-scanning electron microscopy, FIB-SEM for revealing the three-dimensional features of osteocytic cytoplasmic processes in metaphyseal (immature) and diaphyseal (mature) trabeculae. Tibiae of eight-week-old male mice were fixed with aldehyde solution, and treated with block staining prior to FIB-SEM observation. While two-dimensional backscattered SEM images showed osteocytes’ cytoplasmic processes in a fragmented fashion, three-dimensional reconstructions of FIB-SEM images demonstrated that osteocytes in primary metaphyseal trabeculae extended their cytoplasmic processes randomly, thus maintaining contact with neighboring osteocytes and osteoblasts. In contrast, diaphyseal osteocytes extended thin cytoplasmic processes from their cell bodies, which ran perpendicular to the bone surface. In addition, these osteocytes featured thick processes that branched into thinner, transverse cytoplasmic processes; at some point, however, these transverse processes bend at a right angle to run perpendicular to the bone surface. Osteoblasts also possessed thicker cytoplasmic processes that branched off as thinner processes, which then connected with cytoplasmic processes of neighboring osteocytes. Thus, FIB-SEM is a useful technology for visualizing the three-dimensional structures of osteocytes and their cytoplasmic processes.


FIB-SEM Osteocyte Osteoblast Three-dimensional structure Bone 



This study was partially supported by the Grants-in Aid for Scientific Research of JSPS (Hasegawa T and Amizuka N) and the Nanotechnology Platform Program of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan (Hasegawa T).

Compliance with ethical standards

Ethical approval

The animal protocols used in this study were approved by the Hokkaido University Guidelines for Animal Experimentation (approval No. 15-0041).

Conflict of interest

No potential conflicts of interest exist.

Supplementary material

Supplementary material 1 (AVI 38299 KB)

Supplementary material 2 (MP4 5633 KB)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Tomoka Hasegawa
    • 1
    Email author
  • Tomomaya Yamamoto
    • 2
  • Hiromi Hongo
    • 1
  • Zixuan Qiu
    • 1
  • Miki Abe
    • 1
  • Takuma Kanesaki
    • 3
  • Kawori Tanaka
    • 3
  • Takashi Endo
    • 4
  • Paulo Henrique Luiz de Freitas
    • 5
  • Minqi Li
    • 6
  • Norio Amizuka
    • 1
  1. 1.Developmental Biology of Hard Tissue, Graduate School of Dental Medicine and Faculty of Dental MedicineHokkaido UniversitySapporoJapan
  2. 2.Japan Self-Defense Forces Hanshin HospitalKawanishiJapan
  3. 3.Carl Zeiss Microscopy Co., Ltd.TokyoJapan
  4. 4.Laboratory of Nano-Micro Material Analysis, Joint-use Facilities, Faculty of EngineeringHokkaido UniversitySapporoJapan
  5. 5.Department of DentistryFederal University of Sergipe at LagartoLagartoBrazil
  6. 6.Division of Basic Science of Stomatology, The School of StomatologyShandong UniversityJinanChina

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