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Cytological Analyses by Advanced Electron Microscopy

  • Takako M. Ichinose
  • Atsuko H. IwaneEmail author
Chapter

Abstract

Recently several microscopy techniques have revealed the dynamics and structure of intracellular material such as organelles and supramolecular proteins for a better understanding of the fundamental properties of a cell. Electron microscopy provides information on extremely small biological specimens, providing images of several target molecules and organelles, including mitochondria and plastids, and their surrounding intracellular environments. Technical development using several advanced electron microscopes has been advanced to obtain 3D structural modeling. Among them, cryo-TEM and tomography techniques are useful for visualization of fine structures, but the observation area is limited. Conversely, “FIB-SEM” theoretically is not affected by the thickness of the sample, and FIB-SEM and reconstruction are effective for 3D structural modeling of C. merolae cells (2–5 microns) during cell division at the whole cell level. Another technique, UHVEM tomography, visualizes a detailed 3D structure of the light-harvesting complex on the surface of the thylakoid membrane in the plastid and the micro-compartments (cristae) in mitochondria. Furthermore, fine whole cell 3D structural model using cryo-STEM tomography, which is obtained under physical fixation and without staining, instead of chemical fixation, is compared with the models of FIB-SEM and 3D reconstruction.

Keywords

FIB-SEM SBF-SEM EM-tomography EM (electron microscopy) Block-face imaging Cryo-TEM Cryo-TEM tomography Cyanidioschyzon merolae Physical fixation 

Abbreviations

3D

Three-dimensional

ATUM-SEM

Automated tape-collecting ultramicrotome

BSE

Backscattered electron

CMOS

Complementary metal oxide semiconductor

EM

Electron microscopy

FIB-SEM

Focused ion beam SEM

HAADF

High-angle annular dark field

HPF

High-pressure freezing

LM

Light microscopy

SBF-SEM

Serial block-face scanning electron microscopy

SBF-SEM

Serial block-face SEM

SEM

Scanning electron microscopy

STEM

Scanning transmission electron microscopy

TEM

Transmission electron microscopy

UHVEM

Ultra-high-voltage electron microscopy

Notes

Acknowledgments

We thank K. Ohta, Ph.D. (Kurume University) and T. Nishida, Ph.D. (Osaka University) for FIB-SEM operation/discussion and UHVEM operation, respectively.

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Copyright information

© Springer Nature Singapore Pte Ltd. 2017

Authors and Affiliations

  1. 1.Cell Field Structure, Quantitative Biology CenterRikenHigashi-HiroshimaJapan
  2. 2.Special Research Promotion Group, Graduate School of Frontier BiosciencesOsaka UniversitySuitaJapan

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