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Rapid Method for Electron Tomographic Reconstruction and Three-Dimensional Modeling of the Murine Synapse Using an Automated Fiducial Marker-Free System

Published online by Cambridge University Press:  06 August 2013

Hyun-wook Kim ,
Seung Hak Oh ,
Namkug Kim ,
Eiko Nakazawa  and
Im Joo Rhyu
Hyun-wook Kim
Affiliation:
Department of Anatomy, College of Medicine, Korea University, 126-1 Anam-dong 5ga, Seongbuk-gu, Seoul 136-705, Korea
Seung Hak Oh
Affiliation:
Department of Anatomy, College of Medicine, Korea University, 126-1 Anam-dong 5ga, Seongbuk-gu, Seoul 136-705, Korea
Namkug Kim
Affiliation:
Department of Radiology, Research Institute of Radiology, Asan Medical Center, University of Ulsan College of Medicine, 388-1 Pungnap-dong, Songpa-gu, Seoul 138-736, Korea
Eiko Nakazawa
Affiliation:
Hitachi High-Technologies Corporation, Nanotechnology Products Business Group, Naka Division, 24-14, Nishi-Shimbashi 1-chome, Minato-ku, Tokyo 105-8717, Japan
Im Joo Rhyu*
Affiliation:
Department of Anatomy, College of Medicine, Korea University, 126-1 Anam-dong 5ga, Seongbuk-gu, Seoul 136-705, Korea
*
*Corresponding author. E-mail: irhyu@korea.ac.kr
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Abstract

Electron tomography (ET) has recently afforded new insights into neuronal architecture. However, the tedious process of sample preparation, image acquisition, alignment, back projection, and additional segmentation process of ET repels beginners. We have tried Hitachi's commercial packages integrated with a Hitachi H-7650 TEM to examine the potential of using an automated fiducial-less approach for our own neuroscience research. Semi-thick sections (200–300 nm) were cut from blocks of fixed mouse (C57BL) cerebellum and prepared for ET. Sets of images were collected automatically as each section was tilted by 2° increments (±60°). “Virtual” image volumes were computationally reconstructed in three dimension (3D) with the EMIP software using either the commonly used “weighted back-projection” (WBP) method or “topography-based reconstruction” (TBR) algorithm for comparison. Computed tomograms using the TBR were more precisely reconstructed compared with the WBP method. Following reconstruction, the image volumes were imported into the 3D editing software A-View and segmented according to synaptic organization. The detailed synaptic components were revealed by very thin virtual image slices; 3D models of synapse structure could be constructed efficiently. Overall, this simplified system provided us with a graspable tool for pursuing ET studies in neuroscience.

Keywords

electron tomographyfiducialWBP algorithmTBR algorithmA-View3D reconstructionsynapse
Type
Research Article
Information
Microscopy and Microanalysis , Volume 19 , Supplement S5: IUMAS , August 2013 , pp. 182 - 187
Copyright
Copyright © Microscopy Society of America 2013 

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