Hostname: page-component-586b7cd67f-t8hqh Total loading time: 0 Render date: 2024-11-24T05:01:47.474Z Has data issue: false hasContentIssue false
  • English
  • Français

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: [email protected]
Get access

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 

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Baba, N. & Katayama, E. (2008). A novel “ghost”-free tomographic image reconstruction method applicable to rotary-shadowed replica specimens. Ultramicroscopy 108(3), 239–255.Google Scholar
Baumeister, W., Grimm, R. & Walz, J. (1999). Electron tomography of molecules and cells. Trends Cell Biol 9(2), 81–85.Google Scholar
Cao, M., Takaoka, A., Zhang, H.B. & Nishi, R. (2010). An automatic method of detecting and tracking fiducial markers for alignment in electron tomography. J Electron Microsc (Tokyo) 60(1), 39–46.Google Scholar
Chen, X., Winters, C.A. & Reese, T.S. (2008). Life inside a thin section: Tomography. J Neurosci 28(38), 9321–9327.Google Scholar
Donohoe, B.S., Mogelsvang, S. & Staehelin, L.A. (2006). Electron tomography of ER, golgi and related membrane systems. Methods 39(2), 154–162.Google Scholar
Henderson, R. & Unwin, P.N. (1975). Three-dimensional model of purple membrane obtained by electron microscopy. Nature 257(5521), 28–32.Google Scholar
Kremer, J.R., Mastronarde, D.N. & McIntosh, J.R. (1996). Computer visualization of three-dimensional image data using IMOD. J Struct Biol 116(1), 71–76.Google Scholar
Lee, K.J., Park, C.H. & Rhyu, I.J. (2005). Efficient three-dimensional reconstruction of synapse with high-voltage electron microscopy. J Electron Microsc (Tokyo) 54(2), 139–141.Google Scholar
Liu, Y., Penczek, P.A., McEwen, B.F. & Frank, J. (1995). A marker-free alignment method for electron tomography. Ultramicroscopy 58(3-4), 393–402.Google Scholar
McIntosh, R., Nicastro, D. & Mastronarde, D. (2005). New views of cells in 3D: An introduction to electron tomography. Trends Cell Biol 15(1), 43–51.Google Scholar
Morone, N., Fujiwara, T., Murase, K., Kasai, R.S., Ike, H., Yuasa, S., Usukura, J. & Kusumi, A. (2006). Three-dimensional reconstruction of the membrane skeleton at the plasma membrane interface by electron tomography. J Cell Biol 174(6), 851–862.Google Scholar
Nickell, S., Forster, F., Linaroudis, A., Net, W.D., Beck, F., Hegerl, R., Baumeister, W. & Plitzko, J.M. (2005). TOM software toolbox: Acquisition and analysis for electron tomography. J Struct Biol 149(3), 227–234.Google Scholar
Park, I.S., Han, J.W., Lee, K.J., Lee, N.J., Lee, W.T., Park, K.A. & Rhyu, I.J. (2006). Evaluation of morphological plasticity in the cerebella of basketball players with MRI. J Kor Med Sci 21(2), 342–346.Google Scholar
Penczek, P., Marko, M., Buttle, K. & Frank, J. (1995). Double-tilt electron tomography. Ultramicroscopy 60(3), 393–410.Google Scholar
Shoop, R.D., Esquenazi, E., Yamada, N., Ellisman, M.H. & Berg, D.K. (2002). Ultrastructure of a somatic spine mat for nicotinic signaling in neurons. J Neurosci 22(3), 748–756.Google Scholar
Siksou, L., Rostaing, P., Lechaire, J.P., Boudier, T., Ohtsuka, T., Fejtova, A., Kao, H.T., Greengard, P., Gundelfinger, E.D., Triller, A. & Marty, S. (2007). Three-dimensional architecture of presynaptic terminal cytomatrix. J Neurosci 27(26), 6868–6877.Google Scholar
Sougrat, R., Bartesaghi, A., Lifson, J.D., Bennett, A.E., Bess, J.W., Zabransky, D.J. & Subramaniam, S. (2007). Electron tomography of the contact between T cells and SIV/HIV-1: Implications for viral entry. PLoS Pathog 3(5), e63.Google Scholar
Suloway, C., Shi, J., Cheng, A., Pulokas, J., Carragher, B., Potter, C.S., Zheng, S.Q., Agard, D.A. & Jensen, G.J. (2009). Fully automated, sequential tilt-series acquisition with Leginon. J Struct Biol 167(1), 11–18.Google Scholar
Urban, E., Jacob, S., Nemethova, M., Resch, G.P. & Small, J.V. (2010). Electron tomography reveals unbranched networks of actin filaments in lamellipodia. Nat Cell Biol 12(5), 429–435.Google Scholar
Zheng, Q.S., Braunfeld, M.B., Sedat, J.W. & Agard, D.A. (2004). An improved strategy for automated electron microscopic tomography. J Struct Biol 147(2), 91–101.Google Scholar