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  Cryo FIB-SEM: Volume imaging of cellular ultrastructure in native frozen specimens

Schertel, A., Snaidero, N., Han, H., Ruhwedel, T., Laue, M., Grabenbauer, M., et al. (2013). Cryo FIB-SEM: Volume imaging of cellular ultrastructure in native frozen specimens. Journal of Structural Biology, 184(2), 355-360. doi:10.1016/j.jsb.2013.09.024.

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 Creators:
Schertel, Andreas1, Author
Snaidero, Nicolas2, Author
Han, Hongmei3, Author              
Ruhwedel, Torben2, Author
Laue, Michael1, Author
Grabenbauer, Markus3, Author              
Moebius, Wiebke2, Author
Affiliations:
1external, ou_persistent22              
2Max Planck Institute of Experimental Medicine, Hermann-Rein-Straße 3, 37075 Göttingen, ou_persistent22              
3Abt. II: Systemische Zellbiologie, Max Planck Institute of Molecular Physiology, Max Planck Society, Otto-Hahn-Str. 11, 44227 Dortmund, DE, ou_1753288              

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Free keywords: CRYOELECTRON MICROSCOPY; ELECTRON-MICROSCOPY; VITREOUS SECTIONS; BLOCK FACES; TOMOGRAPHY; CELLS; RESOLUTION; SAMPLESBiochemistry & Molecular Biology; Biophysics; Cell Biology; Cryo FIB-SEM; High-pressure freezing; Serial block face imaging; 3D reconstruction; Mouse optic nerve; Bacillus subtilis spores;
 Abstract: Volume microscopy at high resolution is increasingly required to better understand cellular functions in the context of three-dimensional assemblies. Focused ion beam (FIB) milling for serial block face imaging in the scanning electron microscope (SEM) is an efficient and fast method to generate such volume data for 3D analysis. Here, we apply this technique at cryo-conditions to image fully hydrated frozen specimen of mouse optic nerves and Bacillus subtilis spores obtained by high-pressure freezing (HPF). We established imaging conditions to directly visualize the ultrastructure in the block face at -150 degrees C by using an in-lens secondary electron (SE) detector. By serial sectioning with a focused ion beam and block face imaging of the optic nerve we obtained a volume as large as X = 7.72 mu m, Y = 5.79 mu m and Z = 3.81 mu m with a lateral pixel size of 7.5 nm and a slice thickness of 30 nm in Z. The intrinsic contrast of membranes was sufficient to distinguish structures like Golgi cisternae, vesicles, endoplasmic reticulum and cristae within mitochondria and allowed for a three-dimensional reconstruction of different types of mitochondria within an oligodendrocyte and an astrocytic process. Applying this technique to dormant B. subtilis spores we obtained volumes containing numerous spores and discovered a bright signal in the core, which cannot be related to any known structure so far. In summary, we describe the use of cryo FIB-SEM as a tool for direct and fast 3D cryo-imaging of large native frozen samples including tissues. (C) 2013 Elsevier Inc. All rights reserved.

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Language(s): eng - English
 Dates: 2013-11
 Publication Status: Published in print
 Pages: 6
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: ISI: 000327171000029
DOI: 10.1016/j.jsb.2013.09.024
 Degree: -

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Title: Journal of Structural Biology
  Other : J. Struct. Biol.
Source Genre: Journal
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Publ. Info: Academic Press
Pages: - Volume / Issue: 184 (2) Sequence Number: - Start / End Page: 355 - 360 Identifier: -