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Reconstructing adhesion structures in tissues by cryo-electron tomography of vitrified frozen sections


Bokstad, Melanie; Sabanay, Helena; Dahan, Idit; Geiger, Benjamin; Medalia, Ohad (2012). Reconstructing adhesion structures in tissues by cryo-electron tomography of vitrified frozen sections. Journal of Structural Biology, 178(2):76-83.

Abstract

Cryo-electron tomography enables three-dimensional insights into the macromolecular architecture of cells in a close-to-life state. However, it is limited to thin specimens, <1.0 μm in thickness, typically restricted to the peripheral areas of intact eukaryotic cells. Analysis of tissue ultrastructure, on the other hand, requires physical sectioning approaches, preferably cryo-sectioning, following which electron tomography (ET) may be performed. Nevertheless, cryo-electron microscopy of vitrified sections is a demanding technique and typically cannot be used to examine thick sections, >80-100 nm, due to surface crevasses. Here, we explore the potential use of cryo-ET of vitrified frozen sections (VFSs) for imaging cell adhesions in chicken smooth muscle and mouse epithelial tissues. By investigating 300-400 nm thick sections, which are collected on the EM grid and re-vitrified, we resolved fine 3D structural details of the membrane-associated dense plaques and flanking caveoli in smooth muscle tissue, and desmosomal adhesions in stratified epithelium. Technically, this method offers a simple approach for reconstructing thick volumes of hydrated frozen sections.

Abstract

Cryo-electron tomography enables three-dimensional insights into the macromolecular architecture of cells in a close-to-life state. However, it is limited to thin specimens, <1.0 μm in thickness, typically restricted to the peripheral areas of intact eukaryotic cells. Analysis of tissue ultrastructure, on the other hand, requires physical sectioning approaches, preferably cryo-sectioning, following which electron tomography (ET) may be performed. Nevertheless, cryo-electron microscopy of vitrified sections is a demanding technique and typically cannot be used to examine thick sections, >80-100 nm, due to surface crevasses. Here, we explore the potential use of cryo-ET of vitrified frozen sections (VFSs) for imaging cell adhesions in chicken smooth muscle and mouse epithelial tissues. By investigating 300-400 nm thick sections, which are collected on the EM grid and re-vitrified, we resolved fine 3D structural details of the membrane-associated dense plaques and flanking caveoli in smooth muscle tissue, and desmosomal adhesions in stratified epithelium. Technically, this method offers a simple approach for reconstructing thick volumes of hydrated frozen sections.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Department of Biochemistry
07 Faculty of Science > Department of Biochemistry
Dewey Decimal Classification:570 Life sciences; biology
Language:English
Date:2012
Deposited On:30 Oct 2012 16:37
Last Modified:21 Nov 2017 16:12
Publisher:Elsevier
ISSN:1047-8477
Publisher DOI:https://doi.org/10.1016/j.jsb.2011.10.013
PubMed ID:22085747

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