Improving the technique of vitreous cryo-sectioning for cryo-electron tomography: Electrostatic charging for section attachment and implementation of an anti-contamination glove box

Pierson, Jason; Fernández, Julio F.; Bos, Erik; Amini, Shoaib; Gnaegi, Helmut; Vos, Matthijn; Bel, Bennie; Adolfsen, Freek; Carrascosa, José L.; Peters, Peter J.

doi:10.1016/j.jsb.2009.10.001

Cited by 81 publications

(64 citation statements)

References 29 publications

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“…In addition to these problems, sections are usually not flat and thus are poorly attached to the support film. This is has been resolved adequately with the application of electrostatic charging (15). In combination with 3D subtomogram averaging, cryo-ET of vitreous sections is therefore currently the only way to quantitatively analyze electron tomographic data from eukaryotic cells at arbitrary regions and produce molecular 3D EM density maps.…”

mentioning

confidence: 99%

The three-dimensional molecular structure of the desmosomal plaque

Al‐Amoudi

Castaño-Díez

Devos

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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The cytoplasmic surface of intercellular junctions is a complex network of molecular interactions that link the extracellular region of the desmosomal cadherins with the cytoskeletal intermediate filaments. Although 3D structures of the major plaque components are known, the overall architecture remains unknown. We used cryoelectron tomography of vitreous sections from human epidermis to record 3D images of desmosomes in vivo and in situ at molecular resolution. Our results show that the architecture of the cytoplasmic surface of the desmosome is a 2D interconnected quasiperiodic lattice, with a similar spatial organization to the extracellular side. Subtomogram averaging of the plaque region reveals two distinct layers of the desmosomal plaque: a lowdensity layer closer to the membrane and a high-density layer further away from the membrane. When combined with a heuristic, allowing simultaneous constrained fitting of the high-resolution structures of the major plaque proteins (desmoplakin, plakophilin, and plakoglobin), it reveals their mutual molecular interactions and explains their stoichiometry. The arrangement suggests that alternate plakoglobin-desmoplakin complexes create a template on which desmosomal cadherins cluster before they stabilize extracellularly by binding at their N-terminal tips. Plakophilins are added as a molecular reinforcement to fill the gap between the formed plaque complexes and the plasma membrane.vitreous sectioning | image processing | genetic algorithms | multi-protein fitting C ell junctions are widely distributed in animal tissues and are most abundant in tissues that are subjected to considerable mechanical stress, such as heart, epidermis, and muscle. These junctions are composed of two main classes of proteins: transmembrane adhesion proteins with an extracellular domain that interacts with either the ECM (e.g., focal adhesion and hemidesmosome) or the extracellular domains of specific transmembrane adhesion proteins from the juxtaposed cell (e.g., adherens junctions and desmosomes); and a cytoplasmic tail that binds to one or more intracellular anchor proteins. These form a distinct plaque on the cytoplasmic face of the plasma membrane and connect the junctional complexes to actin filaments or intermediate filaments (IFs). When they have been assembled, these junctions play essential roles in tissue morphogenesis and maintenance; plaque disruption is a hallmark of many blistering and cancer diseases (1, 2). Biochemical and mutagenesis studies have revealed many interactions between the plaque components and their connections to the extracellular space (3-8). However, the organization and interactions of the constituent proteins in the context of the assembled junctions remain poorly understood. For example, whether or how actin filaments bind in situ to the adherens junctions and how the anchoring proteins in hemidesmosome plaque connect integrin to the IFs is still unclear.In the current study, we present the 3D cytoplasmic surface of the desmosome-the desmosomal plaque-fr...

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mentioning

confidence: 99%

The three-dimensional molecular structure of the desmosomal plaque

Al‐Amoudi

Castaño-Díez

Devos

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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“…A liquid working the same way with the vitreous section has not been found yet, and as such cryo-sections are obtained with a dry knife. Pierson et al [36] implemented a protective glove box in order to reduce the humidity around and within the cryo-ultramicrotome.…”

Section: Cryo-electron Microscopy Of Vitreous Sectionsmentioning

confidence: 99%

“…Moreover, this method is not adequate for proper attachment of cryo-sections to the support film. Pierson et al [36] introduced electrostatic charging for attachment, which is devoid of stamping-related artefacts, but it cannot guarantee uniform attachment of cryo-sections to the carbon film. Consequently, the sections can be affected by the beam exposure and may move during image acquisition [38].…”

Section: Cryo-electron Microscopy Of Vitreous Sectionsmentioning

confidence: 99%

Closer to the native state. Critical evaluation of cryo-techniques for Transmission Electron Microscopy: preparation of biological samples

Mielańczyk

Matysiak

Michalski

et al. 2014

Folia Histochem Cytobiol.

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Abstract:Over the years Transmission Electron Microscopy (TEM) has evolved into a powerful technique for the structural analysis of cells and tissues at various levels of resolution. However, optimal sample preservation is required to achieve results consistent with reality. During the last few decades, conventional preparation methods have provided most of the knowledge about the ultrastructure of organelles, cells and tissues. Nevertheless, some artefacts can be introduced at all stagesofstandard electron microscopy preparation technique. Instead, rapid freezing techniques preserve biological specimens as close as possible to the native state. Our review focuses on different cryo-preparation approaches, starting from vitrification methods dependent on sample size. Afterwards, we discuss Cryo-Electron Microscopy Of VItreous Sections (CEMOVIS) and the main difficulties associated with this technique. Cryo-Focused Ion Beam (cryo-FIB) is described as a potential alternative for CEMOVIS. Another post-processing route for vitrified samples is freeze substitution and embedding in resin for structural analysis or immunolocalization analysis. Cryo-sectioning according to Tokuyasu is a technique dedicated to high efficiency immunogold labelling. Finally, we introduce hybrid techniques, which combine advantages of primary techniques originally dedicated to different approaches. Hybrid approaches permit to perform the study of difficult-to-fix samples and antigens or help optimize the sample preparation protocol for the integrated Laser and Electron Microscopy (iLEM) technique

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“…5,6,7 There are many different specimen holders available for high pressure freezing, your selection will depend on the sample, the type of freezing machine used or the investigative application selected. Here we use Bal-Tec HPM 010 high pressure freezer and cryo ultra microtome model UC6/FC6 from Leica Microsystems.…”

Section: High Pressure Freezing and Cryo Sectioning Of Thicker Cmentioning

confidence: 99%

Electron Cryotomography of Bacterial Cells

Chen

McDowall

Dobro

et al. 2010

JoVE

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While much is already known about the basic metabolism of bacterial cells, many fundamental questions are still surprisingly unanswered, including for instance how they generate and maintain specific cell shapes, establish polarity, segregate their genomes, and divide. In order to understand these phenomena, imaging technologies are needed that bridge the resolution gap between fluorescence light microscopy and higher-resolution methods such as X-ray crystallography and NMR spectroscopy.Electron cryotomography (ECT) is an emerging technology that does just this, allowing the ultrastructure of cells to be visualized in a near-native state, in three dimensions (3D), with "macromolecular" resolution (~4nm).1, 2 In ECT, cells are imaged in a vitreous, "frozen-hydrated" state in a cryo transmission electron microscope (cryoTEM) at low temperature (< -180°C). For slender cells (up to ~500 nm in thickness 3 ), intact cells are plunge-frozen within media across EM grids in cryogens such as ethane or ethane/propane mixtures. Thicker cells and biofilms can also be imaged in a vitreous state by first "high-pressure freezing" and then, "cryo-sectioning" them. A series of two-dimensional projection images are then collected through the sample as it is incrementally tilted along one or two axes. A three-dimensional reconstruction, or "tomogram" can then be calculated from the images. While ECT requires expensive instrumentation, in recent years, it has been used in a few labs to reveal the structures of various external appendages, the structures of different cell envelopes, the positions and structures of cytoskeletal filaments, and the locations and architectures of large macromolecular assemblies such as flagellar motors, internal compartments and chemoreceptor arrays. 1, 2 In this video article we illustrate how to image cells with ECT, including the processes of sample preparation, data collection, tomogram reconstruction, and interpretation of the results through segmentation and in some cases correlation with light microscopy. Video LinkThe video component of this article can be found at

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Improving the technique of vitreous cryo-sectioning for cryo-electron tomography: Electrostatic charging for section attachment and implementation of an anti-contamination glove box

Cited by 81 publications

References 29 publications

The three-dimensional molecular structure of the desmosomal plaque

The three-dimensional molecular structure of the desmosomal plaque

Closer to the native state. Critical evaluation of cryo-techniques for Transmission Electron Microscopy: preparation of biological samples

Electron Cryotomography of Bacterial Cells

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