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Nidau, Switzerland

Bos E.,Netherlands Cancer Institute | Bos E.,National Institute of Metrology of Brazil | Sant Anna C.,National Institute of Metrology of Brazil | Gnaegi H.,Diatome Ltd | And 6 more authors.
Journal of Structural Biology | Year: 2011

Cryo-ultramicrotomy can be used to obtain ultrathin cryo-sections from cryo-fixed or aldehyde-fixed cryo-protected vitreous biologic samples. For immuno-gold EM, cryo-sections are retrieved from the cryo-chamber on a droplet of a pick-up solution (paste-like and almost frozen) to which the sections attach. The sections are then placed on an EM specimen grid at room temperature. This procedure compromises the ultrastructure, resulting in folds, holes, and loss of the original material. In this paper we show the critical influence of humidity, stretching, and relief of compression during thawing of the sections. We show a new lift-up hinge device for semi-automated retrieval of cryo-sections that results in significantly improved section quality. This approach was also applied successfully to vitreous sections from high pressure frozen samples. An important advance is that these vitreous cryo-sections can now successfully be post-fixed and immunolabelled after thawing; this allows cryo-EM comparison with adjacent ribbons of sections still in the frozen hydrated state. These findings call for technical innovations aiming at automated cryo-ultramicrotomy in a fully controlled environment for improved localization of proteins within their 'close to native' cellular context and correlative electron cryo-tomography of consecutive ribbons of sections of one frozen hydrated sample. © 2011 Elsevier Inc. Source


Pierson J.,Netherlands Cancer Institute | Fernandez J.J.,CSIC - National Center for Biotechnology | Fernandez J.J.,University of Almeria | Bos E.,Netherlands Cancer Institute | And 8 more authors.
Journal of Structural Biology | Year: 2010

Cryo-electron tomography of vitreous cryo-sections is the most suitable method for exploring the 3D organization of biological samples that are too large to be imaged in an intact state. Producing good quality vitreous cryo-sections, however, is challenging. Here, we focused on the major obstacles to success: contamination in and around the microtome, and attachment of the ribbon of sections to an electron microscopic grid support film. The conventional method for attaching sections to the grid has involved mechanical force generated by a crude stamping or pressing device, but this disrupts the integrity of vitreous cryo-sections. Furthermore, attachment is poor, and parts of the ribbon of sections are often far from the support film. This results in specimen instability during image acquisition and subsequent difficulty with aligning projection images. Here, we have implemented a protective glove box surrounding the cryo-ultramicrotome that reduces the humidity around and within the microtome during sectioning. We also introduce a novel way to attach vitreous cryo-sections to an EM grid support film using electrostatic charging. The ribbon of vitreous cryo-sections remains in place during transfer and storage and is devoid of stamping related artefacts. We illustrate these improvements by exploring the structure of putative cellular 80S ribosomes within 50 nm, vitreous cryo-sections of Saccharomyces cerevisiae. © 2009 Elsevier Inc. All rights reserved. Source


Sandu A.M.,Materials Innovation Institute M2i | Sandu A.M.,Technical University of Delft | Gnaegi H.,Diatome Ltd | Mulders J.J.L.,FEI Electronic Optics | Zandbergen H.W.,Technical University of Delft
Philosophical Magazine | Year: 2010

Ultramicrotomy is widely regarded as a thin section preparation method for transmission electron microscopy (TEM) investigations. It is shown that ultramicrotomy can also provide a simple path for microstructure analysis and assessment of mechanical properties for a sectioned block-face. Furthermore, electron backscatter diffraction (EBSD) analysis can be applied directly on ultramicrotomed surfaces without any additional polishing or etching. EBSD analysis relates the inherent cutting artefacts to the crystallographic orientations of the grains, hence delivering a rough assessment of their deformation resistance. TEM investigations revealed that crystallographic- related cutting artefacts, which exhibit a wave-like pattern, are the result of the dislocation pile-ups close to the knife-specimen interface. This technique is considered suitable to be coupled with EBSD for three-dimensional microstructure reconstructions when used for serial sectioning of large volumes. © 2010 Taylor & Francis. Source


Stockel S.,TU Chemnitz | Ebert S.,TU Chemnitz | Bottcher M.,TU Chemnitz | Seifert A.,TU Chemnitz | And 6 more authors.
Chemical Vapor Deposition | Year: 2014

We developed aluminium phosphate-coated alumina fibres with significantly improved pull-out properties, which may be suitable for reinforcing advanced inorganic composites qualified for operating temperatures up to 1200°C. Aluminium phosphate layers are generated on the surface of alumina fibres using a continuous chemical vapour deposition (CVD) process: At furnace temperatures between 850°C and 1050°C, within a tube reactor heated by an electrical furnace, the alumina fibres are exposed to gas mixtures of phosphoryl trichloride and oxygen, inducing dense aluminium phosphate layers on the fibre surface. Mini-composites were prepared by embedding the coated fibres into an alumina matrix. © 2014 Wiley-VCH Verlag GmbH & Co. KGaA. Source


Rigort A.,Max Planck Institute of Biochemistry | Bauerlein F.J.B.,Max Planck Institute of Biochemistry | Leis A.,Max Planck Institute of Biochemistry | Gruska M.,Max Planck Institute of Biochemistry | And 7 more authors.
Journal of Structural Biology | Year: 2010

A principal limitation of cryo-transmission electron microscopy performed on cells or tissues is the accessible specimen thickness. This is exacerbated in tomography applications, where the aspect ratio (and thus the apparent specimen thickness) changes considerably during specimen tilting. Cryo-ultramicrotomy is the most obvious way of dealing with this problem; however, frozen-hydrated sections suffer from potentially inconsistent compression that cannot be corrected with certainty, and furthermore, yields of sections that satisfy all of the conditions necessary for tomographic imaging are poor. An alternative approach that avoids mechanical deformations is the use of focused ion beam (FIB) instrumentation, where thinning of the frozen-hydrated specimen occurs through the process of sputtering with heavy ions, typically gallium. Here, we use correlative cryo-fluorescence microscopy to navigate large cellular volumes and to localize specific cellular targets. We show that the selected targets in frozen-hydrated specimens can be accessed directly by focused ion beam milling. We also introduce a novel cryo-planing procedure as a method that could facilitate thinning of large areas of vitreous ice prior to cryo-fluorescence, FIB thinning, and cryo-electron tomography. © 2010 Elsevier Inc. Source

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