The Experts below are selected from a list of 504 Experts worldwide ranked by ideXlab platform
P J Withers - One of the best experts on this subject based on the ideXlab platform.
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3d imaging by serial block face scanning electron microscopy for materials science using Ultramicrotomy
Ultramicroscopy, 2016Co-Authors: T Hashimoto, G. E. Thompson, X Zhou, P J WithersAbstract:Mechanical serial block face scanning electron microscopy (SBFSEM) has emerged as a means of obtaining three dimensional (3D) electron images over volumes much larger than possible by focused ion beam (FIB) serial sectioning and at higher spatial resolution than achievable with conventional X-ray computed tomography (CT). Such high resolution 3D electron images can be employed for precisely determining the shape, volume fraction, distribution and connectivity of important microstructural features. While soft (fixed or frozen) biological samples are particularly well suited for nanoscale sectioning using an ultramicrotome, the technique can also produce excellent 3D images at electron microscope resolution in a time and resource-efficient manner for engineering materials. Currently, a lack of appreciation of the capabilities of Ultramicrotomy and the operational challenges associated with minimising artefacts for different materials is limiting its wider application to engineering materials. Consequently, this paper outlines the current state of the art for SBFSEM examining in detail how damage is introduced during slicing and highlighting strategies for minimising such damage. A particular focus of the study is the acquisition of 3D images for a variety of metallic and coated systems.
Wolfgang Baumeister - One of the best experts on this subject based on the ideXlab platform.
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opening windows into the cell focused ion beam milling for cryo electron tomography
Current Opinion in Structural Biology, 2013Co-Authors: Elizabeth Villa, Miroslava Schaffer, Jürgen M. Plitzko, Wolfgang BaumeisterAbstract:Cryo-electron tomography (CET) is ideally suited for bridging the resolution gap between molecular and cellular structural studies. However, CET is limited to a sample thickness under 500 nm, which is thinner than most cells. Here, we review a method for preparing cells for CET using focused-ion-beam milling, a technique commonly used in materials science. Adapted to cryogenic conditions, FIB milling can be applied to various cell types to produce samples thin enough for CET that do not present the artefacts typical to other preparation techniques, for example, cryo-Ultramicrotomy, effectively opening windows into intact cells. Samples can be produced routinely and reproducibly. The data obtained from CET can be used for structural studies in situ, or to do quantitative cell biology studies, in which cells can be observed at the molecular level under different physiological conditions.
Miroslava Schaffer - One of the best experts on this subject based on the ideXlab platform.
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opening windows into the cell focused ion beam milling for cryo electron tomography
Current Opinion in Structural Biology, 2013Co-Authors: Elizabeth Villa, Miroslava Schaffer, Jürgen M. Plitzko, Wolfgang BaumeisterAbstract:Cryo-electron tomography (CET) is ideally suited for bridging the resolution gap between molecular and cellular structural studies. However, CET is limited to a sample thickness under 500 nm, which is thinner than most cells. Here, we review a method for preparing cells for CET using focused-ion-beam milling, a technique commonly used in materials science. Adapted to cryogenic conditions, FIB milling can be applied to various cell types to produce samples thin enough for CET that do not present the artefacts typical to other preparation techniques, for example, cryo-Ultramicrotomy, effectively opening windows into intact cells. Samples can be produced routinely and reproducibly. The data obtained from CET can be used for structural studies in situ, or to do quantitative cell biology studies, in which cells can be observed at the molecular level under different physiological conditions.
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Ultramicrotomy in the esem a versatile method for materials and life sciences
Journal of Microscopy, 2009Co-Authors: Armin Zankel, Miroslava Schaffer, B Kraus, Peter Poelt, Elisabeth IngolicAbstract:We here present the results of the first materials science analyses obtained with the prototype of a serial block-face sectioning and imaging tool, 3Viewtrade mark of Gatan, Inc (Pleasanton, CA, U.S.A.). It is a specially designed ultramicrotome operating in situ within an environmental scanning electron microscope originally developed for life science research. The microtome removes thin slices from the sample and the environmental scanning electron microscope images each new block surface of the specimen (serial block-face scanning electron microscopy). The Schottky emitter (FEG) of the microscope delivers high spatial resolution and has the advantage of stable performance and high durability. The slice thickness can typically be selected between 50 and 100 nm. It is possible to cut hundreds of slices and simultaneously acquire images with Digital Micrographtrade mark Model 700 (Gatan, Inc.). This article outlines the set-up and describes the automated process. The preparation of specimens for in situ Ultramicrotomy is explained and the parameters for good image quality are discussed. In addition, special operative and analytic features of the controlling software are presented. Three different technical materials and one botanical specimen were analyzed delivering first results of this method for materials science and for botany.
Melanie Poinsot - One of the best experts on this subject based on the ideXlab platform.
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Amorphous Calcium Carbonate Granules Form Within an Intracellular Compartment in Calcifying Cyanobacteria
Frontiers in Microbiology, 2018Co-Authors: Marine Blondeau, Feriel Skouri-panet, Jean-michel Guigner, Céline Férard, Melanie Poinsot, Martin Sachse, Claire Boulogne, Cynthia Gillet, Jennyfer Miot, Karim BenzeraraAbstract:The recent discovery of cyanobacteria forming intracellular amorphous calcium carbonate (ACC) has challenged the former paradigm suggesting that cyanobacteria-mediated carbonatogenesis was exclusively extracellular. Yet, the mechanisms of intracellular biomineralization in cyanobacteria and in particular whether this takes place within an intracellular microcompartment, remain poorly understood. Here, we analyzed six cyanobacterial strains forming intracellular ACC by transmission electron microscopy. We tested two different approaches to preserve as well as possible the intracellular ACC inclusions: (i) freeze-substitution followed by epoxy embedding and room-temperature Ultramicrotomy and (ii) high-pressure freezing followed by cryo-Ultramicrotomy, usually referred to as cryo-electron microscopy of vitreous sections (CEMOVIS). We observed that the first method preserved ACC well in 500-nm-thick sections but not in 70-nm-thick sections. However, cell ultrastructures were difficult to clearly observe in the 500-nm-thick sections. In contrast, CEMOVIS provided a high preservation quality of bacterial ultrastructures, including the intracellular ACC inclusions in 50-nm-thick sections. ACC inclusions displayed different textures, suggesting varying brittleness, possibly resulting from different hydration levels. Moreover, an electron dense envelope of ∼2.5 nm was systematically observed around ACC granules in all studied cyanobacterial strains. This envelope may be composed of a protein shell or a lipid monolayer, but not a lipid bilayer as usually observed in other bacteria forming intracellular minerals. Overall, this study evidenced that ACC inclusions formed and were stabilized within a previously unidentified bacterial microcompartment in some species of cyanobacteria.
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Data_Sheet_1_Amorphous Calcium Carbonate Granules Form Within an Intracellular Compartment in Calcifying Cyanobacteria.PDF
2018Co-Authors: Marine Blondeau, Feriel Skouri-panet, Jean-michel Guigner, Céline Férard, Melanie Poinsot, Martin Sachse, Claire Boulogne, Cynthia Gillet, Jennyfer Miot, Karim BenzeraraAbstract:The recent discovery of cyanobacteria forming intracellular amorphous calcium carbonate (ACC) has challenged the former paradigm suggesting that cyanobacteria-mediated carbonatogenesis was exclusively extracellular. Yet, the mechanisms of intracellular biomineralization in cyanobacteria and in particular whether this takes place within an intracellular microcompartment, remain poorly understood. Here, we analyzed six cyanobacterial strains forming intracellular ACC by transmission electron microscopy. We tested two different approaches to preserve as well as possible the intracellular ACC inclusions: (i) freeze-substitution followed by epoxy embedding and room-temperature Ultramicrotomy and (ii) high-pressure freezing followed by cryo-Ultramicrotomy, usually referred to as cryo-electron microscopy of vitreous sections (CEMOVIS). We observed that the first method preserved ACC well in 500-nm-thick sections but not in 70-nm-thick sections. However, cell ultrastructures were difficult to clearly observe in the 500-nm-thick sections. In contrast, CEMOVIS provided a high preservation quality of bacterial ultrastructures, including the intracellular ACC inclusions in 50-nm-thick sections. ACC inclusions displayed different textures, suggesting varying brittleness, possibly resulting from different hydration levels. Moreover, an electron dense envelope of ∼2.5 nm was systematically observed around ACC granules in all studied cyanobacterial strains. This envelope may be composed of a protein shell or a lipid monolayer, but not a lipid bilayer as usually observed in other bacteria forming intracellular minerals. Overall, this study evidenced that ACC inclusions formed and were stabilized within a previously unidentified bacterial microcompartment in some species of cyanobacteria.
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Biomineralization Patterns of Intracellular Carbonatogenesis in Cyanobacteria: Molecular Hypotheses
Minerals, 2016Co-Authors: Isabel Margaret-oliver, Nithavong Cam, Thomas Boudier, Marine Blondeau, Eric Leroy, Julie Cosmidis, Feriel Skouri-panet, Jean-michel Guigner, Céline Férard, Melanie PoinsotAbstract:The recent discovery of intracellular carbonatogenesis in several cyanobacteria species has challenged the traditional view that this process was extracellular and not controlled. However, a detailed analysis of the size distribution, chemical composition and 3-D-arrangement of carbonates in these cyanobacteria is lacking. Here, we characterized these features in Candidatus Gloeomargarita lithophora C7 and Candidatus Synechococcus calcipolaris G9 by conventional transmission electron microscopy, tomography, Ultramicrotomy, and scanning transmission X-ray microscopy (STXM). Both Ca. G. lithophora C7 and Ca. S. calcipolaris G9 formed numerous polyphosphate granules adjacent or engulfing Ca-carbonate inclusions when grown in phosphate-rich solutions. Ca-carbonates were scattered within Ca. G. lithophora C7 cells under these conditions, but sometimes arranged in one or several chains. In contrast, Ca-carbonates formed at cell septa in Ca. S. calcipolaris G9 and were segregated equally between daughter cells after cell division, arranging as distorted disks at cell poles. The size distribution of carbonates evolved from a positively to a negatively skewed distribution as particles grew. Conventional Ultramicrotomy did not preserve Ca-carbonates explaining partly why intracellular calcification has been overlooked in the past. All these new observations allow discussing with unprecedented insight some nucleation and growth processes occurring in intracellularly calcifying cyanobacteria with a particular emphasis on the possible involvement of intracellular compartments and cytoskeleton.
Marine Blondeau - One of the best experts on this subject based on the ideXlab platform.
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Amorphous Calcium Carbonate Granules Form Within an Intracellular Compartment in Calcifying Cyanobacteria
Frontiers in Microbiology, 2018Co-Authors: Marine Blondeau, Feriel Skouri-panet, Jean-michel Guigner, Céline Férard, Melanie Poinsot, Martin Sachse, Claire Boulogne, Cynthia Gillet, Jennyfer Miot, Karim BenzeraraAbstract:The recent discovery of cyanobacteria forming intracellular amorphous calcium carbonate (ACC) has challenged the former paradigm suggesting that cyanobacteria-mediated carbonatogenesis was exclusively extracellular. Yet, the mechanisms of intracellular biomineralization in cyanobacteria and in particular whether this takes place within an intracellular microcompartment, remain poorly understood. Here, we analyzed six cyanobacterial strains forming intracellular ACC by transmission electron microscopy. We tested two different approaches to preserve as well as possible the intracellular ACC inclusions: (i) freeze-substitution followed by epoxy embedding and room-temperature Ultramicrotomy and (ii) high-pressure freezing followed by cryo-Ultramicrotomy, usually referred to as cryo-electron microscopy of vitreous sections (CEMOVIS). We observed that the first method preserved ACC well in 500-nm-thick sections but not in 70-nm-thick sections. However, cell ultrastructures were difficult to clearly observe in the 500-nm-thick sections. In contrast, CEMOVIS provided a high preservation quality of bacterial ultrastructures, including the intracellular ACC inclusions in 50-nm-thick sections. ACC inclusions displayed different textures, suggesting varying brittleness, possibly resulting from different hydration levels. Moreover, an electron dense envelope of ∼2.5 nm was systematically observed around ACC granules in all studied cyanobacterial strains. This envelope may be composed of a protein shell or a lipid monolayer, but not a lipid bilayer as usually observed in other bacteria forming intracellular minerals. Overall, this study evidenced that ACC inclusions formed and were stabilized within a previously unidentified bacterial microcompartment in some species of cyanobacteria.
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Data_Sheet_1_Amorphous Calcium Carbonate Granules Form Within an Intracellular Compartment in Calcifying Cyanobacteria.PDF
2018Co-Authors: Marine Blondeau, Feriel Skouri-panet, Jean-michel Guigner, Céline Férard, Melanie Poinsot, Martin Sachse, Claire Boulogne, Cynthia Gillet, Jennyfer Miot, Karim BenzeraraAbstract:The recent discovery of cyanobacteria forming intracellular amorphous calcium carbonate (ACC) has challenged the former paradigm suggesting that cyanobacteria-mediated carbonatogenesis was exclusively extracellular. Yet, the mechanisms of intracellular biomineralization in cyanobacteria and in particular whether this takes place within an intracellular microcompartment, remain poorly understood. Here, we analyzed six cyanobacterial strains forming intracellular ACC by transmission electron microscopy. We tested two different approaches to preserve as well as possible the intracellular ACC inclusions: (i) freeze-substitution followed by epoxy embedding and room-temperature Ultramicrotomy and (ii) high-pressure freezing followed by cryo-Ultramicrotomy, usually referred to as cryo-electron microscopy of vitreous sections (CEMOVIS). We observed that the first method preserved ACC well in 500-nm-thick sections but not in 70-nm-thick sections. However, cell ultrastructures were difficult to clearly observe in the 500-nm-thick sections. In contrast, CEMOVIS provided a high preservation quality of bacterial ultrastructures, including the intracellular ACC inclusions in 50-nm-thick sections. ACC inclusions displayed different textures, suggesting varying brittleness, possibly resulting from different hydration levels. Moreover, an electron dense envelope of ∼2.5 nm was systematically observed around ACC granules in all studied cyanobacterial strains. This envelope may be composed of a protein shell or a lipid monolayer, but not a lipid bilayer as usually observed in other bacteria forming intracellular minerals. Overall, this study evidenced that ACC inclusions formed and were stabilized within a previously unidentified bacterial microcompartment in some species of cyanobacteria.
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Biomineralization Patterns of Intracellular Carbonatogenesis in Cyanobacteria: Molecular Hypotheses
Minerals, 2016Co-Authors: Isabel Margaret-oliver, Nithavong Cam, Thomas Boudier, Marine Blondeau, Eric Leroy, Julie Cosmidis, Feriel Skouri-panet, Jean-michel Guigner, Céline Férard, Melanie PoinsotAbstract:The recent discovery of intracellular carbonatogenesis in several cyanobacteria species has challenged the traditional view that this process was extracellular and not controlled. However, a detailed analysis of the size distribution, chemical composition and 3-D-arrangement of carbonates in these cyanobacteria is lacking. Here, we characterized these features in Candidatus Gloeomargarita lithophora C7 and Candidatus Synechococcus calcipolaris G9 by conventional transmission electron microscopy, tomography, Ultramicrotomy, and scanning transmission X-ray microscopy (STXM). Both Ca. G. lithophora C7 and Ca. S. calcipolaris G9 formed numerous polyphosphate granules adjacent or engulfing Ca-carbonate inclusions when grown in phosphate-rich solutions. Ca-carbonates were scattered within Ca. G. lithophora C7 cells under these conditions, but sometimes arranged in one or several chains. In contrast, Ca-carbonates formed at cell septa in Ca. S. calcipolaris G9 and were segregated equally between daughter cells after cell division, arranging as distorted disks at cell poles. The size distribution of carbonates evolved from a positively to a negatively skewed distribution as particles grew. Conventional Ultramicrotomy did not preserve Ca-carbonates explaining partly why intracellular calcification has been overlooked in the past. All these new observations allow discussing with unprecedented insight some nucleation and growth processes occurring in intracellularly calcifying cyanobacteria with a particular emphasis on the possible involvement of intracellular compartments and cytoskeleton.
