scholarly journals A modified lysosomal organelle mediates nonlytic egress of reovirus

2020 ◽  
Vol 219 (7) ◽  
Author(s):  
Isabel Fernández de Castro ◽  
Raquel Tenorio ◽  
Paula Ortega-González ◽  
Jonathan J. Knowlton ◽  
Paula F. Zamora ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Electron Tomography ◽  
Microvascular Endothelial Cells ◽  
Brain Microvascular Endothelial Cells ◽  
Basal Surface ◽  
Reovirus Infection ◽  
Transmission Electron ◽  
Membrane Bound ◽  
Viral Inclusions ◽  
Virus Egress

Mammalian orthoreoviruses (reoviruses) are nonenveloped viruses that replicate in cytoplasmic membranous organelles called viral inclusions (VIs) where progeny virions are assembled. To better understand cellular routes of nonlytic reovirus exit, we imaged sites of virus egress in infected, nonpolarized human brain microvascular endothelial cells (HBMECs) and observed one or two distinct egress zones per cell at the basal surface. Transmission electron microscopy and 3D electron tomography (ET) of the egress zones revealed clusters of virions within membrane-bound structures, which we term membranous carriers (MCs), approaching and fusing with the plasma membrane. These virion-containing MCs emerged from larger, LAMP-1–positive membranous organelles that are morphologically compatible with lysosomes. We call these structures sorting organelles (SOs). Reovirus infection induces an increase in the number and size of lysosomes and modifies the pH of these organelles from ∼4.5–5 to ∼6.1 after recruitment to VIs and before incorporation of virions. ET of VI–SO–MC interfaces demonstrated that these compartments are connected by membrane-fusion points, through which mature virions are transported. Collectively, our results show that reovirus uses a previously undescribed, membrane-engaged, nonlytic egress mechanism and highlights a potential new target for therapeutic intervention.

Three-Dimensional Dispersion of Nano-Fillers in Soft Composite as Revealed by Transmission Electron Microscopy/Electron Tomography (3D-TEM)

2006 ◽  
Vol 514-516 ◽  
pp. 353-358 ◽  
Author(s):  
Shinzo Kohjiya
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Carbon Black ◽  
Natural Rubber ◽  
Structural Information ◽  
Electron Tomography ◽  
Nano Composites ◽  
Nano Structure ◽  
Transmission Electron ◽  
Particulate Silica

. Generally rubber products are a typical soft material, and a composite of a nano-filler (typically, carbon black or particulate silica) and a rubber (natural rubber and various synthetics are used). The properties of these soft nano-composites have been well known to depend on the dispersion of the nano-filler in the rubbery matrix. The most powerful tool for the elucidation of it has been transmission electron microscopy (TEM). The microscopic techniques are based on the projection of 3-dimensional (3D) body on a plane (x, y plane), thus the structural information along the thickness (z axis) direction of the sample is difficult to obtain. This paper describes our recent results on the dispersion of carbon black (CB) and particulate silica in natural rubber (NR) matrix observed by TEM combined with electron tomography (3D-TEM) technique, which enabled us to obtain images of 3D nano-structure of the sample. Thus, 3D images of CB and silica in NR matrix are visualized and analyzed in this communication. These results are precious ones for the design of soft nano-composites, and the technique will become an indispensable one in nanotechnology.

scholarly journals Recent Advances in Transmission Electron Microscopy for Materials Science at the EMAT Lab of the University of Antwerp

Materials ◽  
2018 ◽  
Vol 11 (8) ◽  
pp. 1304 ◽  
Author(s):  
Giulio Guzzinati ◽  
Thomas Altantzis ◽  
Maria Batuk ◽  
Annick De Backer ◽  
Gunnar Lumbeeck ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Materials Science ◽  
Electron Tomography ◽  
High Resolution Imaging ◽  
Rapid Progress ◽  
Transmission Electron ◽  
The World ◽  
Resolution Imaging ◽  
The University

The rapid progress in materials science that enables the design of materials down to the nanoscale also demands characterization techniques able to analyze the materials down to the same scale, such as transmission electron microscopy. As Belgium’s foremost electron microscopy group, among the largest in the world, EMAT is continuously contributing to the development of TEM techniques, such as high-resolution imaging, diffraction, electron tomography, and spectroscopies, with an emphasis on quantification and reproducibility, as well as employing TEM methodology at the highest level to solve real-world materials science problems. The lab’s recent contributions are presented here together with specific case studies in order to highlight the usefulness of TEM to the advancement of materials science.

scholarly journals Infection With a Novel Rickettsiella Species in Emperor Scorpions (Pandinus imperator)

2020 ◽  
Vol 57 (6) ◽  
pp. 858-870
Author(s):  
Sushan Han ◽  
Aníbal G. Armién ◽  
Janet E. Hill ◽  
Champika Fernando ◽  
Dan S. Bradway ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Rapid Progression ◽  
Protein Coding ◽  
Hematopoietic Organ ◽  
Transmission Electron ◽  
History Of ◽  
Gross Lesions ◽  
Membrane Bound ◽  
Methenamine Silver

Rickettsiella infection was diagnosed in 4 adult emperor scorpions ( Pandinus imperator) from 2 different collections over a 3-year period. One case had a 2-day history of weakness, failure to lift the tail, or respond to stimulation, with rapid progression to death. The other 3 cases were found dead. There were no gross lesions, but histologically the hemolymphatic vasculature and sinuses, presumed hematopoietic organ, heart, midgut and midgut diverticula, nerves, and skeletal muscle were infiltrated with phagocytic and granular hemocytes with necrosis. Phagocytic hemocytes contained abundant intracellular microorganisms that were Fite’s acid-fast-positive, Macchiavello-positive, variably gram-positive or gram-negative, and Grocott’s methenamine silver-negative. By transmission electron microscopy, hemocytes contained numerous phagocytic vacuoles with small dense bacterial forms (mean 0.603 × 0.163 μm) interspersed with large bacterial forms (mean 1.265 × 0.505 μm) and few intermediary forms with electron-dense nucleoids and membrane-bound crystalline arrays (average 4.72 μm). Transmission electron microscopy findings were consistent with bacteria of the family Coxiellaceae. Based on sequencing the 16S ribosomal RNA gene, the identity was confirmed as Rickettsiella, and phylogenetic analysis of protein-coding genes gidA, rspA, and sucB genes suggested the emperor scorpion pathogen as a new species. This study identifies a novel Rickettsiella causing infection in emperor scorpions and characterizes the unique pathological findings of this disease. We suggest this organism be provisionally named Rickettsiella scorpionisepticum.

Computer-Controlled Transmission Electron Microscopy: Automated Tomography

2001 ◽  
Vol 7 (S2) ◽  
pp. 968-969
Author(s):  
Theo van der Krift ◽  
Ulrike Ziese ◽  
Willie Geerts ◽  
Bram Koster
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
User Interfaces ◽  
Electron Tomography ◽  
Imaging Method ◽  
Optical Elements ◽  
Computer Screen ◽  
Transmission Electron ◽  
Computer Controlled ◽  
Electron Microscopes

The integration of computers and transmission electron microscopes (TEM) in combination with the availability of computer networks evolves in various fields of computer-controlled electron microscopy. Three layers can be discriminated: control of electron-optical elements in the column, automation of specific microscope operation procedures and display of user interfaces. The first layer of development concerns the computer-control of the optical elements of the transmission electron microscope (TEM). Most of the TEM manufacturers have transformed their optical instruments into computer-controlled image capturing devices. Nowadays, the required controls for the currents through lenses and coils of the optical column can be accessed by computer software. The second layer of development is aimed toward further automation of instrument operation. For specific microscope applications, dedicated automated microscope-control procedures are carried out. in this paper, we will discuss our ongoing efforts on this second level towards fully automated electron tomography. The third layer of development concerns virtual- or telemicroscopy. Most telemicroscopy applications duplicate the computer-screen (with accessory controls) at the microscope-site to a computer-screen at another site. This approach allows sharing of equipment, monitoring of instruments by supervisors, as well as collaboration between experts at remote locations.Electron tomography is a three-dimensional (3D) imaging method with transmission electron microscopy (TEM) that provides high-resolution 3D images of structural arrangements. with electron tomography a series of images is acquired of a sample that is tilted over a large angular range (±70°) with small angular tilt increments.

Three-Dimensional Imaging of Shear Band Produced by ECAP Process in Al-Ag Alloy

2006 ◽  
Vol 503-504 ◽  
pp. 603-608
Author(s):  
Koji Inoke ◽  
Kenji Kaneko ◽  
Z. Horita
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Electron Tomography ◽  
Shear Bands ◽  
Three Dimensional ◽  
Angular Pressing ◽  
Ecap Process ◽  
Transmission Electron ◽  
Scanning Transmission ◽  
The Matrix

A significant change in microstructure occurs during the application of severe plastic deformation (SPD) such as by equal-channel angular pressing (ECAP). In this study, intense plastic strain was imposed on an Al-10.8wt%Ag alloy by the ECAP process. The amount of strain was controlled by the numbers of passes. After 1 pass of ECAP, shear bands became visible within the matrix. With increasing numbers of ECAP passes, the fraction of shear bands was increased. In this study, the change in microstructures was examined by three-dimensional electron tomography (3D-ET) in transmission electron microscopy (TEM) or scanning transmission electron microscopy (STEM). With this 3D-ET method, it was possible to conduct a precise analysis of the sizes, widths and distributions of the shear bands produced by the ECAP process. It is demonstrated that the 3D-ET method is promising to understand mechanisms of microstructural refinement using the ECAP process.

Phenethyl Isothiocyanate (PEITC) Regulates Autophagy in Chronic Lymphocytic Leukemia.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2906-2906
Author(s):  
Jemimah Adams ◽  
R Gitendra Wickremasinghe ◽  
Archibald G Prentice ◽  
Jonathan C. Strefford ◽  
Andrew Duncombe ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Annexin V ◽  
Lymphocytic Leukemia ◽  
Phenethyl Isothiocyanate ◽  
Autophagosome Formation ◽  
Double Membrane ◽  
Transmission Electron ◽  
Membrane Bound ◽  
Autophagy Pathway

Abstract Abstract 2906 Chronic Lymphocytic leukemia (CLL) is currently incurable using conventional therapies. CLL cells can evade killing by various therapeutic strategies. However the precise mechanisms are currently unknown. Autophagy is regulated by a complex system of proteins, and is used by both normal and malignant cells as a protective mechanism against cellular stress induced by starvation, hypoxia, reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress. In malignant cells autophagy was shown to promote tumorigenesis and/or resistance to chemotherapy. Therefore we hypothesized that autophagy may play a role in CLL biology. Autophagy can also promote cell death when stress signals are elevated above a particular threshold for a prolonged period of time. In this study we investigated the basal expression levels of autophagy specific genes and the effect of autophagy specific inhibitors (Bafilomycin, 3-methyladenine and hydroxychloroquine) and inducers (Phenethyl isothiocyanate) on CLL survival. Phenethyl isothiocyanate (PEITC) is about to enter clinical trials for CLL (NCT00968461). We have investigated induction of components of the autophagic pathway following treatment of CLL cells in vitro with a range of chemical inhibitors. Immunoblotting was carried out to investigate components of the autophagy pathway using phosphorylation state-specific and pan-reactive antibodies. Bafilomycin (BAF), 3-methyladenine (3-MA) and hydroxychloroquine (HCQ) toxicity towards CLL samples were evaluated by Annexin V/PI staining, MTT assay and immunoblotting for cleavage of the caspase 3 substrate poly(ADP ribose) polymerase (PARP) from its 116KDa to its 85KDa form. PEITC was used at concentrations between 2.5 and 25μM to investigate its effect on signaling. Autophagy was quantitated by immunoblotting of LC3-I and LC3-II. Lipidation of LC3 from LC3-I to LC3-II is a surrogate marker of autophagy and is essential for autophagasome formation. Immunoblotting was also performed for ATG3, ATG5 and ATG7, key components of the autophagy pathway. Monodansylcadaverine (MDC) was used with immunofluorescence and FACS analysis to investigate increases in autophagasome formation. Transmission electron microscopy (TEM) was used to confirm double membrane bound autophagosomes. Co-immunoprecipitation was used to evaluate if Beclin-1 was sequestered by Bcl-2 preventing autophagy. Its release from Bcl-2 enables Beclin-1 to interact with other autophagy specific proteins and initiates autophagasome formation. LC3-I was lipidated to LC3-II (p=0.019) and ATG3 (p=0.021) was upregulated to a greater extent in CLL samples compared with normal B-cell controls at basal levels. This suggested that autophagy was active to a greater extent in CLL samples compared with normal individuals. In addition Beclin was dissociated from Bcl-2 in CLL samples indicating that autophagy was active. Autophagy appears to be a pro-survival mechanism in untreated CLL cells as inhibiting basal levels of autophagy with autophagy inhibitors BAF (50–200nM), 3-MA (5–10mM) and hydroxychlorquine (5–10μM) resulted in CLL apoptosis as shown by MTT, Annexin V/PI analysis and PARP cleavage. Interestingly augmenting autophagy was also capable of inducing apoptosis in CLL samples. Treatment with PEITC caused an increase in punctate staining using MDC which is suggestive of autophagosome formation. We went on to determine that PEITC further induced LC3-II lipidation using immunoblotting and showed a substantial increase in overall LC3 protein expression. PEITC also induced the expression of ATG3, a key protein in the autophagy pathway. We then evaluated autophagosome formation using TEM (Figure 1). Our data showed greater numbers of autophagosomes in the PEITC treated samples compared to the untreated controls. Therefore autophagy in CLL sits on a knife-edge, such that perturbations that either increase pro- death or decrease pro-survival autophagy signals can result in CLL cell death, depending on the duration and intensity of the signal. Figure 1. Transmission electron microscopy of CLL cells CLL cells were treated with 10μM PEITC. Double membrane bound organelles were found in the CLL cells after treatment which were not present in the no addition control (depicted by the arrows). These organelles are autophagsomes. Magnification (left picture) ruler is 500nM, (right picture) ruler is 100nM Figure 1. Transmission electron microscopy of CLL cells . / CLL cells were treated with 10μM PEITC. Double membrane bound organelles were found in the CLL cells after treatment which were not present in the no addition control (depicted by the arrows). These organelles are autophagsomes. Magnification (left picture) ruler is 500nM, (right picture) ruler is 100nM Disclosures: No relevant conflicts of interest to declare.

Three-Dimensional Chemistry of Multiphase Nanomaterials by Energy-Filtered Transmission Electron Microscopy Tomography

2012 ◽  
Vol 18 (5) ◽  
pp. 1118-1128 ◽  
Author(s):  
Lucian Roiban ◽  
Loïc Sorbier ◽  
Christophe Pichon ◽  
Pascale Bayle-Guillemaud ◽  
Jacques Werckmann ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Point Of View ◽  
Analysis Tool ◽  
Shell Nanoparticles ◽  
3D Nanostructures ◽  
Transmission Electron ◽  
Silica Alumina

AbstractA three-dimensional (3D) study of multiphase nanostructures by chemically selective electron tomography combining tomographic approach and energy-filtered imaging is reported. The implementation of this technique at the nanometer scale requires careful procedures for data acquisition, computing, and analysis. Based on the performances of modern transmission electron microscopy equipment and on developments in data processing, electron tomography in the energy-filtered imaging mode is shown to be a very appropriate analysis tool to provide 3D chemical maps at the nanoscale. Two examples highlight the usefulness of analytical electron tomography to investigate inhomogeneous 3D nanostructures, such as multiphase specimens or core-shell nanoparticles. The capability of discerning in a silica-alumina porous particle the two different components is illustrated. A quantitative analysis in the whole specimen and toward the pore surface is reported. This tool is shown to open new perspectives in catalysis by providing a way to characterize precisely 3D nanostructures from a chemical point of view.

Cryo-transmission electron microscopy of Ag nanoparticles grown on an ionic liquid substrate

2010 ◽  
Vol 25 (7) ◽  
pp. 1264-1271 ◽  
Author(s):  
Dalaver H. Anjum ◽  
Rebecca M. Stiger ◽  
James J. Finley ◽  
James F. Conway
Keyword(s):  
Electron Microscopy ◽  
Ionic Liquid ◽  
Electron Tomography ◽  
Silver Nanostructures ◽  
Electron Dose ◽  
Transmission Electron ◽  
Induced Motion ◽  
Novel Method ◽  
Individual Nanoparticles

We report a novel method of growing silver nanostructures by cathodic sputtering onto an ionic liquid (IL) and our visualization by transmission cryo-electron microscopy to avoid beam-induced motion of the nanoparticles. By freezing the IL suspension and controlling electron dose, we can assess properties of particle size, morphology, crystallinity, and aggregation in situ and at high detail. We observed round silver nanoparticles with a well-defined diameter of 7.0 ± 1.5 nm that are faceted with crystalline cubic structures and ˜80% of the particles have multiply twinned faults. We also applied cryo-electron tomography to investigate the structure of the nanoparticles and to directly visualize the IL wetting around them. In addition to particles, we observed nanorods that appear to have assembled from individual nanoparticles. Reexamination of the samples after 4–5 days from initial preparation showed significant changes in morphology, and potential mechanisms for this are discussed.

scholarly journals From tissue retrieval to electron tomography: nanoscale characterization of the interface between bone and bioactive glass

2021 ◽  
Vol 18 (182) ◽  
pp. 20210181
Author(s):  
Chiara Micheletti ◽  
Pedro Henrique Silva Gomes-Ferreira ◽  
Travis Casagrande ◽  
Paulo Noronha Lisboa-Filho ◽  
Roberta Okamoto ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Bioactive Glass ◽  
Focused Ion Beam ◽  
Electron Tomography ◽  
Ion Beam ◽  
Three Dimensions ◽  
Transmission Electron Microscopy Analysis ◽  
Dental Procedures ◽  
Transmission Electron

The success of biomaterials for bone regeneration relies on many factors, among which osseointegration plays a key role. Biogran (BG) is a bioactive glass commonly employed as a bone graft in dental procedures. Despite its use in clinical practice, the capability of BG to promote osseointegration has never been resolved at the nanoscale. In this paper, we present the workflow for characterizing the interface between newly formed bone and BG in a preclinical rat model. Areas of bone–BG contact were first identified by backscattered electron imaging in a scanning electron microscope. A focused ion beam in situ lift-out protocol was employed to prepare ultrathin samples for transmission electron microscopy analysis. The bone–BG gradual interface, i.e. the biointerphase, was visualized at the nanoscale with unprecedented resolution thanks to scanning transmission electron microscopy. Finally, we present a method to view the bone–BG interface in three dimensions using electron tomography.

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