Studying cytokinesis and midbody remnants using correlative light/scanning EM

2017 ◽  
pp. 239-251 ◽  
Author(s):  
S. Frémont ◽  
A. Echard
Keyword(s):  
Scanning Em

The ultrastructure of the double membrane-bound bodies and endoplasmic reticulum in serial sections of wheat spot mosaic-affected wheat plants

1990 ◽  
Vol 48 (3) ◽  
pp. 694-695
Author(s):  
M. H. Chen ◽  
C. Hiruki
Keyword(s):  
Endoplasmic Reticulum ◽  
High Resolution ◽  
Membrane Structure ◽  
Mosaic Disease ◽  
Serial Sections ◽  
Thin Sections ◽  
Double Membrane ◽  
Southern Alberta ◽  
Membrane Bound ◽  
Scanning Em

Wheat spot mosaic disease was first discovered in southern Alberta, Canada, in 1956. A hitherto unidentified disease-causing agent, transmitted by the eriophyid mite, caused chlorosis, stunting and finally severe necrosis resulting in the death of the affected plants. Double membrane-bound bodies (DMBB), 0.1-0.2 μm in diameter were found to be associated with the disease.Young tissues of leaf and root from 4-wk-old infected wheat plants were fixed, dehydrated, and embedded in Spurr’s resin. Serial sections were collected on slot copper grids and stained. The thin sections were then examined with a Hitachi H-7000 TEM at 75 kV. The membrane structure of the DMBBs was studied by numbering them individually and tracing along the sections to see any physical connection with endoplasmic reticulum (ER) membranes. For high resolution scanning EM, a modification of Tanaka’s method was used. The specimens were examined with a Hitachi Model S-570 SEM in its high resolution mode at 20 kV.

Morphilogy and Ultrastructure of in Vitro Cultures of Aortic Endothelial Cells Interacting with Antibodies

1979 ◽  
Author(s):  
S. Korach ◽  
D. Ngo
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Vascular Endothelial Cells ◽  
Intercellular Junctions ◽  
Cell Types ◽  
Endothelial Damage ◽  
Antibody Concentration ◽  
High Yields ◽  
Scanning Em

Adult pig aortas, sectioned longitudinally, were incubated in 0.1% collagenase-PBS (15 mn, 37°C). Gentle scraping of the lumenal surface resulted in high yields (3-4 x 106 cell/aorta) of viable endothelial cells, essentially devoid of other cell types by morphological and immunochemical (F VIII-antigen) criteria. Confluent monolayers were incubated for various times (5 mn to 1 wk) with decomplemented rabbit antisera raised against pig endothelial cells. Changes in cell morphology appeared to depend on antibody concentration rather than on duration of contact with antiserum. High concentrations of antiserum (5 to 20%) led to cytoplasmic shredding, bulging of cells and extensive vacuolization, whereas at lower concentrations, cells appeared almost normal. Transmission EM studies by the indirect immunoperoxydase method showed antibodies reacting with unfixed cells to be distributed all over the upper cell surface, in the outer parts of intercellular junctions, and within numerous pinocytotic vesicles. Much weaker reactions could also be seen at the lower cell surface. When viewed under the Scanning EM, antiserum-treated endothelial cells also disclosed antibody concentration-dependent bulging and release of cells from their substrate. In vitro studies of gradual modifications of vascular endothelial cells acted upon by antibodies should provide a better understanding of the structural and biochemical processes underlying endothelial damage and detachment.

scholarly journals Batch Production of High-Quality Graphene Grids for Cryo-EM: Cryo-EM Structure of Methylococcus capsulatus Soluble Methane Monooxygenase Hydroxylase

2021 ◽  
Author(s):  
Eungjin Ahn ◽  
byungchul Kim ◽  
uhn-soo Cho
Keyword(s):  
Protein Denaturation ◽  
Methane Monooxygenase ◽  
Atomic Layer ◽  
Water Interface ◽  
Methylococcus Capsulatus ◽  
Force Microscopy ◽  
Voltage Electron ◽  
Soluble Methane Monooxygenase ◽  
Air Water Interface ◽  
Scanning Em

Cryogenic electron microscopy (cryo-EM) has become a widely used tool for determining protein structure. Despite recent advances in instruments and algorithms, sample preparation remains a major bottleneck for several reasons, including protein denaturation at the air/water interface and the presence of preferred orientations and nonuniform ice layers. Graphene, a two-dimensional allotrope of carbon consisting of a single atomic layer, has recently attracted attention as a near-ideal support film for cryo-EM that can overcome these challenges because of its superior properties, including mechanical strength and electrical conductivity. Graphene minimizes background noise and provides a stable platform for specimens under a high-voltage electron beam and cryogenic conditions. Here, we introduce a reliable, easily implemented, and reproducible method of producing 36 graphene-coated grids at once within 1.5 days. The quality of the graphene grids was assessed using various tools such as scanning EM, Raman spectroscopy, and atomic force microscopy. To demonstrate their practical application, we determined the cryo-EM structure of Methylococcus capsulatus soluble methane monooxygenase hydroxylase (sMMOH) at resolutions of 2.9 and 2.4 angstrom using Quantifoil and graphene-coated grids, respectively. We found that the graphene-coated grid has several advantages; for example, it requires less protein, enables easy control of the ice thickness, and prevents pro-tein denaturation at the air/water interface. By comparing the cryo-EM structure of sMMOH with its crystal structure, we revealed subtle yet significant geometrical differences at the non-heme di-iron center, which may better indicate the active site configuration of sMMOH in the resting/oxidized state.

scholarly journals High-precision targeting workflow for volume electron microscopy

2021 ◽  
Vol 220 (9) ◽  
Author(s):  
Paolo Ronchi ◽  
Giulia Mizzon ◽  
Pedro Machado ◽  
Edoardo D’Imprima ◽  
Benedikt T. Best ◽  
...  
Keyword(s):  
Focused Ion Beam ◽  
Single Cells ◽  
Ion Beam ◽  
Mouse Mammary Gland ◽  
Fluorescence Signal ◽  
3D Objects ◽  
Scanning Em ◽  
Automated Data Acquisition ◽  
Volume Electron Microscopy ◽  
Volume Em

Cells are 3D objects. Therefore, volume EM (vEM) is often crucial for correct interpretation of ultrastructural data. Today, scanning EM (SEM) methods such as focused ion beam (FIB)–SEM are frequently used for vEM analyses. While they allow automated data acquisition, precise targeting of volumes of interest within a large sample remains challenging. Here, we provide a workflow to target FIB-SEM acquisition of fluorescently labeled cells or subcellular structures with micrometer precision. The strategy relies on fluorescence preservation during sample preparation and targeted trimming guided by confocal maps of the fluorescence signal in the resin block. Laser branding is used to create landmarks on the block surface to position the FIB-SEM acquisition. Using this method, we acquired volumes of specific single cells within large tissues such as 3D cultures of mouse mammary gland organoids, tracheal terminal cells in Drosophila melanogaster larvae, and ovarian follicular cells in adult Drosophila, discovering ultrastructural details that could not be appreciated before.

Endothelial gaps: time course of formation and closure in inflamed venules of rats

1997 ◽  
Vol 272 (1) ◽  
pp. L155-L170 ◽  
Author(s):  
P. Baluk ◽  
A. Hirata ◽  
G. Thurston ◽  
T. Fujiwara ◽  
C. R. Neal ◽  
...  
Keyword(s):  
Substance P ◽  
Light Microscopy ◽  
Half Life ◽  
Time Course ◽  
Gap Formation ◽  
Gap Closure ◽  
Silver Nitrate Staining ◽  
Plasma Leakage ◽  
Scanning Em ◽  
Endothelial Gaps

In the rat trachea, substance P causes rapid but transient plasma leakage. We sought to determine how closely the number, morphology, and size of endothelial gaps correspond to the time course of this leakage. Endothelial gaps were examined by scanning electron microscopy (EM), by transmission EM, or by light microscopy after silver nitrate staining. Substance P-induced leakage of the particulate tracer Monastral blue peaked at 1 min but decreased with a half-life of 0.3 min. The number of silver-stained gaps also peaked at 1 min then decreased significantly more slowly (half-life 1.9 min) than the leakage. Scanning EM revealed two types of endothelial gaps, designated vertical gaps and oblique slits. Vertical gaps predominated at peak leakage, whereas oblique slits became more common as the leakage diminished. Measurements of the mean diameter of vertical gaps made by light microscopy, scanning EM, and transmission EM were all in the range of 0.36-0.47 micron. Fingerlike endothelial cell processes that appeared during gap formation became shorter as the leakage diminished (mean length: 1.44 microns at 1 min compared with 1.06 microns at 3 min after substance P), suggesting a role in gap closure. We conclude that the plasma leakage occurring immediately after an inflammatory stimulus results from the rapid formation of endothelial gaps. Multiple factors, including alterations in gap morphology, gap closure, and changes in driving force, are likely to participate in the rapid decrease in the leakage.

scholarly journals Structure and Assembly of Intracellular Mature Vaccinia Virus: Isolated-Particle Analysis

2001 ◽  
Vol 75 (22) ◽  
pp. 11034-11055 ◽  
Author(s):  
Gareth Griffiths ◽  
Roger Wepf ◽  
Thomas Wendt ◽  
Jacomine Krijnse Locker ◽  
Marek Cyrklaff ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Vaccinia Virus ◽  
Viral Entry ◽  
Particle Analysis ◽  
Assembly Process ◽  
Novel Approach ◽  
Complex Topology ◽  
Scanning Em ◽  
Mature Virus

ABSTRACT In a series of papers, we have provided evidence that during its assembly vaccinia virus is enveloped by a membrane cisterna that originates from a specialized, virally modified, smooth-membraned domain of the endoplasmic reticulum (ER). Recently, however, Hollinshead et al. (M. Hollinshead, A. Vanderplasschen, G. I. Smith, and D. J. Vaux, J. Virol. 73:1503–1517, 1999) argued against this hypothesis, based on their interpretations of thin-sectioned material. The present article is the first in a series of papers that describe a comprehensive electron microscopy (EM) analysis of the vaccinia Intracellular Mature Virus (IMV) and the process of its assembly in HeLa cells. In this first study, we analyzed the IMV by on-grid staining, cryo-scanning EM (SEM), and cryo-transmission EM. We focused on the structure of the IMV particle, both after isolation and in the context of viral entry. For the latter, we used high-resolution cryo-SEM combined with cryofixation, as well as a novel approach we developed for investigating vaccinia IMV bound to plasma membrane fragments adsorbed onto EM grids. Our analysis revealed that the IMV is made up of interconnected cisternal and tubular domains that fold upon themselves via a complex topology that includes an S-shaped fold. The viral tubules appear to be eviscerated from the particle during viral infection. Since the structure of the IMV is the result of a complex assembly process, we also provide a working model to explain how a specialized smooth-ER domain can be modulated to form the IMV. We also present theoretical arguments for why it is highly unlikely that the IMV is surrounded by only a single membrane.

scholarly journals Scanning EM studies of normal human lens fibres and fibres from nuclear cataracts

Eye ◽  
1991 ◽  
Vol 5 (1) ◽  
pp. 86-92 ◽  
Author(s):  
R J Stirling ◽  
P G Griffiths
Keyword(s):  
Human Lens ◽  
Normal Human ◽  
Scanning Em

Scanning Em study of the living cyrtocrinid Holopus rangii (Echinodermata, Crinoidea) and implications for its functional morphology

1992 ◽  
Vol 66 (4) ◽  
pp. 665-675 ◽  
Author(s):  
Stephen K. Donovan
Keyword(s):  
Functional Morphology ◽  
Living Animal ◽  
Sem Study ◽  
Scanning Em ◽  
Hard Substrates

The unusual extant crinoid Holopus rangii d'Orbigny lacks a column, cements directly to hard substrates, has a conical to tubular dorsal cup in which plate sutures cannot be discerned, and has 10 tapered arms that are able to enroll swiftly into a “fist-like” configuration when disturbed. The first SEM study of H. rangii is presented herein. The gross morphology and skeletal microstructure of this species display many characteristics that bear an obvious relationship to the functional morphology of the living animal. Dorsal cups show a range of morphologies, reminiscent of those found in acorn barnacles and other tubular encrusters. During arm enrollment, the adjacent arms abut and form an impervious seal. Of the two specimens dissected, one had a regenerating arm.

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