Assessment of different fixation protocols on the presence of membrane-bound vesicles in Caco-2 cells: A multidimensional view by means of correlative light and 3-D transmission electron microscopy

Micron ◽  
2014 ◽  
Vol 67 ◽  
pp. 20-29 ◽  
Author(s):  
Gerald Shami ◽  
Delfine Cheng ◽  
Jeffrey Henriquez ◽  
Filip Braet
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Transmission Electron ◽  
Membrane Bound

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.

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.

The ultrastructure of the skeleton and skeletogenic tissues of the temperate coral Caryophyllia smithii

1990 ◽  
Vol 70 (2) ◽  
pp. 295-310 ◽  
Author(s):  
Martin D'A.A. Le Tissier
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Organic Matrix ◽  
Secretory Vesicles ◽  
Intercellular Spaces ◽  
Transmission Electron ◽  
Temperate Coral ◽  
Zooxanthellate Coral ◽  
Membrane Bound ◽  
Calicoblastic Ectoderm

The skeleton and calicoblastic ectoderm of the scleractinian non-zooxanthellate coral Caryophyllia smithii were investigated by light microscopy, scanning and transmission electron microscopy. Except for some costal spines, the skeleton was fasciculate. Fasciculi were made up of bundles of crystalline needles, each crystalline needle consisting of a number of linear series of small (<1 μm) rounded crystals. Fractured skeletons showed the fasciculi to be arranged into layers and that within some septa, theca and costal spines there were spaces that contained neither mineral nor organic matter. These spaces could also be found at the growing edges of septa and theca. Demineralization of the skeleton revealed an organic matrix whose configuration mirrored the architecture of the skeleton. In areas of the skeleton where deposition was occurring the overlying calicoblastic ectoderm was relatively thin with prominent intercellular spaces and secretory vesicles. In contrast, over non-depositing areas the calicoblastic ectoderm was thick and contained residual bodies, nematocysts and membrane-bound granules. The results are compared and contrasted with those from scleractinian corals that have endosymbiotic zooxanthellae.

scholarly journals FRTL-5 Rat Thyroid Cells Release Thyroglobulin Sequestered in Exosomes: A Possible Novel Mechanism for Thyroglobulin Processing in the Thyroid

2016 ◽  
Vol 2016 ◽  
pp. 1-7 ◽  
Author(s):  
Pavel Vlasov ◽  
Sonia Q. Doi ◽  
Donald F. Sellitti
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Western Blotting ◽  
Thyroid Stimulating Hormone ◽  
Cellular Proteins ◽  
Thyroid Cells ◽  
Normal Thyroid ◽  
Transmission Electron ◽  
Membrane Bound ◽  
Rat Thyroid

Exosomes are 30–100 nm, membrane-bound vesicles containing specific cellular proteins, mRNAs, and microRNAs that take part in intercellular communication between cells. A possible role for exosomes in thyroid function has not been fully explored. In the present study, FRTL-5 rat thyroid cells were grown to confluence and received medium containing either thyroid stimulating hormone (TSH), exogenous bovine thyroglobulin (bTg), or neither additive for 24 or 48 hours followed by collection of spent medium and ultracentrifugation to isolate small vesicles. Transmission electron microscopy and Western blotting for CD9 indicated the presence of exosomes. Western blotting of exosome extract using a monoclonal anti-Tg antibody revealed a Tg-positive band at ~330 kDa (the expected size of monomeric Tg) with a higher density in TSH-treated cells compared to that in untreated cells. These results are the first to show that normal thyroid cells in culture produce exosomes containing undegraded Tg.

Virus-Like and Membrane-Bound Particles in the Venom Apparatus of a Parasitoid Wasp (Hymenoptera:Braconidae)

1981 ◽  
Vol 39 ◽  
pp. 610-611
Author(s):  
Karthryn M. Edson
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Reproductive System ◽  
Inner Core ◽  
Parasitoid Wasp ◽  
Accessory Gland ◽  
Secretory Cells ◽  
Transmission Electron ◽  
Membrane Bound ◽  
Secretory Apparatus

Successful parasitism of a host by a parasitoid wasp may be aided by secretions from the venom apparatus, an accessory gland of the female parasitoid's reproductive system. In the present study, transmission electron microscopy of the venom apparatus of Meteorus leviventris reveals virus-like and membrane-bound particles which may influence successful parasitism of the host. This is the first evidence of such particles within the venom apparatus of a parasitoid.The venom apparatus of M. leviventris consists of a venom reservoir with two highly ramified gland filaments attached to it by a common duct.TEM of the secretory cells of the gland filaments reveals particles approximately 50 nm in diameter contained within a cytoplasmic stroma (Fig. 1). These virus-like particles (VLP) have a dense inner core and a hexagonal congifuration. Similar particles are found free within the cytoplasm or associated with vacuoles (Fig. 2).Secretory cells of the gland filaments contain a secretory apparatus which consists of an array of microvilli converging on a central lumen (Fig. 3).

Techniques for Transmission Electron Microscopy of Fiber-Matrix Interfaces in Aluminum Alloy-Boron Composites by Ion Erosio

1971 ◽  
Vol 29 ◽  
pp. 204-205
Author(s):  
G. G. Shaw
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Aluminum Alloy ◽  
Electron Beam ◽  
Pure Aluminum ◽  
Ion Milling ◽  
Transmission Electron ◽  
Fiber Matrix ◽  
Ion Erosion ◽  
Row Of Fibers

The morphology and composition of the fiber-matrix interface can best be studied by transmission electron microscopy and electron diffraction. For some composites satisfactory samples can be prepared by electropolishing. For others such as aluminum alloy-boron composites ion erosion is necessary.When one wishes to examine a specimen with the electron beam perpendicular to the fiber, preparation is as follows: A 1/8 in. disk is cut from the sample with a cylindrical tool by spark machining. Thin slices, 5 mils thick, containing one row of fibers, are then, spark-machined from the disk. After spark machining, the slice is carefully polished with diamond paste until the row of fibers is exposed on each side, as shown in Figure 1.In the case where examination is desired with the electron beam parallel to the fiber, preparation is as follows: Experimental composites are usually 50 mils or less in thickness so an auxiliary holder is necessary during ion milling and for easy transfer to the electron microscope. This holder is pure aluminum sheet, 3 mils thick.

Direct Observation of Heat Exchanger Deposits by Cross Sectioning

1967 ◽  
Vol 25 ◽  
pp. 364-365
Author(s):  
R. W. Anderson ◽  
D. L. Senecal
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Heat Exchanger ◽  
Direct Observation ◽  
Cross Sections ◽  
Preparation Method ◽  
Specimen Preparation ◽  
Flowing Water ◽  
Transmission Electron ◽  
Deposit Layer

A problem was presented to observe the packing densities of deposits of sub-micron corrosion product particles. The deposits were 5-100 mils thick and had formed on the inside surfaces of 3/8 inch diameter Zircaloy-2 heat exchanger tubes. The particles were iron oxides deposited from flowing water and consequently were only weakly bonded. Particular care was required during handling to preserve the original formations of the deposits. The specimen preparation method described below allowed direct observation of cross sections of the deposit layers by transmission electron microscopy.The specimens were short sections of the tubes (about 3 inches long) that were carefully cut from the systems. The insides of the tube sections were first coated with a thin layer of a fluid epoxy resin by dipping. This coating served to impregnate the deposit layer as well as to protect the layer if subsequent handling were required.

Phase Transformations in Ti-7w/oMo-3w/oMe Alloy

1974 ◽  
Vol 32 ◽  
pp. 514-515
Author(s):  
S. Fujishiro
Keyword(s):  
Electron Microscopy ◽  
Mechanical Properties ◽  
Transmission Electron Microscopy ◽  
Tensile Strength ◽  
Mechanical Processing ◽  
Ray Method ◽  
X Ray ◽  
Transmission Electron ◽  
Water Quench ◽  
Alpha Beta

The mechanical properties of three titanium alloys (Ti-7Mo-3Al, Ti-7Mo- 3Cu and Ti-7Mo-3Ta) were evaluated as function of: 1) Solutionizing in the beta field and aging, 2) Thermal Mechanical Processing in the beta field and aging, 3) Solutionizing in the alpha + beta field and aging. The samples were isothermally aged in the temperature range 300° to 700*C for 4 to 24 hours, followed by a water quench. Transmission electron microscopy and X-ray method were used to identify the phase formed. All three alloys solutionized at 1050°C (beta field) transformed to martensitic alpha (alpha prime) upon being water quenched. Despite this heavily strained alpha prime, which is characterized by microtwins the tensile strength of the as-quenched alloys is relatively low and the elongation is as high as 30%.

Two- or three-way observations of the identical cell elements within the same sections by LM, TEM and/or SEM

1978 ◽  
Vol 36 (2) ◽  
pp. 82-83 ◽  
Author(s):  
Nakazo Watari ◽  
Yasuaki Hotta ◽  
Yoshio Mabuchi
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Fine Structure ◽  
Light Microscopy ◽  
Thin Sections ◽  
Transmission Electron ◽  
Identical Cell ◽  
Electron Microscopes ◽  
Scanning Electron

It is very useful if we can observe the identical cell elements within the same sections by light microscopy (LM), transmission electron microscopy (TEM) and/or scanning electron microscopy (SEM) sequentially, because, the cell fine structure can not be indicated by LM, while the color is; on the other hand, the cell fine structure can be very easily observed by EM, although its color properties may not. However, there is one problem in that LM requires thick sections of over 1 μm, while EM needs very thin sections of under 100 nm. Recently, we have developed a new method to observe the same cell elements within the same plastic sections using both light and transmission (conventional or high-voltage) electron microscopes.In this paper, we have developed two new observation methods for the identical cell elements within the same sections, both plastic-embedded and paraffin-embedded, using light microscopy, transmission electron microscopy and/or scanning electron microscopy (Fig. 1).

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