Cells grown on glass coverslips were fixed for 30 min by incubation with pre-warmed 2.5% glutaraldehyde/2% sucrose in 50 mM sodium cacodylate buffer (CaCo)(50 mM cacodylate, 50 mM KCl, 2.6 mM CaCl2, 2.6 mM MgCl2, pH-7.4). After 3 washes with 50 mM CaCo buffer, cells were incubated with 2% osmium tetroxide/50 mM CaCo for 40 min on ice, washed with water 3 times and treated with 0.5% uranyl acetate for 30 min. After 30 min rinsing with water, cells were progressively dehydrated with increasing concentrations of ethanol (40% to 100%) and finally infiltrated in a polymerizing Epon/araldite resin (Araldite 502/Embed 812 kit; Electron Microscopy Sciences) for 72 h at 60°C. Embedded cells were sectioned into 70-nm-thick slices by using an Ultracut UCT microtome (Leica) and a diamond knife (Diatome). After counterstaining with 3% uranyl acetate in 70% methanol for 5 min and 2% lead citrate in water for 2 min, cells were examined either with an EM-10 transmission electron microscope (Zeiss) with a built-in MegaView camera (Olympus) or with a JEOL JEM-1400 transmission electron microscope (Jeol Ltd., Tokyo, Japan). Quantification of virion number, size, and distribution was done manually or using macros developed for the Fiji software package.
Megaview camera
Manufactured by Olympus
Sourced in Japan
The MegaView camera is a high-resolution imaging system designed for laboratory applications. It features a large sensor that captures detailed images at high resolutions.
Automatically generated - may contain errors
Lab products found in correlation
Em 10 transmission electron microscope, by Zeiss (4 mentions)
Diamond knife, by Electron Microscopy Sciences (3 mentions)
Ultracut uct microtome, by Leica (3 mentions)
Araldite 502 embed 812 kit, by Electron Microscopy Sciences (3 mentions)
Jem 1400 transmission electron microscope, by JEOL (2 mentions)
Agr1043, by Agar Scientific (2 mentions)
Ags162, by Agar Scientific (2 mentions)
Morgagni 268d tem, by Thermo Fisher Scientific (1 mentions)
Ultraview ers spinning disc, by PerkinElmer (1 mentions)
Te2000 e, by Nikon (1 mentions)
Jem 1010 transmission electron microscope, by Olympus (1 mentions)
10 protocols using megaview camera
1
Transmission Electron Microscopy Imaging of Cells
2
Electron Microscopy of Mitochondria
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Cells grown on glass coverslips were washed twice with pre-warmed PBS and fixed by 30 min incubation with 2.5% glutaraldehyde/2% sucrose in 50 mM sodium cacodylate buffer (CaCo) supplemented with 50 mM KCl, 2.6 mM MgCl2 and 2.6 mM CaCl2. After three washes with 50 mM CaCo, cells were incubated with 2% osmium tetroxide/50 mM CaCo for 40 min on ice, washed with water three times, and treated with 0.5% uranyl acetate for 30 min. After 30 min rinsing with water, cells were progressively dehydrated with increasing concentrations of ethanol (40% to 100%) and finally infiltrated in a polymerizing Epon/araldite resin (Araldite 502/Embed 812 kit; Electron Microscopy Sciences) for 72 hr at 60°C. Embedded cells were sectioned into 65-nm slices by using an Ultracut UCT microtome (Leica) and a diamond knife (Diatome). After counterstaining with 3% uranyl acetate in 70% methanol for 5 min and 2% lead citrate in water for 2 min, cells were examined with an EM-10 transmission electron microscope (Zeiss) with a built-in MegaView camera (Olympus). The length of mitochondria was measured using the ImageJ software package.
3
Characterization of Cobalt-Substituted Nanoparticles
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For the evaluation of shape and size of the resulting particles, TEM images were acquired for the samples of series 2. One sample for each cobalt substitution rate x was used as a dry powder and suspended in micropure water at a concentration of 5 mg/mL. The sample was deposited on a carbon support film on a 400-mesh copper grid manufactured by Quantifoil Micro Tools (Großlöbichau, Germany). The films were hydrophilized in an Argon plasma produced by a Diener Electronics (Ebhausen, Germany) plasma oven for 120 s prior to sample deposition. An amount of 10 μL of the suspension was then placed on the film, the excess blotted off using filter paper, and allowed to air dry. TEM images were acquired with a 200 kV FEI Tecnai G2 20 (Hillboro, OR, USA) using a 1 k × 1 k Olympus MegaView camera (Münster, Gemany) with the acceleration voltage set to 120 kV.
4
Nanoemulsion Characterization and Cytotoxicity
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Particle size, polydispersity and z-potential of nanoemulsions were determined by dynamic-light-scattering (DLS) and phase-analysis light scattering (PALS) according to Artiga-Artigas et al. [18 (link)]. To avoid multiple scattering effects, samples were diluted prior to analysis with Milli-Q water (1:10).
Nanoemulsions were observed by negative-staining electron microscopy as a direct measurement of their droplet size and shape as reported by Artiga-Artigas et al. [18 (link)]. The grids were observed in a transmission electron microscope Morgagni 268D TEM (FEI Company, Netherlands) with a CCD Mega-View camera (Olympus, Tokyo, Japan).
Cells THP-1 (human leukemia monocytic cell line) and HT-29 (human colon cancer cell line) were used to evaluate the cytotoxicity of coatings. Cell culture maintenance and cell viability were evaluated as described previously [4 (link)].
Nanoemulsions were observed by negative-staining electron microscopy as a direct measurement of their droplet size and shape as reported by Artiga-Artigas et al. [18 (link)]. The grids were observed in a transmission electron microscope Morgagni 268D TEM (FEI Company, Netherlands) with a CCD Mega-View camera (Olympus, Tokyo, Japan).
Cells THP-1 (human leukemia monocytic cell line) and HT-29 (human colon cancer cell line) were used to evaluate the cytotoxicity of coatings. Cell culture maintenance and cell viability were evaluated as described previously [4 (link)].
5
Ultrastructural Analysis of Mycobacterial Infection in Zebrafish
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Mycobacteria in tail fin of 5 dpf zebrafish larvae as previously described (43 (link)). Wild type zebrafish larvae were infected with ~250 CFU M. marinum or M. avium at 2 dpf. At 3 dpi, the infected larvae were fixed in 2% glutaraldehyde and 2% paraformaldehyde in sodium cacodylate buffer (pH 7.2) for 3 h at room temperature after anesthetized properly. Subsequently, fixed samples were kept at 4°C for a further 16 h fixation. The next day, the samples were fixed in 1% osmium tetroxide in sodium cacodylate buffer (with 15mgr Potassium Ferrocyanide/ml) for 1 h at room temperature. All samples were kept in epoxy resin (Agar Scientific, AGR1043) for 16 h after the dehydration through a series of ethanol. Ultrathin sections were collected on Formvar coated 200 mesh or one hole copper grids (Agar Scientific, AGS162) stained with 2% uranyl acetate in 50% ethanol and lead citrate for 10 min each. The samples were imaged on a JEM- JEOL 1400 transmission electron microscope (Tokyo, Japan), which was equipped with an Olympus Megaview camera (Tokyo, Japan).
6
Correlating Mitochondrial Ultrastructure and Morphology
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Huh7 cells stably expressing YFP-Sec61β and mito-mTurquoise2 were grown in 6-cm diameter dishes (Mattek) containing gridded coverslips and infected with DVs at a MOI of 1. Three days post infection, cells were washed twice and fixed by 20 min incubation with PBS containing 4% paraformaldehyde at room temperature. After several times washing with PBS, cells were examined with an Ultraview ERS spinning disc (PerkinElmer Life Sciences) on a Nikon TE2000-E inverted confocal microscope. Cells of interest were selected, and 0.13-μm optical sections in the cyan and YFP channels were acquired. The corresponding coordinates were also recorded using transmitted light with a differential interference contrast configuration. Cells were prepared and embedded for TEM analysis as previously described, and blocks were trimmed around the cell of interest according to the recorded coordinates. Ultrathin sections were prepared and examined with an EM-10 transmission electron microscope (Zeiss) containing a built-in MegaView camera (Olympus). Using ultrastructural and fluorescent patterns of mitochondria morphology and distribution, EM images and corresponding confocal microscopy Z-stacks were superimposed with the Photoshop CS5.1 software package (Adobe) and used for correlation analysis.
7
Zebrafish Larvae Ultrastructure Analysis
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Before being used for electron microscopy the zebrafish larvae were anesthetized with 200 µg/ml tricaine, imaged alive by CLSM and afterwards immediately fixated in 2% glutaraldehyde and 2% paraformaldehyde in sodium cacodylate buffer (pH 7.2) for 3 h at room temperature followed by fixation for 16 h at 4 °C. Postfixation was performed in 1% osmium tetroxide in sodium cacodylate buffer for 1 h at room temperature. After dehydration through a graded series of ethanol all specimens were kept in epoxy resin (Agar Scientific, AGR1043) for 16 h before embedding. Ultrathin sections were collected on Formvar coated 200 mesh or one hole copper grids (Agar Scientific, AGS162) stained with 2% uranyl acetate in 50% ethanol and lead citrate for 10 min each. Electron microscopy images were obtained with a JEOL JEM-1010 transmission electron microscope (Tokyo, Japan) equipped with an Olympus Megaview camera (Tokyo, Japan).
8
Transmission Electron Microscopy Imaging of Cells
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Cells grown on glass coverslips were fixed for 30 min by incubation with pre-warmed 2.5% glutaraldehyde/2% sucrose in 50 mM sodium cacodylate buffer (CaCo)(50 mM cacodylate, 50 mM KCl, 2.6 mM CaCl2, 2.6 mM MgCl2, pH-7.4). After 3 washes with 50 mM CaCo buffer, cells were incubated with 2% osmium tetroxide/50 mM CaCo for 40 min on ice, washed with water 3 times and treated with 0.5% uranyl acetate for 30 min. After 30 min rinsing with water, cells were progressively dehydrated with increasing concentrations of ethanol (40% to 100%) and finally infiltrated in a polymerizing Epon/araldite resin (Araldite 502/Embed 812 kit; Electron Microscopy Sciences) for 72 h at 60°C. Embedded cells were sectioned into 70-nm-thick slices by using an Ultracut UCT microtome (Leica) and a diamond knife (Diatome). After counterstaining with 3% uranyl acetate in 70% methanol for 5 min and 2% lead citrate in water for 2 min, cells were examined either with an EM-10 transmission electron microscope (Zeiss) with a built-in MegaView camera (Olympus) or with a JEOL JEM-1400 transmission electron microscope (Jeol Ltd., Tokyo, Japan). Quantification of virion number, size, and distribution was done manually or using macros developed for the Fiji software package.
9
Electron Microscopy of Trichoplax Eggs
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For electron microscopy, 24e39 h old wild-type, and TcKnk-1, TcRtv, and TcLac-2 knockdown eggs were collected, dechorionated in 4% hypochlorite (w/v), and fixed for 1 h in 5 ml heptane and 5 ml of a 2.5% gluteraldehyde (v/v) and 2% paraformaldehyde (w/v) solution in 0.1 M cacodylate buffer (pH 7.4). Subsequently, eggs were removed from this solution, washed with 70% ethanol to remove the heptane, and fixed for another hour in the same fixative without heptane. After fixation, specimens were washed (3 Â 10 min) in cacodylate buffer and placed for 1 h in 1% osmium tetraoxide (w/v). Then, specimens were dehydrated with increasing concentrations of ethanol and embedded in Agar100. Sections of about 70 nm thickness were contrasted with uranyl acetate and lead citrate, and studied with a JEOL 1010 transmission microscope coupled to an Olympus MegaView camera.
10
Immunolabeling and TEM Imaging of Protein
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The material was adsorbed to carbon-and pioloform-coated 300 mesh copper grids (Science Services GmbH) for 10 min at RT, and remaining liquid was drained using a whatman paper.
Immunolabeling against the C-terminal Strep-tag II was performed as described in (Fogeron et al. 2015b (link)). Samples were examined by using a Zeiss EM-10 transmission electron microscope (Zeiss, Goettingen, Germany) with a built in Mega View camera (Olympus;
Tokyo, Japan).
Immunolabeling against the C-terminal Strep-tag II was performed as described in (Fogeron et al. 2015b (link)). Samples were examined by using a Zeiss EM-10 transmission electron microscope (Zeiss, Goettingen, Germany) with a built in Mega View camera (Olympus;
Tokyo, Japan).
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