Phage lysates were concentrated before Transmission electron microscopy (TEM) analysis by centrifugation at 21,000 × g for 1 h, the pellet was resuspended with 0.1 M ammonium acetate (Thermo Fisher Scientific, United Kingdom), centrifuged at 21,000 × g for 1 h and resuspended with a 0.1 M ammonium acetate solution. The highly concentrated phages (1011 PFU/ml) were negatively stained with 1% uranyl acetate (w/v) for 10 s and applied to 3 mm carbon coated copper grids (Agar Scientific Ltd., United Kingdom). Phages were examined with an EOL 1220 (JEOL UK Ltd., United Kingdom) ran at 80 kV and images were acquired by SIS Megaview III camera with analysis software (Olympus Soft Imaging Solutions, Germany) (Ackermann, 2009 (link)). TEM analysis was conducted by Dr. Ali Ali and Natalie Allcock, Core Biotechnology Services, University of Leicester, United Kingdom.
Sis megaview 3 camera
Manufactured by Olympus
Sourced in Italy
The SIS Megaview III camera is a high-performance digital camera designed for scientific imaging applications. It features a large sensor with high resolution, allowing for the capture of detailed images. The camera is optimized for a range of microscopy techniques and can be integrated with various microscope systems.
Automatically generated - may contain errors
Lab products found in correlation
Uc6 ultramicrotome, by Leica (2 mentions)
Transmission electron microscope, by Philips (1 mentions)
Uranyl acetate, by Thermo Fisher Scientific (1 mentions)
Em uc6 ultramicrotome, by Agar Scientific (1 mentions)
Analysis pro software, by Olympus (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Glutaraldehyde, by Merck Group (1 mentions)
Osmium tetroxide, by TAAB (1 mentions)
Uranyl acetate, by TAAB (1 mentions)
Agar 100, by Agar Scientific (1 mentions)
Em208s transmission electron microscope, by Thermo Fisher Scientific (1 mentions)
8 protocols using sis megaview 3 camera
1
Phage Concentration and Transmission Electron Microscopy
2
Ultrastructural Analysis of Mitochondrial Morphology
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The HT22 cells were seeded in 75 cm2 flasks at a density of 2 × 106 cells/well and left to adhere for 24 hours. After treatments, the cells were washed, prefixed in situ with 2.5% glutaraldehyde in 0.1 mol/L phosphate buffer for 15 minutes, gently scraped and centrifuged at 300 x g for 10 min. The pellets were fixed in 2.5% glutaraldehyde for an additional 45 minutes at room temperature. After two washes in 0.1 mol/L phosphate buffer, the cells were postfixed for 1 hour in 1% OsO4, dehydrated in a graded series of increasing concentrations of ethanol and embedded in araldite. Thin sections were counterstained with uranyl acetate and lead citrate. Ultrastructural analysis was performed with a transmission electron microscope (Philips) and imaged with an SIS MegaView III camera (Soft Imaging System).36 (link) Mitochondria were analyzed for major/minor axis using the NIH‐Image J software plugin for mitochondrial morphology. The parameters obtained from these analyses were then used to calculate the Aspect Ratio (AR), that is, the ratio between the major and minor axes of the ellipse equivalent to the mitochondrion. The final values are indicative of mitochondrial elongation and fragmentation.37
3
Visualizing Peptide-RNA Condensates
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Peptide/RNA condensates were prepared at a final concentration of 2 mM and 1 mg mL−1 for the peptide and poly-U, respectively, in 10 mM Tris-HCl buffer pH 7.5 at 25 °C. An aliquot of 10 μL of the sample solution was applied to a carbon-coated grid and incubated for 2 min. Excess solution was removed by blotting the grid with a piece of filter paper, followed by staining with 10 μL of 2% (w/v) uranyl acetate solution for 2 min. After blotting excess stain solution, the grid was left to air-dry. The negatively stained sample was imaged by a JEM-1400Plus TEM operating at 80 kV. Images were recorded using a SIS Megaview III camera, iTEM imaging platform (Olympus).
4
Ultrastructural Analysis of Pancreatic Tissue
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For electron microscopy, thinly sliced sections of the pancreas (∼1 mm thick) were fixed for 1 h in 4% (w/v) paraformaldehyde and 2.5% (v/v) glutaraldehyde (both from Sigma) at room temperature before additional fixation in 1% (w/v) osmium tetroxide and further staining in 0.5% (w/v) uranyl acetate (both from TAAB Laboratory Equipment Ltd, Reading, UK). The sections were then dehydrated through a series of graded ethanol concentrations, before flat embedding in Agar 100 epoxy resin. After polymerising for 2 days, ultrathin sections (60–80 nm) were cut on a Leica EM UC6 ultramicrotome and transferred onto formvar-coated 200 μm mesh copper grids (Agar Scientific, Stansted, UK). These were counterstained with 4% uranyl acetate and Reynolds lead citrate before visualisation at 100 kV on a FEI Technai 120 KV G2 Spirit BioTWIN electron microscope (FEI, Eindhoven, The Netherlands). Digital images were taken up to × 87 000 magnification and captured using a SIS Megaview III camera (Olympus, Hamburg, Germany) and analysed using AnalySIS Pro software (Olympus).
5
Visualizing SARS-CoV-2 Infection in Vero Cells
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Vero cells (8.6 × 106 cells) in 100 mm tissue culture dishes were infected at an MOI of 10 PFU/cell and processed for transmission electron microscopy (TEM) experiments [27 (link)]. Infected cells were processed 18 h post-infection. Samples were examined using an FEI Tecnai 12 electron microscope; images were obtained with an SIS Megaview III camera (Olympus, Tokyo, Japan).
6
Electron Microscopy Analysis of OmpA Oligomers
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Samples were prepared of OmpA171 and OmpA325 under monomer conditions (1 μM protein in 600 mM urea) or self-associating conditions (3 μM protein in 300 mM urea or 10 μM protein in 800 mM urea). All mixtures were prepared in 20 mM Tris, pH 8. Self-associating samples were incubated for 16 h at 25°C. Negatively stained samples were prepared for transmission electron microscopy (TEM) using freshly ionized formvar/carbon-coated copper grids. The protein solutions were adsorbed to the grids for 5 minutes, then washed six times with water. The grids were stained with 2% uranyl acetate for 1 minute before removing excess stain. The samples were observed in an FEI Tecnai 12 TWIN electron microscope operating at 100 kV. Images were captured using an SIS Megaview III camera (Olympus). Width measurements of observed structures were made with ImageJ.
7
Ultrastructural Analysis of Cell Monolayers
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Cell monolayers were fixed in 2.5% glutaraldehyde, 2% paraformaldehyde, 2 mM CaCl2 in 0.1 M sodium cacodylate buffer, pH 7.4, overnight at 4 °C, and processed according to Perry and Gilbert. Cells were washed in buffer and post-fixed with 1% OsO4 in 0.1 M sodium cacodylate buffer for 1 h at RT, treated with 1% tannic acid in 0.05 M cacodylate buffer for 30 min and rinsed in 1% sodium sulphate in 0.05 M cacodylate buffer for 10 min. Postfixed specimens were washed, dehydrated through a graded series of ethanol solutions (30–100% ethanol) and embedded in Agar 100 (Agar Scientific, Monterotondo, RM, Italy). Ultrathin sections, obtained by a UC6 ultramicrotome (Leica), were stained with uranyl acetate and Reynold’s lead citrate and examined at 100 kV by a Philips EM 208S Transmission Electron Microscope (FEI-Thermo Fisher) equipped with an acquisition system Megaview III SIS camera (Olympus-SIS, Milan, Italy).
8
Ultrastructural Analysis of ARF3 Mutants
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COS-1 cells were seeded at 2 × 105 in six-well plates and transfected with ARF3 WT, K127E and D93N for 48 h. Cells were then fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) 1 h at RT, washed in buffer and post-fixed in 1% OsO4 in 0.1 M sodium cacodylate buffer for 1 h at RT. Post-fixed specimens were washed in buffer, dehydrated through a graded series of ethanol solutions (30–100% ethanol) and embedded in Agar Resin Kit (Agar Scientific, R1031). Ultrathin sections, obtained by a UC6 ultramicrotome (Leica), were stained with uranyl acetate and Reynolds’ lead citrate and examined at 100 kV by a Philips EM 208S transmission electron microscope (FEI-Thermo Fisher) equipped with acquisition system Megaview III SIS camera (Olympus-SIS Milan, Italy) and iTEM3 software.
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