orius sc2001, by Ametek - Product details

1

Negative Staining of Nanoparticles for TEM

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Transmission electron microscopy (TEM) imaging was performed on a JEOL 2100 microscope operating at an acceleration voltage of 200 kV and equipped with a CCD Camera Orius SC2001 from Gatan.
The samples were mounted onto freshly glow-discharged carbon-coated copper grids (Agar Scientific, Essex, UK) at a concentration ranging from 0.5 to 1 g /L. After that, the grids were blotted with filter paper and immersed for 10 s into a phosphotungstic acid (PTA) solution at 0.75 wt% for negative staining. Then, the grids were blotted again and dried under vacuum for 1 min.
For PTA solution, 37.5 mg of PTA was dissolved in boiling distilled water (5 mL). The pH was adjusted to 7.0 by adding a few drops of 5 M NaOH under continuous stirring. The PTA solution was then filtered through a 0.2 μm filter.

Fetsch C., Gaitzsch J., Messager L., Battaglia G, & Luxenhofer R. (2016). Self-Assembly of Amphiphilic Block Copolypeptoids – Micelles, Worms and Polymersomes. Scientific Reports, 6, 33491.

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2

Characterization of Nanoparticle Structures

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TEM analysis was performed using a FEI Tecnai G2 Spirit electron microscope and/or a JEOL 2100 operating at 200 kV equipped with a CCD camera Orius SC2001 from Gatan. Copper grids were glow discharged and the sample was adsorbed onto the grid. The sample was then stained with 0.75 wt% phosphotungstic acid (PTA) adjusted to pH 7.4 with NaOH. All the TEM analyses were carried out with dried samples.
DLS analyses were carried out using a Zetasizer Nano ZS (Malvern Ltd.) at a copolymer concentration of 0.25 mg/mL. DLS measurements were based on 12−14 runs, 10-second sub-runs. Samples were analysed at 25 °C with a scattering angle of 173° and a 633 nm HeNe laser based on a material refractive index (RI) of 1.59, a dispersant refractive index of 1.330 and a viscosity of 0.89.

Robertson J.D., Rizzello L., Avila-Olias M., Gaitzsch J., Contini C., Magoń M.S., Renshaw S.A, & Battaglia G. (2016). Purification of Nanoparticles by Size and Shape. Scientific Reports, 6, 27494.

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3

Ultrastructural Analysis of Liver and Cells

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For ultrastructural analysis in ultrathin sections, small pieces (∼1 mm³) of liver or cell monolayers were fixed for at least 1 h in a mixture of 2.5% formaldehyde prepared from paraformaldehyde powder, and 0.1% glutaraldehyde in 0.05 M cacodylate buffer pH 7.3 to which 0.1% picric acid (trinitrophenol) and 0.03% CaCl₂ were added. Then they were washed in 0.1 M cacodylate buffer and the cells were scraped off and processed further as a pellet. Then the samples were post-fixed in 1% OsO₄ in 0.1 M cacodylate buffer pH 7.3 for 1 h, washed with distilled water, and en bloc stained with 2% aqueous uranyl acetate for 20 min at 60°C. The samples were dehydrated in ethanol, processed through propylene oxide, and embedded in Poly/Bed 812 (Polysciences, Warrington, PA, USA). Semi-thin sections 1 µm thick were cut and stained with toluidine blue. Ultrathin sections were cut on Leica EM UC7 ultramicrotome (Leica Microsystems, Buffalo Grove, IL, USA), stained with lead citrate, and examined in a JEOL JEM-1400 transmission electron microscope at 80 kV. Images were acquired either on film or on bottom-mounted CCD camera Orius SC2001 (Gatan, Pleasanton, CA, USA).

Fagg W.S., Liu N., Patrikeev I., Saldarriaga O.A., Motamedi M., Popov V.L., Stevenson H.L, & Fair J.H. (2021). Endoderm and Hepatic Progenitor Cells Engraft in the Quiescent Liver Concurrent with Intrinsically Activated Epithelial-to-Mesenchymal Transition. Cell Transplantation, 30, 0963689721993780.

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4

Ultrastructural Analysis of ZIKV Infection

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Vero cells were infected with single-round infectious E103A HA-NS2A ZIKV (derived from trans-complementation as shown in Fig. 2E) at an MOI of 1. At 24 h p.i., cells were fixed with fixative (50 mM cacodylate buffer [pH 7.3], 2.5% formaldehyde, 0.1% glutaraldehyde, 0.01% picric acid, 0.03% CaCl₂) for 2 h at room temperature and then washed twice with 100 mM cacodylate buffer. The monolayers were scraped off and centrifuged at 12,000 rpm for 5 min to pellet cells. The pellets were postfixed with 1% OsO₄ in 100 mM cacodylate buffer, washed with electron microscopy (EM)-grade water, and en bloc stained with 2% aqueous uranyl acetate in water for 20 min at 60°C. The pellets were dehydrated in ethanol series (from 50% to 100%) and processed through propylene oxide before being embedded in Poly/Bed 812 (Polysciences). Ultrathin sections (70 nm) were cut on a Leica EM UC7 ultramicrotome (Leica Microsystems), stained with 0.4% lead citrate in water for 3 min, and examined with a Philips CM100 transmission electron microscope at 60 kV. Digital images were acquired with a bottom-mounted charge-coupled device (CCD) camera, Orius SC2001 (Gatan, Pleasanton, CA).

Zhang X., Xie X., Xia H., Zou J., Huang L., Popov V.L., Chen X, & Shi P.Y. (2019). Zika Virus NS2A-Mediated Virion Assembly. mBio, 10(5), e02375-19.

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5

Electron Microscopy of Mosquito Saliva

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Methods were modified from Théry et al. (2006) [42 (link)]. 10uL of pooled saliva from uninfected (mock) and DENV2 infected mosquitoes was directly absorbed onto formvar copper grids (Electron Microscopy Sciences) for 10 min and excess saliva blotted away. Next, grids were fixed by floating on a mixture of 2% glutaraldehyde (Electron Microscopy Sciences) and 2% paraformaldehyde (Electron Microscopy Sciences) for 10 min and excess fixative blotted away. Grids were then floated on a solution of uranyl acetate (Electron Microscopy Services) and 0.15M oxalic acid (Sigma-Aldrich) at a 1:1 ratio for 5 min and excess uranyl oxalate blotted away. Lastly, grids were embedded by floating on a solution containing 2% methyl cellulose (Sigma-Aldrich, 25 centripose, M-6385) and 4% uranyl acetate (Electron Microscopy Services) at a ratio of 9:1 for 10 min on ice. Grids were examined in a JEOL JEM-1400 (USA) transmission electron microscope at 80 kV. Images were acquired on a bottom-mounted CCD camera Orius SC2001 (Gatan, Pleasanton, CA).

Yeh S.C., Strilets T., Tan W.L., Castillo D., Medkour H., Rey-Cadilhac F., Serrato-Pomar I.M., Rachenne F., Chowdhury A., Chuo V., Azar S.R., Singh M.K., Hamel R., Missé D., Kini R.M., Kenney L.J., Vasilakis N., Marti-Renom M.A., Nir G., Pompon J, & Garcia-Blanco M.A. (2023). The anti-immune dengue subgenomic flaviviral RNA is present in vesicles in mosquito saliva and is associated with increased infectivity. PLOS Pathogens, 19(3), e1011224.

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6

Ultrastructural Analysis of Cell Mitochondria

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To obtain ultrastructural analysis of cells in ultrathin sections, cells were fixed for at least 1 hour in a mixture of 2.5% formaldehyde prepared from paraformaldehyde powder, and 0.1% glutaraldehyde in 0.05M cacodylate buffer pH 7.3 to which 0.01% picric acid and 0.03% CaCl₂ were added. Cell monolayers were washed in 0.1 M cacodylate buffer, were scraped off and processed further as a pellet. The pellets were post-fixed in 1% OsO₄ in 0.1M cacodylate buffer pH 7.3 for 1 hour, washed with distilled water and en bloc stained with 2% aqueous uranyl acetate for 20 min at 60°C. The cell pellets were dehydrated in ethanol, processed through propylene oxide and embedded in Poly/Bed 812 (Polysciences, Warrington, PA). Ultrathin sections were cut on Leica EM UC7 ultramicrotome (Leica Microsystems, Buffalo Grove, IL), stained with lead citrate and examined in a JEM-1400 (JEOL USA, Peabody, MA) transmission electron microscope at 80 kV. Digital images were acquired with a bottom-mounted CCD camera Orius SC200 1 (Gatan, Pleasanton, CA). Mitochondrial size was quantified using ImageJ software.

Cao J., Verma S.K., Jaworski E., Mohan S., Nagasawa C.K., Rayavara K., Sooter A., Miller S.N., Holcomb R.J., Powell M.J., Ji P., Elrod N.D., Yildirim E., Wagner E.J., Popov V., Garg N.J., Routh A.L, & Kuyumcu-Martinez M.N. (2021). RBFOX2 is critical for maintaining alternative polyadenylation patterns and mitochondrial health in rat myoblasts. Cell reports, 37(5), 109910.

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7

Transmission Electron Microscopy Analysis of Nanoparticles

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TEM analysis was performed using a JEOL 2100 operating at 200 kV equipped with a CCD camera Orius SC2001 from Gatan. Copper grids were glow discharged and the sample was adsorbed onto the grid. The sample was then stained with 0.75 wt% phosphotungstic acid (PTA) raised to pH 7.4 with NaOH. The images of tubes and spheres were analyzed by ImageJ 1.47 software (https://imagej.nih.gov/ij/) and used to investigate the morphometric parameters of the nanoparticles. 100 random nanoparticles were measured to obtain: mean size, area, perimeter, circularity and aspect ratio. Note that circularity values near 1 indicate a perfect cycle, whereas close to 0 indicate a high sharpness degree.

Scarpa E., De Pace C., Joseph A.S., de Souza S.C., Poma A., Liatsi-Douvitsa E., Contini C., De Matteis V., Martí J.S., Battaglia G, & Rizzello L. (2020). Tuning cell behavior with nanoparticle shape. PLoS ONE, 15(11), e0240197.

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8

Ultrastructural Analysis of Cell Mitochondria

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To obtain ultrastructural analysis of cells in ultrathin sections, cells were fixed for at least 1 hour in a mixture of 2.5% formaldehyde prepared from paraformaldehyde powder, and 0.1% glutaraldehyde in 0.05M cacodylate buffer pH 7.3 to which 0.01% picric acid and 0.03% CaCl₂ were added. Cell monolayers were washed in 0.1 M cacodylate buffer, were scraped off and processed further as a pellet. The pellets were post-fixed in 1% OsO₄ in 0.1M cacodylate buffer pH 7.3 for 1 hour, washed with distilled water and en bloc stained with 2% aqueous uranyl acetate for 20 min at 60°C. The cell pellets were dehydrated in ethanol, processed through propylene oxide and embedded in Poly/Bed 812 (Polysciences, Warrington, PA). Ultrathin sections were cut on Leica EM UC7 ultramicrotome (Leica Microsystems, Buffalo Grove, IL), stained with lead citrate and examined in a JEM-1400 (JEOL USA, Peabody, MA) transmission electron microscope at 80 kV. Digital images were acquired with a bottom-mounted CCD camera Orius SC200 1 (Gatan, Pleasanton, CA). Mitochondrial size was quantified using ImageJ software.

Cao J., Verma S.K., Jaworski E., Mohan S., Nagasawa C.K., Rayavara K., Sooter A., Miller S.N., Holcomb R.J., Powell M.J., Ji P., Elrod N.D., Yildirim E., Wagner E.J., Popov V., Garg N.J., Routh A.L, & Kuyumcu-Martinez M.N. (2021). RBFOX2 is critical for maintaining alternative polyadenylation patterns and mitochondrial health in rat myoblasts. Cell reports, 37(5), 109910.

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Orius sc2001

Lab products found in correlation

8 protocols using orius sc2001

Negative Staining of Nanoparticles for TEM

Characterization of Nanoparticle Structures

Ultrastructural Analysis of Liver and Cells

Ultrastructural Analysis of ZIKV Infection

Electron Microscopy of Mosquito Saliva

Ultrastructural Analysis of Cell Mitochondria

Transmission Electron Microscopy Analysis of Nanoparticles

Ultrastructural Analysis of Cell Mitochondria

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