TEM micrographs were obtained using an FEI Spirit TEM (Hillsboro, USA) operated at 120 kV. A 400-mesh Formvar® carbon-coated copper grid was glow-discharged in a vacuum evaporator (Denton, Moorestown, New Jersey) for 30 s. The sample was prepared by placing 2 μL of sonicated CaCO3 nanoparticles solution onto the grid and wicking off the excess sample with filter paper after 30 s. Alternatively, for EtOH or DMSO solvent-based solutions, 3 μL of particle solution were placed on the grid and left to dry out at room temperature or with the aid of a heat gun.
Spirit tem
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Spirit TEM is a transmission electron microscope (TEM) designed for use in research and industrial laboratories. It provides high-resolution imaging and analysis capabilities for a wide range of samples. The Spirit TEM is a core piece of equipment for advanced materials characterization and life science applications.
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Lab products found in correlation
Vitrobot mark 4, by Thermo Fisher Scientific (1 mentions)
Ab 6573, by Abcam (1 mentions)
Eagle ccd camera, by Thermo Fisher Scientific (1 mentions)
Eponate 12, by Ted Pella (1 mentions)
Em uc6 ultramicrotome, by Leica (1 mentions)
Bac to bac system, by Thermo Fisher Scientific (1 mentions)
Benzonase, by Merck Group (1 mentions)
Glutaraldehyde, by TAAB (1 mentions)
Formaldehyde, by TAAB (1 mentions)
Diamond knife, by Electron Microscopy Sciences (1 mentions)
11 protocols using spirit tem
1
TEM Imaging of CaCO3 Nanoparticles
2
Cryo-EM Structure of 30S-ABCE1 Complex
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Freshly prepared sample was applied to 2 nm pre-coated Quantifoil R3/3 holey carbon supported grids and vitrified using a Vitrobot Mark IV (FEI Company) and visualized on a Spirit TEM (FEI Company) with about 20e− Å−2 at a nominal magnification of × 105,000 with a nominal defocus between −1 μm and −3.5 μm. Automatic particle detection was performed by the programme SIGNATURE49 (link). Initial in silico sorting of the data set consisting of 54,800 particles in total was performed using the SPIDER software package49 (link). Classes were obtained by competitive projection matching in SPIDER50 (link)51 (link). The final 30S·ABCE1 data set contained 19,500 particles and the final resolution was 17 Å (Fourier shell correlation 0.5).
For interpretation of the 30S·ABCE1 electron density at a molecular level, the models for the Pyrococcus furiosus 30S subunit (4V6U)52 (link) and ribosome-bound ABCE1 in (3J15)8 (link) were fitted as rigid bodies using UCSF Chimera. The FeS cluster domain was repositioned by a rotation of ∼160° around a hinge (residues 76–78) into an unaccounted electron density near ribosomal protein S12. This repositioning results in a close contact between lysine 60 of ABCE1 (Lys64 in P. furiosus) and lysine 40 of S12 and is consistent with above described XL-MS data.
For interpretation of the 30S·ABCE1 electron density at a molecular level, the models for the Pyrococcus furiosus 30S subunit (4V6U)52 (link) and ribosome-bound ABCE1 in (3J15)8 (link) were fitted as rigid bodies using UCSF Chimera. The FeS cluster domain was repositioned by a rotation of ∼160° around a hinge (residues 76–78) into an unaccounted electron density near ribosomal protein S12. This repositioning results in a close contact between lysine 60 of ABCE1 (Lys64 in P. furiosus) and lysine 40 of S12 and is consistent with above described XL-MS data.
3
Immunogold Labeling of Diaphragm Catalase
4
TEM Imaging of Copper-Chitosan Nanoparticles
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TEM micrographs were obtained using an FEI Spirit TEM (Hillsboro, USA) operated at 120kV using a 400-mesh Formvar ® carbon-coated copper grid. The Cu–chitosan NPs sample was prepared by vortexing and placing 2.0l of the sonicated colloidal solution onto the grid, using a 10 ml disposable pipet to re-suspend the sample, making EM grids (carbon-coated 400-mesh copper grids) directly on the specimen and using the filter to wick away specimen droplets, specimen–side up in the specimen petri dish.
5
Visualization of RAD-51 and RFS-1/RIP-1 Complexes
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RAD-51 and RFS-1/RIP-1 in indicated concentrations were incubated with 250 nM (in nucleotides) 150-mer poly(dT) ssDNA in 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 2 mM MgCl2, 2 mM ATP for 5 min °C. For negative staining, Quantifoil R2/2, 2 nm carbon, 400 Cu mesh grids were glow discharged for 30 s at 25 mA with a K100X glow discharger (EMS), 4 μL of sample was added to the grid left for 1 min. Excess sample was blotted away leaving a thin film. Then the grid was dipped into buffer solution twice and dipped twice into 2% uranyl acetate solution, blotting in between. Negative stain EM data were acquired on Tecnai Spirit TEM operated at 120 kV, equipped with a n FEI Eagle CCD camera.
6
Ultrastructural Analysis of hiPSC-Derived Cardiomyocytes
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Human iPSC-derived cardiomyocyte monolayers were grown on Permanox Chamber slides (Lab Tek), and hiPSC-CMs were cultured on aligned nanofiber patches. The cells were then fixed in 2.5% glutaraldehyde in 0.1M phosphate buffer with 0.1M sucrose pH = 7.4 for 1 hour. They were then post fixed in 1% osmium tetroxide for 30 minutes, en bloc stained in 2% uranyl acetate for 30 minutes and dehydrated in a graded series of ethanol. HPMA (hydroxy-propyl methacrylate) was used as an intermediate between ethanol and Eponate12 (Ted Pella Inc.) epoxy resin. Final embedding was in Eponate12 in inverted Beem (Ted Pella Inc.) capsules and polymerized overnight at 70°C. Capsules with cells were trimmed and 70 nm thick sections were obtained using a Leica EM UC6 ultra-microtome and a diamond knife. Observations were made using a FEI Spirit TEM at 80 kV and images captured with an AMT camera.
7
Transmission Electron Microscopy Imaging
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Ultrathin sections (70 nm) were collected on 300 mesh copper grids. Samples were post-stained with either Sato’s lead solution only (cultured cells) or with 2% uranyl acetate and Sato’s lead solution (mouse brain slices). Sections were imaged on an FEI Spirit TEM at 80 kV equipped with a 2k × 2k Tietz CCD camera.
8
Recombinant AAV2 Virus-Like Particle Production
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The AAV2L336C substitution was created within the AAV2 cap gene encoding all three viral proteins, VP1, VP2, and VP3, as previously described5 (link). A recombinant baculovirus, encoding the AAV2 cap gene, with the L336C substitution, was created using the Bac-to-Bac system (Thermo Fisher). A plaque purified and titered baculovirus stock was used to infect Sf9 insect cells, at a multiplicity of infectivity of 5 to generate virus-like particles (VLPs). The harvested pellet (from lysed cells and polyethylene glycol precipitated supernatant) was freeze/thawed three times with Benzonase (EMD Millipore Cat#712053) treatment. After the third thaw, the resulting clarified supernatant was purified using a step iodixanol gradient followed by anion exchange46 (link) and then dialyzed into 50 mM HEPES, pH 7.4 with 2 mM MgCl2 and 150 mM NaCl. The sample concentration was determined by optical density assuming an extinction coefficient of 1.7 mg mL−1 cm−1) for AAV2 VLPs. The VLP purity and integrity were confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and negative stain EM on an FEI Spirit TEM, respectively.
9
Conventional and Immunoelectron Microscopy Workflow
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For conventional EM analysis, MEFs were cultured on collagen‐coated plastic coverslips and fixed in mixed solution of 2.5% glutaraldehyde (TAAB, Berkshire, England) and 2.0% formaldehyde (TAAB, Berkshire, England) in 0.1 M sodium phosphate buffer pH 7.4 (phosphate buffer) for 2 h. The cells were washed in the same buffer five times and post‐fixed in 1% osmium tetroxide (Agar) in phosphate buffer for 1 hour and then dehydrated and embedded in Epon 812 (Agar) according to a standard procedure.29 (link) Ultrathin sections were stained with uranyl acetate and lead citrate and observed under an FEI Technical Spirit TEM. Images were recorded with a Gatan CCD camera (Gatan US 1000X‐U Camera 2000 kV). For immunoelectron microscopy analysis, cells were fixed with 4% formaldehyde solution (TAAB, Berkshire, England) in phosphate buffer for 2 hours on ice. The pre‐embedding gold enhancement immunogold method was used as described previously.29 (link), 30 (link)
10
Drosophila Larval CNS Ultrastructure
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The data volume used was the whole central nervous system described in [2 (link)]. The specimen was a 6 hour old female Drosophila melanogaster, in the L1 larval stage. It is comprised of 4850 sections, each 50nm thick, cut with a Diatome diamond knife. Each section was imaged at 3.8 × 3.8nm resolution using an FEI Spirit TEM. The images were montaged and registered using the nonlinear elastic method described in [43 (link)].
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