Protein and peptide samples were prepared for TEM in 96-well plates as described above for ThT assays, except that ThT dye was not added (instead an equal volume of PBS pH 7.4 was added). Samples were spotted onto freshly glow-discharged carbon-coated formvar grids (Ted Pella Inc) and allowed to adsorb for 3 to 4 min before wicking off excess liquid with Whatman filter paper. The grids were then washed twice with milliQ water, followed by staining with 2% (w/v) uranyl acetate for 2 min, wicked off, and the grids allowed to fully dry before imaging. Grids were imaged using either a T12 or T20 electron microscope (FEI).
T20 electron microscope
Manufactured by Thermo Fisher Scientific
The T20 electron microscope is a versatile imaging tool used for high-resolution analysis of materials and biological samples. It employs a focused beam of electrons to capture detailed images of specimens at the nanoscale level. The T20 offers advanced imaging capabilities and is designed for use in research, development, and quality control applications.
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9 protocols using t20 electron microscope
1
Protein Sample Preparation for TEM
2
Negative Stain Electron Microscopy
3
Negative Stain Electron Microscopy of Env-C3d Fusion
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The purified Env-C3d fusion trimers were analyzed by negative stain electron microscopy (NS-EM). A 3- µl aliquot containing ~0.01 mg/ml of the sample was applied for 15 s onto a carbon-coated 400 Cu-mesh grid that had been glow- discharged at 30 mA for 30 s, then negatively stained with 0.7% uranyl formate for 45 s. Data were collected using an FEI T20 electron microscope operating at 200 kV, with an electron dose of ~45 e-/Å2 and a magnification of 80,000 × that resulted in a pixel size of 2.74 Å at the specimen plane. Images were acquired with an Eagle 2k × 2k CCD camera (FEI) using a nominal defocus of 1000 nm and the Serial EM software (56 (link)). Particles were selected from the micrographs and extracted, and a reference-free 2D class averages were obtained using RELION 2.1.0 (57 (link)).
4
Negative Stain Electron Microscopy
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The fibril sample (3 μL) was spotted onto a freshly glow-discharged carbon-coated electron microscopy grid. After 1 minute, 6 μL uranyl acetate (2% in aqueous solution) was applied to the grid for 1 minutes. The excessive stain was remove by a filter paper. Another 6μL uranyl acetate was applied to the grid and immediately removed. The samples were imaged using an FEI T20 electron microscope.
5
Nanomaterial Characterization via TEM
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Samples were prepared as in the ESEM assay, including the PchNPs concentrations. The pellets were resuspended in sterile distilled water, 4 μL of the sample was deposited onto a carbon-coated copper grid (Cu200 mesh) and left to dry in air for several hours at room temperature. TEM analysis was carried out in a TECNAI T20 electron microscope (FEI) working at 60 kV.
6
Negative Staining Procedure for Electron Microscopy
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Particles in the early intermediate density fraction were pelleted at 192,000 g. Pellets were resuspended in 7 µl PBS and were added on top of carbon-coated 300 mesh, copper grids and incubated for 3 min at room temperature. The grids were then washed two times in 50 µl water and stained with 2% uranyl acetate. Imaging was performed on a T20 electron microscope (FEI) operated at 200 keV. Images were recorded on a CCD Eagle camera (FEI).
7
Electron Microscopy of Viral Particles
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VLPs and the pseudotyped VSV were collected and purified as previously described. The virions were adsorbed on a Formvar carbon-coated copper grid by floating it on a drop of sample suspension for 15 min and then fixing using 2% formaldehyde/2% glutaraldehyde solution in 0.1 M cacodylate buffer. The grids were blotted, and then negatively stained with 1% aqueous uranyl acetate and viewed using a FEI T20 electron microscope.
8
Negative Stain Electron Microscopy of Purified Trimers
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The purified trimers were analyzed by negative stain electron microscopy (NS-EM). A 3 µl aliquot containing ~0.01 mg/mL of the sample was applied for 15 s onto a carbon-coated 400 Cu-mesh grid that had been glowing discharged at 30 mA for 30 s, then negatively stained with 0.7% uranyl formate for 45 s. Data were collected using a FEI T20 electron microscope operating at 200 kV, with an electron dose of ~45 e−/Å2 and a magnification of 80,000× that resulted in a pixel size of 2.74Å at the specimen plane. Images were acquired with an Eagle 2k × 2k CCD camera (FEI) using a nominal defocus of 1,000 nm and the SerialEM software (26 (link)). Particles were selected from the micrographs, extracted, and a reference-free 2D class averages were obtained using RELION 2.1.0 (27 (link)).
9
Electron Microscopy of EVs and RNPs
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EV and RNP were separated by density gradient centrifugation as described above, and resuspended in 15 µl PBS. 3 µl aliquots of either EV or RNP were added on top of carbon-coated 300 mesh, copper grids, and incubated for 2 min at room temperature. The grids were then washed two times in 50 µl PBS and stained with 2% uranyl acetate. Imaging was performed on a T20 electron microscope (FEI) operated at 200 keV. Images were recorded on a CCD Eagle camera (FEI).
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