3 µL of MRS2 (0.02 mg/mL) was applied on a glow discharged carbon-coated 400 square mesh copper grid (EMS) and incubate for 1 min. The grid was then blotted by filter paper (Whatman) and washed once with 3 µL of Nano-W negative staining solution (Nanoprobes) followed by incubation with 3 µL of Nano-W for 1 min. The grid was blotted to remove excess staining solution and air-dried by waving. Images was recorded using an FEI Tecnai T20 TEM operated at 200 kV with a direct electron detector K2 Summit (Gatan Inc). Data was collected using SerialEM48 (link) at a nominal magnification of 25,000x, a pixel size of 3.04 Å/px, and defocus range between −1.5 µm and −2.5 µm. A total of 459 images was collected. Image processing was performed using cisTEM v1.0.049 (link). 215,448 particles were picked from the micrographs after CTF estimation, extracted with a box of 108 px (~328 Å) and analyzed by 2D classification.
Tecnai t20 tem
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Tecnai T20 TEM is a transmission electron microscope (TEM) designed for high-resolution imaging and analysis of a wide range of materials. It features a LaB6 electron source, advanced optics, and versatile imaging and analytical capabilities. The Tecnai T20 TEM provides users with the tools to perform detailed structural and compositional studies at the nanoscale level.
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Lab products found in correlation
Filter paper, by Cytiva (1 mentions)
K2 summit, by Ametek (1 mentions)
K3 camera, by Ametek (1 mentions)
Titan krios, by Thermo Fisher Scientific (1 mentions)
Em gp2, by Leica (1 mentions)
R gent se em, by Aurion (1 mentions)
Quanta 250 feg esem, by Thermo Fisher Scientific (1 mentions)
300 mesh carbon coated copper grids, by Agar Scientific (1 mentions)
Titan3 60 300, by Thermo Fisher Scientific (1 mentions)
X pert powder diffractometer, by Malvern Panalytical (1 mentions)
Leap 4000x si instrument, by Cameca (1 mentions)
Ivas 3, by Cameca (1 mentions)
Amicon ultra 0.5 centrifugal filter 10 kda, by Merck Group (1 mentions)
Exorneasy serum plasma kit, by Qiagen (1 mentions)
Qiazol, by Qiagen (1 mentions)
Nb5000 fib sem, by Hitachi (1 mentions)
Infinity 3, by Teledyne (1 mentions)
Eclipse ts100, by Teledyne (1 mentions)
Ultracut uc7 microtome, by Leica (1 mentions)
Uc6 ultramicrotome, by Leica (1 mentions)
18 protocols using tecnai t20 tem
1
Cryo-EM Imaging of Protein Complexes
2
Cryo-EM Structural Analysis of MRS2 Protein
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3 μl purified MRS2 at approximately 0.5 mg/ml was applied to a glow-discharged 400-mesh R 1.2/1.3 Cu grid (Quantifoil). The cryo grids were blotted for 6 s at 4°C and 95%, and plunge-frozen into liquid ethane using a Leica EM GP2 (Leica) and stored in liquid nitrogen. The grids were screened using on an FEI Tecnai T20 TEM before data collection.
Cryo-EM datasets were acquired with SerialEM48 (link) using a Titan Krios (FEI, now ThermoFisher Scientific) operated at 300 keV and equipped with an energy filter and K3 camera (Gatan Inc.). Movies of 50 frames with a dose of 1 e−/Å2 per frame (50 e−/Å2 total dose) were recorded at a nominal magnification of 105,000x, corresponding to a physical pixel size of 0.83 Å/px (super-resolution pixel size 0.415 Å/px) in CDS mode at a dose rate of 10 e−/px/s and a defocus range of −0.7 to −2.0 µm. In total, 3,991 and 9,656 movies were collected for MRS2-Mg2+ and MRS2-EDTA, respectively (Supplementary Table 1 ).
Cryo-EM datasets were acquired with SerialEM48 (link) using a Titan Krios (FEI, now ThermoFisher Scientific) operated at 300 keV and equipped with an energy filter and K3 camera (Gatan Inc.). Movies of 50 frames with a dose of 1 e−/Å2 per frame (50 e−/Å2 total dose) were recorded at a nominal magnification of 105,000x, corresponding to a physical pixel size of 0.83 Å/px (super-resolution pixel size 0.415 Å/px) in CDS mode at a dose rate of 10 e−/px/s and a defocus range of −0.7 to −2.0 µm. In total, 3,991 and 9,656 movies were collected for MRS2-Mg2+ and MRS2-EDTA, respectively (
3
Electron Tomographic Analysis of IMPDH2 Inhibition
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Inclusions created by inhibited IMPDH2 protein known as Rings & Rods structures (Smigova et al., 2011 (link)), immunogold labeled by the pre-embedding technique, were subjected to an electron tomographic experiment. Samples were processed according to (Smigova et al., 2011 (link)). Briefly, human Hep2 cells were treated by IMPDH inhibitor ribavirin. The cells were fixed in formaldehyde, permeabilized and immunolabeled with the primary anti-IMPDH2 antibody (12948-1-AP, Proteintech, Manchester, United Kingdom) and the secondary antibody conjugated with ultrasmall gold (Aurion, Wageningen, The Netherlands). After the pre-embedding, the ultrasmall colloidal gold particles were silver enhanced, the immunolabeled cells were postfixed with glutaraldehyde, incubated with R-GENT SE-EM (Aurion), dehydrated in ethanol, embedded into Araldite/Embed 812 and polymerized. Single-axis tomographic dataset was acquired at a Tecnai T20 TEM (FEI, Hillsboro, OR, USA) equipped with a 200 kV LaB6 gun and a 2 K Gatan Ultrascan 1000 CCD at the ICBP, Prague. Projections were recorded within the ±65° range at 1° step at the magnification of 7800×, reconstructions were computed by WBP in IMOD (Kremer et al., 1996 (link)). Small sub-volumes containing a few silver-enhanced colloidal gold particles from this reconstruction were examined in order to test the effect of angular filtering in their neighborhood.
4
Nanoparticle Characterization by TEM and SEM
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1–2 μL of sample was let to dry on a carbon grid. TEM imaging was performed with a FEI Tecnai T20 TEM operating at an acceleration voltage of 200 kV. SEM imaging was performed with a FEI Quanta 250 FEG-ESEM. 20 mL of water was filtered through the membranes prior to SEM imaging in order to remove any salts and loosely bound nanoparticles.
5
Exosome Visualization via Transmission Electron Microscopy
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For TEM observation, a moderate amount of exosome suspension was placed on carbon-coated 300 mesh copper grids (Agar Scientific Ltd., Stansted, UK) for 2 min. The samples were fixed in 3% paraformaldehyde at 4 °C overnight and 1% osmium tetroxide at pH 7.2 for 1 h at room temperature (RT). After washing in double-distilled water for three times, samples were stained with uranyl acetate for 10 min and lead citrate for 5 min at RT. After air-drying, exosomes can be viewed with a FEI Tecnai T20 TEM, operated at 120 kV.
6
Lorentz TEM and Kerr Imaging of Magnetic Films
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The L-TEM images shown here were acquired using an FEI Tecnai T20 TEM operated at 200 kV in Lorentz mode, with the objective lens only weakly excited52 . Kerr imaging was carried out in an Evico microscope. The perpendicular magnetic anisotropy of the deposited films was confirmed by polar Kerr microscopy showing square hysteresis loops (for example, that in Fig. 3i ).
7
Structural Characterization of Mixed Powder
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XRD was performed on a PANalytical X’Pert powder diffractometer, with Cu source . For these measurements, a graded Bragg-Brentano HD, with a 1/4° divergent and 1/2° anti-scattered slits, and a 5-mm anti-scatter slit together with a Soller slit (with an opening radius of 0.04), in the incident and the diffracted beam side, were used. A continuous scan from 5° to 120° was performed on the sample using a step size of 0.008° with a 40-s time per step. STEM combined with high-angle annular dark-field imaging and EDX analysis with a Super-X EDX detector was performed in the double-corrected Linköping FEI Titan3 60-300 operated at 300 kV. SAED was performed on an FEI Tecnai T20 TEM operated at 200 kV. The specimens were prepared by embedding the ground-mixed powder in a Cu grid with C film.
8
Immunogold labeling of bacterial EspA
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Bacterial samples were fixed on ice with 1% paraformaldehyde/PBS for an hour and washed three times in PBS for 5 min each time. Sample suspension droplets (5 μl) were placed onto Formvar/Carbon nickel‐coated support grids, which had been pretreated with a 0.1% poly‐L‐lysine solution. Bacterial suspensions were left to settle for 20 min, the grids were floated sample on 0.05 M glycine/PBS droplets for 5 min and then rinsed three times with 3% BSA/PBS for 5 min. Bacterial samples were placed onto an α‐EspA (1:100 dilution) for an hour, washed six times on 3% BSA/PBS for 5 min each and then placed onto droplets of secondary GAR 10 nm gold for an hour. Samples were washed six times in 3% BSA/PBS for 5 min each, rinsed with PBS six times for 3 min, fixed on 1% glutaraldehyde/PBS for 5 min, washed six times in distilled water and then left to dry. Immunostained bacterial samples were viewed on a FEI Tecnai T20 TEM running at 200 kV and dm4/tiff images were captured using Gatan Digital Imaging software.
9
Correlative TEM and APT Analysis
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The nanostructure of the NC-SS and distribution of the alloying elements and impurities were investigated by using correlative TEM and APT on the same APT sample. Blanks with a size of 0.5 × 0.5 × 15 mm3 cut from bulk material were electropolished with a standard two-step electropolishing technique to produce APT needle samples. A needle sample was loaded on a specially designed APT sample TEM holder with the maximum tilt angle of ± 70°. TEM examinations were performed by using an FEI Tecnai-T20 TEM at an operation voltage of 200 kV. Subsequent APT characterization of the same needle sample was conducted on a Cameca LEAP 4000X SI instrument, at a specimen base temperature of 40 K, under UV laser pulsing at a pulse laser energy of 40 pJ, a pulse frequency of 250 kHz and a target evaporation rate of 0.5% per pulse. APT data reconstruction and statistical analyses were performed by using a commercial software (Cameca IVAS®3.6.12).
10
Exosome Isolation and Characterization from Plasma
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For every patient, 1 mL of plasma was used. EVs were isolated by affinity-based binding to spin columns via an exoRNeasy Serum/Plasma Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Briefly, melted plasma was mixed with binding buffer and added to the exoEasy membrane affinity spin column. Samples were subjected to ultrafiltration using an Amicon Ultra-0.5 Centrifugal Filter 10 kDa (Merck Millipore, Germany) to reduce the eluate volume to 50 µL and exchange the buffer with phosphate buffer saline (PBS). For transmission electron microscopy (TEM), the size distribution measurement, and western blotting, the EVs were eluted with 400 μL of XE elution buffer, according to previous publications [29 (link)]. For TEM, ultrathin sections (100 nm) were cut using a LeicaUC6 ultra-microtome and post-stained with uranyl acetate for 10 min and with lead citrate for 5 min at room temperature before observation in a FEI Tecnai T20 TEM, operated at 120 kV. For EV RNA isolation, EVs were lysed on the column using QIAzol (Qiagen), and total RNA was then eluted and purified, as per other publications [30 (link)].
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