Cryo-TEM samples are prepared in a controlled environment vitrification system (CEVS) or using the commercial environmentally controlled automated Vitrobot (FEI, Netherlands), always at a controlled temperature (25 °C) and at saturation. A 6 µL drop of the suspension is placed on a 200-mesh TEM copper grid covered with a perforated carbon film. To remove excess solution and produce a thin liquid film the drop is blotted, manually in the CEVS and automatically in the Vitrobot. The blotted sample is then plunged into liquid ethane (−183 °C) to form a vitrified specimen and transferred to liquid nitrogen (−196 °C) for storage. Vitrified specimens are examined at temperatures below −175 °C using a Gatan 626 cryo holder either in a Tecnai T12 G2 TEM (FEI, Netherlands) or a Philips CM120 TEM operating at 120 kV. Images are recorded on a Gatan MultiScan 791 camera or Gatan UltraScan 1000 using the DigitalMicrograph software (Gatan, U.K.) in the low-dose imaging mode to minimize beam exposure and electron-beam radiation damage, as described.35 ,38
Tecnai t12 g2 tem
Manufactured by Thermo Fisher Scientific
Sourced in Netherlands
The Tecnai T12 G2 TEM is a transmission electron microscope (TEM) designed and manufactured by Thermo Fisher Scientific. It is used for high-resolution imaging and analysis of samples at the nanoscale level.
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4 protocols using tecnai t12 g2 tem
1
Cryo-EM Sample Preparation Protocol
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Cryo-TEM Vitrified Specimen Preparation
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Cryo-TEM vitrified specimens were prepared in a controlled environment vitrification system at 25 °C and 100% relative humidity. The procedure is described in detail elsewhere38 . About 3 μl of the sample was applied onto a perforated (lacey) carbon film-coated 200 mesh copper TEM grid. Excess liquid was blotted with filter paper, and the specimen was immediately plunged into liquid ethane at its freezing point (−183 °C). The vitrified specimens were 240 transferred to a 626 Gatan cryo-holder, using it tansfer station, and imaged at 120 kV acceleration voltage in an FEI Tecnai T12 G2 TEM, using low-dose imaging to minimize electron-beam radiation damage. Typical electron exposures were 10 to 15 e− Å−2. The specimens were kept in the TEM at about −175 °C. Images were digitally recorded with a Gatan US1000 high-resolution (2it × 2high-resolution (2charge-coupled device camera, using the Digital Micrograph software.
3
Transmission Electron Microscopy of Nanoparticles
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TEM images of the HEC coated nanoparticles were obtained with the use of a Philips CM20 Analytical TEM at 80 kV accelerating voltage. Samples were prepared by placing a drop of nanoparticles in aqueous suspension onto carbon coated grids for 1 minute and then exposed to a 1% uranyl acetate solution before being dried and placed in the instrument.
Vitrified specimens were prepared at a controlled temperature and at water saturation in the Vitrobot (FEI, Netherlands), and kept in liquid nitrogen until examination. Cryo-TEM analysis was done with a Tecnai T12 G2 TEM (FEI, Netherlands) operating at 120 kV. Images were recorded digitally on a Gatan UltraScan 1000 camera using the DigitalMicrograph software (Gatan, U.K.). Images are recorded in the low-dose imaging mode to minimize beam exposure and electron-beam radiation damage.20,21 (link)
Vitrified specimens were prepared at a controlled temperature and at water saturation in the Vitrobot (FEI, Netherlands), and kept in liquid nitrogen until examination. Cryo-TEM analysis was done with a Tecnai T12 G2 TEM (FEI, Netherlands) operating at 120 kV. Images were recorded digitally on a Gatan UltraScan 1000 camera using the DigitalMicrograph software (Gatan, U.K.). Images are recorded in the low-dose imaging mode to minimize beam exposure and electron-beam radiation damage.20,21 (link)
4
Vitrification of Biological Samples for TEM
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Samples were prepared in a closed chamber at a controlled temperature and at water saturation. A 5- to 6-μl drop of each suspension was placed on a 200-mesh transmission electron microscope (TEM) copper grid covered with a perforated carbon film. The drop was blotted, and the sample was plunged into liquid ethane (−183°C) to form a vitrified specimen and then transferred to liquid nitrogen (−196°C) for storage. Vitrified specimens were examined at temperatures less than −175°C in a Talos F200C with field emission gun operated at 200 kV or a Tecnai T12 G2 TEM (FEI, Netherlands). Images were recorded on a cooled Falcon IIIEC (FEI) direct detection device by TIA software attached to the Talos or a Gatan MultiScan 791 camera by Digital Micrograph software (Gatan, UK) on the Tecnai TEM. Volta PhasePlate was used for contrast enhancement. Images are recorded in the low-dose imaging mode to minimize beam exposure and electron beam radiation damage using laboratory procedures.
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