Specimens were vitrified in liquid ethane and analyzed in a TEM microscope at low temperature. The vitrification process was performed in an FEI Vitrobot: a 3-µl drop of an aqueous suspension of the material was placed on a TEM Quantifoil carbon grid, excess water blotted away at the Vitrobot with filter paper, and the grid freeze-plunged into liquid ethane. Samples were then transferred under a liquid nitrogen atmosphere to a Gatan TEM cryo-holder equipped with a liquid nitrogen reservoir. TEM images were obtained in a Tecnai T20 (FEI), operated at 200 kV, coupled to a Veleta CCD camera.
Tem cryo holder
Manufactured by Ametek
The TEM cryo-holder is a device designed for use in transmission electron microscopy (TEM) applications. Its core function is to maintain a cryogenic environment for the sample being observed, enabling the study of temperature-sensitive materials and biological specimens.
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Lab products found in correlation
Ultramicrotome, by Leica camera (2 mentions)
Ultrascan ccd camera, by Ametek (2 mentions)
Diatome cryo diamond knife, by Leica camera (2 mentions)
Tecnai t20, by Thermo Fisher Scientific (1 mentions)
Titan tem, by Thermo Fisher Scientific (1 mentions)
Holey carbon support film, by Electron Microscopy Sciences (1 mentions)
Vitrobot mark 3, by Thermo Fisher Scientific (1 mentions)
831 ccd camera, by Ametek (1 mentions)
Ht7700, by Hitachi (1 mentions)
6 protocols using tem cryo holder
1
Cryo-TEM Specimen Vitrification Protocol
2
In Situ TEM Characterization of Pd Nanoparticles
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The general experimental conditions are very similar to the previously published papers23 (link),33 (link),38 (link). The TEM grid with Pd particles is cleaned for 1 min in an 3:1 Ar/O2 plasma with RF power of 50 W. The grid is then mounted into a TEM cryo holder (Gatan, Inc.), which has a temperature controller that can allow control of temperature with a precision of ±0.1 K. The Titan TEM (FEI) has ETEM capabilities which allows us to vary the H2 pressure from 4 to ~600 Pa, using a home-built gas cart with mass-flow controllers. In order to avoid condensation of contaminants on the sample during hydrogen gas flow, we use a liquid nitrogen cooled cold finger during our environmental TEM experiments, which minimizes beam-induced hydrocarbon contamination during STEM-EELS and SAED data acquisition at all hydrogen pressures. We obtain our EEL spectra and diffraction while operating the microscope at 80 kV, which gives higher interaction cross-section, and high-resolution TEM images at 300 KV. The H2 (99.9999%, Praxair) pressure in the microscope chamber is monitored using an Edwards Barocell 600 capacitance manometer with a precision of 4%.
3
Cryogenic TEM Imaging Protocol
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TEM grids remain in the dry ice environment until transfer to a Gatan TEM cryo-holder. The cryo-holder is precooled using a cryo-transfer station and the grid is transferred in a liquid nitrogen environment. Imaging is performed at approximately -170 o C and 200 kV, at a spot size selected to avoid damage to the samples from the electron beam. The spot size, which is size of the beam on the sample, ranged from 4 to 6 (arb. units) for minimal damage and depended on the system. The cryogenic temperatures prevent the particles from damaging for tens of seconds while under the electron beam. Size information is gathered using the ImageJ software (NIH).
4
Cryo-TEM Imaging of Phospholipid Blue Phase LCs
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Our approach to imaging of phospholipid assemblies in blue phase LCs was guided by past studies that have reported the use of cryo-TEM for characterization of a range of self-assembled nanostructures in oils51 –55 . Blue phase LCs were prepared by mixing MLC-2142 with 35 % wt/wt chiral dopant S-811. Next, 0.01 % wt/wt PC-C12 (based on blue phase LC mass) was added to the MLC-2142/S-811 mixture using procedures described above in the context of preparing mixtures of phospholipid and 5CB (see above). The LC mixture was held at 46°C (blue phase temperature range is between 44–48°C) for 10 min and then vitrified in liquid nitrogen by using a FEI Vitrobot. Subsequently, cryogenic sectioning was performed at −120°C by using a Diatome cryo-diamond knife mounted on a Leica ultramicrotome with cryogenic sectioning capability. The slice thickness was set to 80–100 nm. The microtomed samples were then transferred onto carbon film-coated TEM grids. Cryo-TEM imaging was carried out on a Tecnai 12 TEM with cryogenic capability. The TEM imaging was operated at 120 kV. A Gatan cryo-TEM holder was used to keep the sample below −170°C during cryo-TEM imaging. The TEM images were collected on a Gatan ultrascan CCD camera.
5
Cryo-TEM Sample Plunge-Freezing Protocol
6
Cryo-TEM Imaging of Phospholipid Blue Phase LCs
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