Cryo-TEM measurements were performed with a Gatan cryo Holder 626 and a FEI Tecnai 20 TEM (200 kV) at approximately −174°C. The images were captured with a Gatan US1000 894 CCD and processed with Leginon software. To maintain the structural integrity of the worm-like micelles, Cryo-TEM samples were prepared in a controlled environment chamber to avoid damage and contamination during sample preparation. The samples were prepared with the following sequence of operations. Five milliliters of the sample was placed onto a perforated polymer film held by tweezers to ensure that the formation of the thin film spanned the mesh hole. After 10 seconds, the samples were immediately immersed into liquid ethane cooled by the nitrogen below its freezing point of −183°C. The samples were then stored in liquid nitrogen to protect against contamination before examination. Several images of each sample were taken, and a representative image was chosen to present.
626 cryo holder
Manufactured by Thermo Fisher Scientific
Sourced in United States
The 626 cryo-holder is a device designed for sample preparation and analysis in cryogenic environments. It provides a stable and controlled temperature environment for specimens during observation or experimentation.
Automatically generated - may contain errors
Lab products found in correlation
Us1000 894 ccd, by Ametek (2 mentions)
Tecnai 20 tem, by Thermo Fisher Scientific (2 mentions)
Fei vitrobot, by Thermo Fisher Scientific (2 mentions)
Tecnai tf20 electron microscope, by Thermo Fisher Scientific (2 mentions)
Falcon 2 direct electron detector, by BD (1 mentions)
Supra 40vp system, by Zeiss (1 mentions)
Analysis software package, by Olympus (1 mentions)
Gp plunger, by Leica (1 mentions)
Talos electron microscope, by Thermo Fisher Scientific (1 mentions)
6 protocols using 626 cryo holder
1
Cryo-TEM Imaging of Worm-like Micelles
2
Cryogenic TEM Sample Preparation
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Specimens for examination were stored in a controlled environment vitrification system with temperature and humidity control functions. The preparation of the cryo-TEM samples was performed with the following sequence of operations. First, 5 mL of the sample was placed onto a perforated polymer film held by tweezers to insure that the formation of the thin film spanned the mesh hole. Second, after 10 seconds, the samples were immediately immersed into liquid ethane just above its freezing point of −183°C. Third, the samples were then stored in liquid nitrogen to protect against contamination before they were moved to a Gatan cryo Holder 626 and examined with a FEI Tecnai 20 TEM (200 kV) at about −174°C. The images were captured with a Gatan US1000 894 CCD and processed with Leginon software.
3
Cryo-EM Sample Preparation Workflow
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Samples were diluted to a final concentration of 20–50 μg ml−1 (20 mM HEPES (pH 8.0), 40 mM KOAc, 5 mM MgCl2, 0.1% trehalose, 2 mM DTT and 0.01% NP-40), and 3 μl of aliquots was applied to freshly glow-discharged Quantifoil R2/1 grids coated with a second layer of thin carbon film. The grids were blotted for 3–4 s at 4 °C in 100% humility, and then plunged into liquid ethane using an FEI Vitrobot (FEI Company). Frozen grids were stored in liquid nitrogen. The grids were first loaded into a Gatan 626 cryo-holder and transferred to an FEI Tecnai TF20 electron microscope to check the quality of the sample vitrification. Then, the grids were transferred to Titan Krios equipped with a field emission source and were operated at 300 kV. Images were recorded on a Falcon 2 direct electron detector at a nominal magnification of × 59,000 with a defocus range of 2–4 μm, resulting in a calibrated sampling of 1.42 Å per pixel. The total accumulated dose rate was set to be 35 e2 per Å2 on the specimen, and the exposure time was 1.5 s. Each image was fractionated into 23 frames.
4
Vitrification and Cryo-EM Imaging of INO80 Complex
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The INO80 fraction was diluted to a final concentration of ~30 μg/ml (20 mM HEPES, pH 8.0, 40 mM KOAc, 5 mM MgCl2, 0.1% trehalose, 2 mM DTT, and 0.01% NP-40) and 3 μl aliquots were applied to freshly glow-discharged Quantifoil R2/1 grids coated with a second layer of thin carbon film. The grids were blotted for 3–4 sec at 4°C in 100% humility, and then plunged into liquid ethane using an FEI Vitrobot (FEI Company). Frozen grids were stored in liquid nitrogen. The grids were first loaded into a Gatan 626 cryo-holder and transferred to an FEI Tecnai TF20 electron microscope to check the quality of the sample vitrification. Then, the grids were transferred to Titan Krios equipped with a field emission source and were operated at 300 kV. Images were recorded on a Falcon II direct electron detector at a nominal magnification of 59000 with a defocus range of 3–5 μm, resulting in a calibrated sampling of 1.42 Å per pixel. The total accumulated dose rate was set to be 35 e2 per Å2 on the specimen, and the exposure time was 1.5 sec. Each image was fractionated into 23 frames.
5
Nanoparticle Characterization by SEM and TEM
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Scanning electron microscopy was carried out using a Zeiss SUPRA 40VP system. Samples were prepared by spreading 2 μL of the nanoparticle suspension in water on a silicon substrate. The sample was dried under ambient conditions and then sputter-coated with a gold-platinum alloy (15 s, 20 mA). Micrographs were acquired using the InLens mode with an accelerating voltage of 10 kV. Particle sizes were measured on micrographs acquired at a magnification of 200 000× using the Olympus Analysis software package. To ensure size measurement accuracy, at least 250 measurements were made per sample. For transmission electron microscopy measurements, a 4 μL aliquot of sample was adsorbed onto a holey carbon-coated grid (Lacey, Tedpella), blotted with Whatman 1 filter paper and plunge-frozen into liquid ethane at −180 °C using a vitrobot (FEI). Frozen grids were transferred onto a Talos electron microscope (FEI) using a Gatan 626 cryo-holder. Electron micrographs were recorded at an accelerating voltage of 200 kV using a low-dose system (50 e− Å−2) and keeping the sample at −175 °C. Defocus values were −3 μm. Micrographs were recorded on a 4 × 4 K Ceta CMOS camera.
6
Cryo-EM Sample Preparation Protocol
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A 4 μL aliquot of sample was adsorbed onto a holey carbon-coated grid (Lacey, USA), blotted with Whatman 1 filter paper and vitrified into liquid ethane at −178 °C using a Leica GP plunger (Leica, Austria). Frozen grids were transferred onto a Talos electron microscope (FEI, USA) using a Gatan 626 cryo-holder. Electron micrographs were recorded at an accelerating voltage of 200 kV and a nominal magnification of 730 00x, using a low-dose system (20 e− Å−2) and keeping the sample at low temperature. Micrographs were recorded on a CETA camera.
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