Samples were prepared by collecting the fumes from an ignited strip of Mg on a standard TEM grid. We tried different TEM grids, lacey carbon grids (Agar, 300 mesh Cu grid), small Si 3 N 4 membranes window (Ted Pella™, 9 windows 0.1*0.1mm with 15nm thick Si 3 N 4 ), and big Si 3 N 4 membrane window (TEMwindows, single 500 µm window with 20nm thick Si3N4) back-covered with a thin (3-5 nm) layer of carbon. This last combination gives the best results, as it allows for a wide tilt angle without the shadow effect from the grid border, and eliminate the electrical charging related drift at high tilt angles (see supplementary).
300 mesh cu grid
Manufactured by Ted Pella
Sourced in United States
300 mesh Cu grids are a type of specimen support made of copper. They are commonly used in electron microscopy applications as a platform to hold thin samples for imaging and analysis.
Automatically generated - may contain errors
Lab products found in correlation
4kx4k fei eagle ccd camera, by Thermo Fisher Scientific (2 mentions)
Eagle slow scan ccd camera, by Thermo Fisher Scientific (1 mentions)
Tecnai g2 spirit biotwin, by Thermo Fisher Scientific (1 mentions)
Vitrobot mark 4, by Thermo Fisher Scientific (1 mentions)
Tecnai 12 g2 twin tem, by Thermo Fisher Scientific (1 mentions)
Lacey substrate, by Ted Pella (1 mentions)
Gatan model 626, by Ametek (1 mentions)
5 protocols using 300 mesh cu grid
1
Fume Collection for TEM Analysis
2
Vitrification and Cryo-TEM Imaging of Vesicles
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The principle of Cryo-TEM of vitrified specimens was previously described (Dubochet et al., 1988) .
The vitrification of the samples was carried out in a homemade vitrification system. The chamber was maintained at 22°C, and the relative humidity was 80%. A 5 µl drop of the sample was deposited onto a lacey carbon film covered with a 300 mesh Cu grid (Ted Pella-USA) rendered hydrophilic using an ELMO glow discharge unit (Cordouan Technologies, Pessac, France). The grid was automatically blotted to form a thin film and plunged in liquid ethane at -190°C, as maintained by liquid nitrogen.
Thus, a vitrified film was obtained in which the native structure of the vesicles was preserved. The grid was mounted onto a cryo holder (Gatan 626-Pleasanton, CA, USA) and observed under low-dose conditions (10 e/A 2 ) in a Tecnai G2 microscope (FEI, Eindhoven, Netherlands) at 200 kV. Images were acquired using an Eagle slow scan CCD camera (FEI).
A c c e p t e d M a n u s c r i p t
The vitrification of the samples was carried out in a homemade vitrification system. The chamber was maintained at 22°C, and the relative humidity was 80%. A 5 µl drop of the sample was deposited onto a lacey carbon film covered with a 300 mesh Cu grid (Ted Pella-USA) rendered hydrophilic using an ELMO glow discharge unit (Cordouan Technologies, Pessac, France). The grid was automatically blotted to form a thin film and plunged in liquid ethane at -190°C, as maintained by liquid nitrogen.
Thus, a vitrified film was obtained in which the native structure of the vesicles was preserved. The grid was mounted onto a cryo holder (Gatan 626-Pleasanton, CA, USA) and observed under low-dose conditions (10 e/A 2 ) in a Tecnai G2 microscope (FEI, Eindhoven, Netherlands) at 200 kV. Images were acquired using an Eagle slow scan CCD camera (FEI).
A c c e p t e d M a n u s c r i p t
3
Transmission Electron Microscopy of Cross-linked CNF
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Cross-linked CNF suspensions were studied with an FEI Tecnai G2 Spirit BioTWIN instrument equipped with a digital camera. The accelerating voltage was 120 kV. For each sample, 10 µL of 0.01 wt % cross-linked CNF suspension was deposited on freshly glow discharged carbon coated 300 mesh Cu grids from Ted Pella Inc. and stained with 2 wt % aqueous uranyl acetate solution.
4
Negative-Staining and Cryo-TEM Imaging
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Negative-staining transmission
electron microscopy (TEM) was performed by applying a drop (3 μL)
of sample to a glow-discharged TEM grid (carbon-supported film on
300-mesh Cu grids, Ted Pella, Ltd.). After 30 s the excess liquid
was blotted, and the grids were stained with 2% uranyl acetate for
30 s and allowed to dry in air. Imaging was carried out using a FEI
Tecnai 12 G2 Twin TEM operated at 120 kV. The images were recorded
by a 4Kx4K FEI Eagle CCD camera using the TIA software. Direct imaging
of samples by cryo-TEM was performed as described elsewhere.39 A 3 μL sample was applied onto a glow-discharged
300-mesh copper TEM grid coated with a holey carbon film (Lacey substrate,
Ted Pella, Ltd.). The excess liquid was blotted and the specimens
were vitrified by rapid plunging into liquid ethane precooled by liquid
nitrogen using a Vitrobot Mark IV (FEI). We then transferred the vitrified
samples into a cryo specimen holder (Gatan model 626; Gatan Inc.)
and imaged them at −177 °C using a Tecnai 12 G2 Twin TEM
(FEI), operated at an acceleration voltage of 120 kV in low-dose mode.
Images were recorded with a 4Kx4K FEI Eagle CCD camera. TIA (Tecnai
Imaging & Analysis) software was used to record the images.
electron microscopy (TEM) was performed by applying a drop (3 μL)
of sample to a glow-discharged TEM grid (carbon-supported film on
300-mesh Cu grids, Ted Pella, Ltd.). After 30 s the excess liquid
was blotted, and the grids were stained with 2% uranyl acetate for
30 s and allowed to dry in air. Imaging was carried out using a FEI
Tecnai 12 G2 Twin TEM operated at 120 kV. The images were recorded
by a 4Kx4K FEI Eagle CCD camera using the TIA software. Direct imaging
of samples by cryo-TEM was performed as described elsewhere.39 A 3 μL sample was applied onto a glow-discharged
300-mesh copper TEM grid coated with a holey carbon film (Lacey substrate,
Ted Pella, Ltd.). The excess liquid was blotted and the specimens
were vitrified by rapid plunging into liquid ethane precooled by liquid
nitrogen using a Vitrobot Mark IV (FEI). We then transferred the vitrified
samples into a cryo specimen holder (Gatan model 626; Gatan Inc.)
and imaged them at −177 °C using a Tecnai 12 G2 Twin TEM
(FEI), operated at an acceleration voltage of 120 kV in low-dose mode.
Images were recorded with a 4Kx4K FEI Eagle CCD camera. TIA (Tecnai
Imaging & Analysis) software was used to record the images.
5
Transmission Electron Microscopy of RGS L106R
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RGS L106R was incubated at a concentration of 8 μM in 50 mM Tris HCl pH 7.4, 150 mM NaCl, 5 mM β-mercaptoethanol at 25°C for 12 hours. The aggregation reaction solution was diluted 10x in the same buffer prior to sample preparation. A drop of 3-5 μL sample was applied to a glow discharged TEM grid (carbon supported film on 300 mesh Cu grids, Ted Pella, Ltd.). After 30 sec the excess liquid was blotted, the grids were stained with 2% uranyl acetate for 30-60 sec, blotted and allowed to dry in air. The samples were examined using FEI Tecnai 12 G2 TWIN TEM operated at 120 kV. The images were recorded by a 4K
x 4K FEI Eagle CCD camera.
x 4K FEI Eagle CCD camera.
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