Cryo-TEM specimens were prepared in a controlled environment box using a vitrification robot (Vitrobot). 60 μL of the microemulsion (the final curcumin concentration was 1% w/w (27.1 mM)) was dropped onto a glow-discharged TEM grid (300-mesh Cu Lacey substrate; Ted Pella Ltd.). Excess was automatically blotted with a filter paper, and the specimen was rapidly plunged into liquid ethane and transferred to liquid nitrogen where it was kept until used. Specimens were analyzed below −175°C using an FEI Tecnai 12G2 TWIN TEM operated at 120 kV in a low-dose mode and with a few micrometers under focus to increase phase contrast. Images were recorded with a Gatan charge-coupled device camera (model 794) and examined using Digital Micrograph software, Version 3.1.
Tecnai 12 g2 twin tem
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Tecnai 12 G2 TWIN TEM is a transmission electron microscope designed for high-resolution imaging of samples. It features a LaB6 electron source and is capable of operating at an accelerating voltage of up to 120 kV. The instrument is equipped with a twin-lens system and provides a range of magnification options to suit various research and analytical needs.
Automatically generated - may contain errors
Lab products found in correlation
Vitrobot mark 4, by Thermo Fisher Scientific (4 mentions)
Lacey substrate, by Ted Pella (2 mentions)
300 mesh cu grid, by Ted Pella (1 mentions)
4kx4k fei eagle ccd camera, by Thermo Fisher Scientific (1 mentions)
Gatan model 626, by Ametek (1 mentions)
Tecnai12 g2 spirit twin tem, by Thermo Fisher Scientific (1 mentions)
Gatan 626, by Ametek (1 mentions)
Eagle ccd camera, by Thermo Fisher Scientific (1 mentions)
6 protocols using tecnai 12 g2 twin tem
1
Cryo-TEM Microemulsion Imaging Protocol
2
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.
3
Cryo-EM Visualization of Protein Samples
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Three representative samples, A3, B3, and C6, and two references,
keratin and PLD, were tested. Samples, excluding PLD, were prepared
at the following procedure: a 3 μL drop of the sample was applied
to a glow-discharged TEM grid (300-mesh Cu grid) coated with a holey
carbon film (Lacey substrate, Ted Pella, Ltd.). The excess liquid
was blotted, and the specimen was vitrified by rapidly plunging into
liquid ethane precooled with liquid nitrogen using Vitrobot Mark IV
(FEI). The vitrified samples were transferred to a cryo-holder (Gatan
model 626) and examined at −177 °C using an FEI Tecnai
12 G2 Spirit TWIN TEM. The images were recorded by a 4 K × 4
K Eagle CCD camera (FEI) at 120 kV in low-dose mode. TEM imaging was
applied for PLD, and a 3 μL drop was applied to a glow-discharged
TEM grid (carbon-supported film on 300-mesh Cu grids, Ted Pella).
After 30 s, the excess liquid was blotted, and the grids were allowed
to dry for 2 h at room temperature before they were observed by an
FEI Tecnai 12 G2 TWIN TEM operated at 120 kV. The images were recorded
by a 4 K × 4 K FEI Eagle CCD camera.
keratin and PLD, were tested. Samples, excluding PLD, were prepared
at the following procedure: a 3 μL drop of the sample was applied
to a glow-discharged TEM grid (300-mesh Cu grid) coated with a holey
carbon film (Lacey substrate, Ted Pella, Ltd.). The excess liquid
was blotted, and the specimen was vitrified by rapidly plunging into
liquid ethane precooled with liquid nitrogen using Vitrobot Mark IV
(FEI). The vitrified samples were transferred to a cryo-holder (Gatan
model 626) and examined at −177 °C using an FEI Tecnai
12 G2 Spirit TWIN TEM. The images were recorded by a 4 K × 4
K Eagle CCD camera (FEI) at 120 kV in low-dose mode. TEM imaging was
applied for PLD, and a 3 μL drop was applied to a glow-discharged
TEM grid (carbon-supported film on 300-mesh Cu grids, Ted Pella).
After 30 s, the excess liquid was blotted, and the grids were allowed
to dry for 2 h at room temperature before they were observed by an
FEI Tecnai 12 G2 TWIN TEM operated at 120 kV. The images were recorded
by a 4 K × 4 K FEI Eagle CCD camera.
4
Cryo-TEM Imaging of Vitrified Samples
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TEM was performed at the Hebrew University's Nano‐Center as follows. Direct imaging of the samples was performed using cryogenic transmission electron microscopy (cryo‐TEM). In this method, 3 μl of the solution was deposited on a glow‐discharged TEM grid (300 mesh Cu Lacey substrate, Ted Pella, Ltd.). Vitrobot Mark IV (FEI) was used to blot the excess liquid in a controlled environment and to vitrify the specimens by a rapid plunging into liquid ethane precooled with liquid nitrogen. The vitrified samples were examined at −177°C using an FEI Tecnai 12 G2 TWIN TEM operated at 120 kV and equipped with a Gatan 626 cold stage. TIA (Tecnai Imaging & Analysis) software was used to record the images in low dose mode on a 4 K × 4 K FEI Eagle CCD (charge‐coupled device) camera. To overcome image internal gradients and noise, we applied a pseudo flat field correction (Fiji, biovoxel) and then local contrast transformation (Fiji, CLAHE) and a smoothing gaussian filter (Sigma – 6px).
5
Cryo-TEM Imaging of TasA Fibers
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A 3 μL droplet of TasA solution was deposited on a glow-discharged TEM grid (300 mesh Cu Lacey substrate grid; Ted Pella). The excess liquid was blotted off with filter paper, and the specimen was rapidly plunged into liquid ethane pre-cooled with liquid nitrogen in a controlled environment using a Vitrobot Mark IV (FEI). The vitrified samples were transferred to a cryo-specimen holder (Gatan 626) and examined at −177 °C using a FEI Tecnai 12 G2 TWIN TEM instrument operated at 120 kV in low-dose mode. Grids were imaged at a few micrometers under focus to increase phase contrast. The images were recorded with a 4K × 4K FEI Eagle charged-couple device (CCD) camera.
TasA fibers that were scanned with cryo-TEM were prepared as follows. TasA were fibers formed by incubating a 200 µg/mL TasA in 20 mM tris, 0.05 M and 1.5 M NaCl at 4 °C for 8 days (preparation method 2, shown below). Bundles of fibers were formed from a 700 µg/mL TasA preparation that was kept in 1.5 M saline extraction buffer [12 (link)] for 10 days at 4 °C (preparation method 1 below). The sample was dialyzed against 20 mM tris, 50 mM NaCl solution prior to cryo-TEM imaging. TasA aggregates in acid formed following the pH adjustment of a TasA solution (200 µg/mL in 20 mM tris, 50 mM NaCl) to 2.5 using formic acid (preparation method 3, shown below).
TasA fibers that were scanned with cryo-TEM were prepared as follows. TasA were fibers formed by incubating a 200 µg/mL TasA in 20 mM tris, 0.05 M and 1.5 M NaCl at 4 °C for 8 days (preparation method 2, shown below). Bundles of fibers were formed from a 700 µg/mL TasA preparation that was kept in 1.5 M saline extraction buffer [12 (link)] for 10 days at 4 °C (preparation method 1 below). The sample was dialyzed against 20 mM tris, 50 mM NaCl solution prior to cryo-TEM imaging. TasA aggregates in acid formed following the pH adjustment of a TasA solution (200 µg/mL in 20 mM tris, 50 mM NaCl) to 2.5 using formic acid (preparation method 3, shown below).
6
Cryo-TEM Imaging of Gold Nanoparticles
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Gold nanoparticles solutions (3 μL) were applied onto a glow-discharged TEM grid (300-mesh Cu grid) coated with a holey carbon film (Lacey substrate, Ted Pella, Ltd., Redding, CA, USA). The excess liquid was blotted, and the specimens were vitrified by a rapid plunging into liquid ethane pre-cooled with liquid nitrogen using Vitrobot Mark IV (FEI). The vitrified samples were examined at −177 °C using FEI Tecnai 12 G2 TWIN TEM (FEI, USA) operated at 120 kV and equipped with a Gatan model 626 cold stage. The images were recorded by a 4k × 4k FEI Eagle CCD camera in low dose mode. TIA software (Tecnai Imaging and Analysis) was used to record the images.
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