The 200 mesh grids covered with perforated carbon film (R1/4 batch of Quantifoil, MicroTools GmbH, Jena, Germany) were cleaned with chloroform and hydrophilised upon 60 s glow discharging at 8 W in a BAL‐TEC MED 020 device (Leica Microsystems, Wetzlar, Germany). After applying aliquots (5 μL) of the dye solution to the grids, the samples were vitrified by automated blotting and plunge freezing into liquid ethane by using an FEI Vitrobot Mark IV device (Thermo Fisher Scientific Inc., Waltham, MA, USA). The vitrified specimens were transferred under liquid nitrogen to an FEI TALOS L120C electron microscope (Thermo Fisher Scientific Inc., Waltham, MA, USA) by using a Gatan cryo‐holder and stage (model 626, Gatan, Inc., Pleasanton, CA, USA). The microscope was equipped with an LaB6 cathode and operated at 120 kV accelerating voltage. Micrographs were acquired on an FEI Ceta CMOS camera (Thermo Fisher Scientific Inc., Waltham, MA, USA) at a nominal magnification of 36 000×, corresponding to a calibrated pixel size of 4.09 Å per pixel.
Baltec med 020 device
Manufactured by Leica Microsystems
Sourced in Germany
The BALTEC MED 020 is a device designed for use in laboratory environments. Its core function is to perform metal coating and deposition processes, which are essential for preparing samples for microscopy and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Tecnai f20 tem, by Thermo Fisher Scientific (3 mentions)
4 k eagle ccd camera, by Thermo Fisher Scientific (3 mentions)
Fei talos l120c electron microscope, by Thermo Fisher Scientific (1 mentions)
Fei vitrobot mark 4, by Thermo Fisher Scientific (1 mentions)
Falcon 3 direct electron detector, by Thermo Fisher Scientific (1 mentions)
Fei talos arctica electron microscope, by Thermo Fisher Scientific (1 mentions)
Talos l120c microscope, by Thermo Fisher Scientific (1 mentions)
7 protocols using baltec med 020 device
1
Cryo-EM Specimen Preparation Protocol
2
Transmission Electron Microscopy Preparation
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Droplets of the corresponding sample solutions (5 μL) were placed for 30 s on hydrophilized (60 s plasma treatment at 8 W using a BAL-TEC MED020 device [Leica Microsystems, Wetzlar, Germany]) carbon-coated copper grid. Excess fluid was removed with a filter paper, and the samples were analyzed in a CM-12 by FEITM with an accelerating voltage of 60 kV.
3
Cryo-ET of Virus Structures
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For cryo-ET of the viruses, perforated carbon film–covered microscopical 200 mesh grids (R1/4 batch of Quantifoil, MicroTools GmbH) were cleaned with chloroform and hydrophilized by 60 s glow discharging at 8 W in a BALTEC MED 020 device (Leica Microsystems), before 4 μl aliquots of the virus solution were applied to the grids. The samples were vitrified by automatic blotting and plunge freezing with a FEI VitrobotMark IV (Thermo Fisher Scientific) using liquid ethane as cryogen. The vitrified specimens were transferred under liquid nitrogen into the autoloader of a FEI TALOS ARCTICA electron microscope (Thermo Fisher Scientific). This microscope is equipped with a high-brightness field-emission gun operated at an acceleration voltage of 200 kV. Single-axis tilt series (±64° at 2° angular increment) were recorded with the Falcon 3 direct electron detector (Thermo Fisher Scientific) using a Volta Phase Plate at 28 K primary magnification with a total dose lower than 100 e−/Å2. Tomogram reconstruction was performed using Thermo Fisher Inspect3D software.
4
Transmission Electron Microscopy of rHA
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The original
solution of rHA was diluted 1:2 with PBS. Then 5 μL of the suspension
was pipetted onto a hydrophilized (by 60 s glow discharging at 8 W
in a BALTEC MED 020 device (Leica Microsystems, Wetzlar, Germany))
Formvar-supported carbon-covered microscopical copper grid (400 mesh).
After 30 s, a piece of filter paper was used to remove excess fluid.
Subsequently, 5 μL of a contrast-enhancing heavy metal staining
solution (1% phosphotungstic acid, pH 7.4) was applied and blotted
again after 45 s. After air-drying, a standard holder was used to
transfer the sample into a Talos L120C microscope (Thermo Fisher Scientific
Inc., Waltham, Massachusetts, USA) equipped with a LaB6-cathode operated at an acceleration voltage of 120 kV. Micrographs
were recorded with a 4k × 4k Ceta 16 M camera at a nominal magnification
of 57000×.
solution of rHA was diluted 1:2 with PBS. Then 5 μL of the suspension
was pipetted onto a hydrophilized (by 60 s glow discharging at 8 W
in a BALTEC MED 020 device (Leica Microsystems, Wetzlar, Germany))
Formvar-supported carbon-covered microscopical copper grid (400 mesh).
After 30 s, a piece of filter paper was used to remove excess fluid.
Subsequently, 5 μL of a contrast-enhancing heavy metal staining
solution (1% phosphotungstic acid, pH 7.4) was applied and blotted
again after 45 s. After air-drying, a standard holder was used to
transfer the sample into a Talos L120C microscope (Thermo Fisher Scientific
Inc., Waltham, Massachusetts, USA) equipped with a LaB6-cathode operated at an acceleration voltage of 120 kV. Micrographs
were recorded with a 4k × 4k Ceta 16 M camera at a nominal magnification
of 57000×.
5
Cryo-EM Imaging of LΔF and LΔF/fMLF Complexes
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Droplets (5
μL) of aqueous samples ofLΔF and LΔF /fMLF at molar ratios of 1:0.25, 1:0.5, and 1:1 in 0.8
M sodium acetate buffer at pH 7.4 were placed on hydrophilized [plasma
treatment using a BALTEC MED 020 device (Leica Microsystems, Wetzlar,
Germany)], perforated carbon filmed grids (Quantifoil Micro Tools
GmbH, Jena, Germany). Excess fluid was blotted off to create an ultrathin
layer (typical thickness 200–300 nm) of the solution, which
spanned the holes of the support film. The prepared samples were immediately
vitrified by propelling the grids into liquid ethane at its freezing
point (−184 °C). The vitrified sample grids were transferred
under liquid nitrogen by the use of a Gatan (Pleasanton, CA, USA)
cryo-holder (model 626) into a Tecnai F20 TEM (FEI company, Oregon,
USA) equipped with FEG and operated at 160 kV acceleration voltage.
Microscopy was carried out at −175 °C sample temperature
using the microscope’s low dose protocol at calibrated primary
magnifications of 50k or 29k. The defocus was set to be 3.98 or 9.81
μm, respectively. Images were recorded by the use of a 4k-Eagle
CCD camera (FEI Company, Oregon, USA) at 2k resolution (binning 2).
μL) of aqueous samples of
M sodium acetate buffer at pH 7.4 were placed on hydrophilized [plasma
treatment using a BALTEC MED 020 device (Leica Microsystems, Wetzlar,
Germany)], perforated carbon filmed grids (Quantifoil Micro Tools
GmbH, Jena, Germany). Excess fluid was blotted off to create an ultrathin
layer (typical thickness 200–300 nm) of the solution, which
spanned the holes of the support film. The prepared samples were immediately
vitrified by propelling the grids into liquid ethane at its freezing
point (−184 °C). The vitrified sample grids were transferred
under liquid nitrogen by the use of a Gatan (Pleasanton, CA, USA)
cryo-holder (model 626) into a Tecnai F20 TEM (FEI company, Oregon,
USA) equipped with FEG and operated at 160 kV acceleration voltage.
Microscopy was carried out at −175 °C sample temperature
using the microscope’s low dose protocol at calibrated primary
magnifications of 50k or 29k. The defocus was set to be 3.98 or 9.81
μm, respectively. Images were recorded by the use of a 4k-Eagle
CCD camera (FEI Company, Oregon, USA) at 2k resolution (binning 2).
6
Vitrification of Protein Samples
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A droplet (5 μL) of the sample solution was placed on hydrophilized (plasma treatment using a BALTEC MED 020 device (Leica Microsystems, Wetzlar, Germany), perforated carbon filmed grids (Quantifoil Micro Tools GmbH, Jena, Germany). The excess fluid was blotted off to create an ultra-thin layer (typical thickness of 200-300 nm) of the solution that spans the holes of the supporting film. Samples were then immediately vitrified by propelling the grids into liquid ethane at its freezing point (-184 °C). The vitrified sample grids were transferred under liquid nitrogen, by the use of a Gatan (Pleasanton,CA, USA) cryo-holder (Model 626), into a Tecnai F20 TEM (FEI company, Oregon, USA) equipped with a field emission gun (FEG) and operated under a 160 kV acceleration voltage. Microscopy was carried out at -175 °C sample temperature using the microscope's low dose protocol at calibrated primary magnifications of 5k and 29k. The defocus was set to 9.81 µm (29k). Images were recorded on a 4k-Eagle CCD camera (FEI Company, Oregon, USA) at 2k resolution (binning 2).
7
Cryogenic Electron Microscopy Sample Preparation
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A droplet (5 mL) of the sample solution was placed on hydrophilized (plasma treatment using a BALTEC MED 020 device (Leica Microsystems, Wetzlar, Germany)), perforated carbon filmed grids (Quantifoil Micro Tools GmbH, Jena, Germany). The excess fluid was removed by blotting to create an ultra-thin layer (typical thickness of 200-300 nm) of the solution, which spans the holes of the support film. The prepared samples were immediately vitrified by propelling the grids into liquid ethane at its freezing point (À184 1C). The vitrified sample grids were transferred under liquid nitrogen by the use of a Gatan (Pleasanton, CA, USA) cryoholder (Model 626) into a Tecnai F20 TEM (FEI company, Oregon, USA) equipped with FEG and operated at 160 kV acceleration voltage. Microscopy was carried out at À175 1C sample temperature using the microscope's low dose protocol at calibrated primary magnifications of 5k and 29k. The defocus was set to be 9.81 mm (29k). Images were recorded by the use of a 4k-Eagle CCD camera (FEI Company, Oregon, USA) at 2k resolution (binning 2).
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