The process of OMV release, the structure of discharged OMVs, and the morphology and integrity of V. vulnificus cells were characterized by cryo-electron microscopy (cryo-EM) and cryo-ET. Non-centrifuged 4 μL aliquots of bacterial cultures in exponential growth phase were combined with 1 μL of 10 nm gold fiducials (Sigma-Aldrich) and plunge frozen on glow-discharged, 200 mesh, copper Quantifoil grids (Quantifoil, Germany) in liquid ethane using a Vitrobot Mark III system (FEI, Hillsboro, OR, United States). The grids were observed in a JEOL JEM-2200FS field emission TEM (FEG-TEM) (JEOL Ltd., Tokyo, Japan) operating at 200 kV and equipped with an in-column energy filter, a DE-20 direct detection device (Direct Electron LP, San Diego, CA, United States), and a 4k × 4k UltraScan 4000 CCD camera (Gatan, Pleasanton, CA, United States). Single axis tilt series were acquired with SerialEM (Mastronarde, 2005 (link)), with an increment of 2° covering -60° to +60°. The cumulative dose was under 150 electrons/Å2 and the defocus was between -4 and -10 μm. The tomograms were reconstructed by r-weighted back-projection using IMOD (Kremer et al., 1996 (link); Mastronarde, 1997 (link)). Volume-rendered segmentations were performed manually using the Amira package (FEI Visualization Sciences Group, Hillsboro, OR, United States).
Ultrascan 4000 4k 4k ccd camera
Manufactured by Ametek
Sourced in United States
The Ultrascan 4000 is a 4k × 4k CCD camera designed for laboratory applications. It features a high-resolution sensor capable of capturing images with a resolution of 4096 × 4096 pixels. The camera is designed for use in various laboratory settings, where high-quality image capture is required.
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Lab products found in correlation
Tecnai g2 spirit, by Ametek (3 mentions)
Tecnai g2 spirit, by Thermo Fisher Scientific (2 mentions)
Tecnai f20, by Thermo Fisher Scientific (2 mentions)
Titan krios tem, by Thermo Fisher Scientific (1 mentions)
400 mesh carbon coated copper grid, by Ted Pella (1 mentions)
Morgagni m268, by Thermo Fisher Scientific (1 mentions)
Jem 2200fs electron microscope, by JEOL (1 mentions)
S200 16 60 gel filtration column, by GE Healthcare (1 mentions)
Malvern zetasizer nano, by Malvern Panalytical (1 mentions)
Filter paper, by Cytiva (1 mentions)
Whatman, by Cytiva (1 mentions)
Morgagni electron microscope, by Thermo Fisher Scientific (1 mentions)
Tecnai t12, by Thermo Fisher Scientific (1 mentions)
15 protocols using ultrascan 4000 4k 4k ccd camera
1
Cryo-EM Characterization of OMV Release
2
Large-volume EM Tomography of BHK Cells
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Two large-volume EM tomography data sets were collected covering an ∼45 μm2 area of a BHK cell through a depth of 2–2.5 μm (Fig S2 ). Images were acquired using a Titan Krios TEM (FEI), operating at 300 kV at room temperature, outfitted with a 4k × 4k UltraScan 4000 CCD camera (Gatan, Inc.). Tilt series were collected with automation using the program SerialEM (Mastronarde, 2003 (link)). First, a low magnification image montage of the entire grid was created to use as a map for marking regions of interest. The area of interest in the cell was visualized and marked on consecutive serial sections. At each section, a tilt series was collected. For each tilt series, images were collected at tilts from +60 to −60 degrees in 2° increments. To increase the area covered by each image, 2 × 2 montage images were collected at each tilt at 14,000× magnification, corresponding to a 0.65 nm/pixel size at the specimen level. Such montage tilt series were collected through 10 serial sections (Tomogram 1) or 7 serial sections (Tomogram 2). Smaller tomograms were collected at a pixel size of 0.8 nm, using a similar collection scheme, without serial sectioning or montaging.
3
Cryo-EM Imaging of Umb1 Particles
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Purified Umb1 particles were diluted to 0.01 mg ml–1 and immediately subject to adsorption to glow-discharged carbon-coated copper grids for 60 s followed by 2% uranyl formate (w/v) staining. Micrographs were recorded using Leginon43 (link) on a 120 KV FEI Tecnai G2 Spirit with a Gatan Ultrascan 4,000 4k × 4k CCD camera at ×67,000 nominal magnification. The defocus ranged from −1.0 to −2.0 µm and the pixel size was 1.6 Å. The parameters of the contrast transfer function (CTF) were estimated using CTFFIND44 (link). All particles were picked in a reference-free manner using DoG Picker45 (link). The particle stack from the micrographs was pre-processed in Relion46 . Particles were re-extracted with a binning factor of 4, resulting in a final box size of 80 pixels and a final pixel size of 6.4 Å. The reference-free 2D classification was performed using CryoSPARC47 (link).
4
Negative Stain EM Imaging of EPN-01
5
Negative-Stain Visualization of PolB1 and Holo-Complexes
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For the negative-stain visualization of the naked PolB1 (∼100 kDa) a glow-discharged carbon-coated grid (CF-300_Cu; EMS, USA) was placed onto 10 μl droplet of 10 μg ml−1 PolB1 and allowed to sit for 1 min, washed with MilliQ water and then stained using 2% uranyl formate solution. Excess liquid was removed by gently touching the grid-side with Whatmann filter paper. A similar procedure was used for the preparation of negative-stain grids of the holo-complexes PolB1:PBP1-ΔC:PBP2:DNA (∼130 kDa) at 10 μg ml−1 (Supplementary Fig. 5a ). Complex with DNA was prepared by incubating the complex with double-stranded DNA at room temperature for 1 h. The mixture was then applied to GE S200 16/60 gel filtration column, the fraction containing protein–DNA complex was used for grid preparation.
A total of 108 images for apo-PolB1 and 400 images for the holoenzyme·DNA PolB1-HE were collected. All data were acquired with a JEOL JEM-2200FS electron microscope operated at 200 kV at a magnification of 90,201 and with underfocus between 1.3–1.7 μm. Images were recorded with an ULTRASCAN 4000, 4 K × 4 K CCD camera (Gatan Inc.) and resulting sampling of 1.66 Å per pixel at the specimen.
A total of 108 images for apo-PolB1 and 400 images for the holoenzyme·DNA PolB1-HE were collected. All data were acquired with a JEOL JEM-2200FS electron microscope operated at 200 kV at a magnification of 90,201 and with underfocus between 1.3–1.7 μm. Images were recorded with an ULTRASCAN 4000, 4 K × 4 K CCD camera (Gatan Inc.) and resulting sampling of 1.66 Å per pixel at the specimen.
6
Cryo-EM Imaging of Frozen Hydrated Samples
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Grids of frozen hydrated samples were transferred into the electron microscope by using the Gatan 626 cryo-transfer system (Gatan, USA). The cryo specimens were imaged in a Tecnai F20 (FEI, Netherlands) electron microscope operated at an acceleration voltage of 200 kV. Low dose images were recorded at a nominal magnification of 80,000 using a Gatan UltraScan 4000 4 k × 4 k CCD camera (Gatan, USA) by an automated data acquisition system, Leginon35 (link).
7
Negative Stain Electron Microscopy of ComM-ATP-ssDNA
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For negative stain electron microscopy, sample was prepared by applying 4 μl of ComM-ATP-ssDNA solution onto a glow-discharged continuous carbon film coated copper grid (EMS) and stained with 0.75% (w/v) uranyl formate. EM micrographs were acquired using a 300 kV JEM-3200FS electron microscopy with 20-eV energy slit under low dose conditions (≤20 e−/Å2) on a Gatan UltraScan 4000 4k × 4k CCD camera. Additional details for EM image collection and analysis are provided in the Supplementary Methods . The EM electron density map has been deposited to EMDataBank.org with the accession number EMD-8575.
8
Structural Visualization of BSMV Using Cryo-EM
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The BSMV samples were diluted to a final concentration of 3 mg/ml in 50 mM Tris–HCl (pH 7.4), 50 mM KCl, 10 mM MgCl2. 3.5 μl of the BSMV was applied to either C-flat grids (r2/2, Protochips) or homemade continuous carbon films, which had been rendered hydrophilic by glow discharge in air. The grids were then blotted and frozen in liquid nitrogen cooled liquid ethane. Low-dose images (20–25 e−/Å2) were manually recorded on a Tecnai Polara EM (FEI) operated at 300 keV, using a Gatan Ultrascan 4,000 4k × 4k CCD camera with an ultra-sensitive phosphor scintillator (Gatan) with a calibrated magnification of 150,000×, giving 1 Å/pixel on the images. A defocus range between 0.7 and 3.0 μm underfocus was used during data collection. Correction for the effects of the contrast transfer function is described in Supplemental Information .
9
Negative Staining of Protein Samples
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Samples were applied to carbon-coated copper grids (square 400 mesh copper grids, 3 mm diameter, Agar Scientific) using the gel-to-grid transfer method. The grids were then washed with 3 μl buffer (100 mM NaCl, 25 mM Tris-HCl pH 7.5), blotted and stained with 3 μl uranyl acetate (2%, pH 4.5) for 2 min. The grids were blotted once more, dried and analyzed under the microscope. A preliminarily examination was carried out on a Tecnai T12 electron microscope at 120 kV to assess grid quality and sample and stain distribution followed by the data collection using Tecnai F20 FEG electron microscope. The images were recorded under low electron-dose conditions using a Gatan Ultrascan 4000, 4k × 4k CCD camera at a nominal magnification of x50 000 (pixel size of 3.6 Å/pixel). A range of defocus values varying from 1.5 to 2.5 μm was used for data collection.
10
Nanoplastic Characterization Protocol
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Nanoplastic suspensions were characterized following temperature pre-treatments. Nanoplastics and aggregate sizes were determined using dynamic light scattering (DLS) and transmission electron microscopy (TEM), while the zeta potential (ZP), which is an estimation of surface charge, was determined from the electrophoretic mobility measured by laser Doppler velocimetry.
The electrophoretic mobility measurements were converted to ZP using the Smoluchowski approximation with the Henry equation (Lowry et al., 2016) . Both DLS and laser Doppler velocimetry measurements were conducted using a Malvern Zetasizer Nano (Malvern Panalytical). For DLS measurements, the Z-average diameter (dh, Z-avg) (cumulants mean diameter), intensity mean diameter (dh, intensity) and volume mean diameter (dh, volume) are reported.
The polydispersity index (PDI) provides an indication of the heterogeneity of aggregate sizes within a suspension and ranges from 0 (monodisperse) to 1 (highly polydisperse). Aggregate morphology and primary particles were visualized using TEM (FEI Technai 120 kV TEM coupled with a Gatan Ultrascan 4000 4k×4k CCD camera. TEM was performed on suspensions deposited onto thin carbon film grids (Pacific Grid-Tech, 300 mesh, 3.05 mm O.D., hole size) and allowed to dry at room temperature after wicking away the excess liquid with a Whatman filter paper.
The electrophoretic mobility measurements were converted to ZP using the Smoluchowski approximation with the Henry equation (Lowry et al., 2016) . Both DLS and laser Doppler velocimetry measurements were conducted using a Malvern Zetasizer Nano (Malvern Panalytical). For DLS measurements, the Z-average diameter (dh, Z-avg) (cumulants mean diameter), intensity mean diameter (dh, intensity) and volume mean diameter (dh, volume) are reported.
The polydispersity index (PDI) provides an indication of the heterogeneity of aggregate sizes within a suspension and ranges from 0 (monodisperse) to 1 (highly polydisperse). Aggregate morphology and primary particles were visualized using TEM (FEI Technai 120 kV TEM coupled with a Gatan Ultrascan 4000 4k×4k CCD camera. TEM was performed on suspensions deposited onto thin carbon film grids (Pacific Grid-Tech, 300 mesh, 3.05 mm O.D., hole size) and allowed to dry at room temperature after wicking away the excess liquid with a Whatman filter paper.
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