Electron tomography of epoxy resin sections of HP frozen, rapidly freeze-substituted ovisacs of A. franciscana was performed using a Tecnai-20 electron microscope at 200 kV equipped with an eucentric goniometer and a single high-tilt holder (FEI, Eindhoven, The Netherlands) Tilt series of digital images within an angular range of 65° to +65° and a tilt increment of 1° were recorded with the help of the Xplore 3D software (FEI company) using a Eagles 4K-CCD camera (FEI Company; chip size: 4.096 × 4.096 pixels). In order to reconstruct the volume of the 200–300 nm thick sections into virtual slices (thickness 0.39–0.46 nm corresponding to a microscope magnification of 29.000× or 25.000× respectively), we used the IMOD software (Boulder Laboratory for 3D Electron Microscopy of Cells, University of Colorado, USA). For 3D-modeling, the structures of interest in each slice were traced with colored contours that were merged in the Z-axis with the help of the Amira 5.3 software (Mercury Computer Systems, Merignac, Cedex, France).
Tecnai 20 electron microscope
Manufactured by Thermo Fisher Scientific
Sourced in Netherlands
The Tecnai 20 is a transmission electron microscope (TEM) manufactured by Thermo Fisher Scientific. It is designed for high-resolution imaging and analysis of materials at the nanoscale. The Tecnai 20 operates at an accelerating voltage of 200 kV and offers a range of advanced imaging modes, including bright-field, dark-field, and high-resolution electron microscopy.
Automatically generated - may contain errors
Lab products found in correlation
4 k eagle ccd camera, by Thermo Fisher Scientific (3 mentions)
Xplore3d software, by Thermo Fisher Scientific (1 mentions)
Single high tilt holder, by Thermo Fisher Scientific (1 mentions)
Em uc7 ultramicrotome, by Leica Microsystems (1 mentions)
Ultrascan 1000 ccd camera, by Ametek (1 mentions)
Xl30 sfeg, by Thermo Fisher Scientific (1 mentions)
Sp8 confocal microscope, by Leica camera (1 mentions)
Solarus plasma cleaner, by Ametek (1 mentions)
Jsm 6310, by JEOL (1 mentions)
Ultracut uct microtome, by Leica camera (1 mentions)
13 protocols using tecnai 20 electron microscope
1
3D Ultrastructural Analysis of Artemia Ovisacs
2
Transmission Electron Microscopy of OMVs
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The bacterial culture sample taken before filtration and centrifugation and the isolated EcO83-OMVs were imaged with TEM. The bacterial culture was centrifuged at 5 000 × g and the pellet was washed twice with PBS before microscopy. EcO83-OMVs were dissolved in NaCl. Ultrastructural visualization was carried out by single-droplet negative stain. Briefly, 5 µl of each OMV preparation were left to adhere on formvar- and carbon-coated grids for 20 s. Simultaneous addition of 5—10 µl 1% uranylacetate and absorption of liquid was repeated twice to contrast the samples. Dried grids were imaged in an FEI Tecnai20 electron microscope (FEI, Eindhoven, NL) equipped with a 4 K Eagle-CCD camera.
3
Ultrastructural Changes in Ischemic Microvessels
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Transient focal cerebral ischemia was induced in one Trpv4–/– and one WT mouse. At 6 h post-tMCAO (Kurisu et al., 2016 (link)), mice were transcardially perfused with 4% paraformaldehyde with 3.4% sucrose. Brain slices including the right striatum were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer at 4°C overnight, post-fixed with 1% OsO4 in 0.1 M cacodylate buffer on ice for 2 h, dehydrated in a graded ethanol series and propylene oxide, and embedded in epoxy resin. Ultrathin sections were cut at 80-nm thickness with an EM UC7 ultramicrotome (Leica Microsystems, Wetzlar, Germany) and stained with 2% uranyl acetate followed by a mixture of lead nitrate-lead acetate-lead citrate. The sections were observed with a Tecnai 20 electron microscope (FEI Co., Eindhoven, Netherlands) operated at an accelerating voltage of 200 kV and micrographs were recorded at a magnification of 5,000× for at least 10 microvasculatures.
4
Electron Microscopy Analysis of Viral Capsid Assembly
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Vero parental or Vero+BirA cell lines were seeded in 35 mm plates and infected at an MOI = 10 for 1.5 h at 37 °C before transfer into 1.2 mL fresh medium containing 50 µM biotin. At 7.5 hpi, the cells were washed once with serum-free medium and then fixed with 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium cacodylate buffer for 30 min at room temperature. The Einstein Analytical Imaging Facility then processed the samples by postfixing with 1% osmium tetroxide and 2% uranyl acetate, dehydrating with ethanol, and lifting the monolayer from the dish by propylene oxide. The samples were pelleted and embedded, and thin sections were stained with uranyl acetate followed by lead citrate. Images were taken on a JEOL 1200EX electron microscope at 80 kV and assembled using Adobe Photoshop software. For negative stain analysis of CLP assembly reactions, 5 µL of the 100 µL reaction was applied to glow-discharged carbon-coated copper grids and stained with 1% v/v uranyl acetate. Grids were air dried and then viewed on a FEI Tecnai 20 electron microscope at 120 kV.
5
Characterization of Carbon Nanotubes by TEM
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The CNTs were dispersed in DMEM cell culture medium at 1 mg/ml dilution and treated with ultrasound for 20 min. 5 μl of this solution was placed on a carbon coated copper grid that had previously been treated with a PELCO easiGlow Glow Discharge device. After 1 min incubation, the solution was withdrawn using non hardened microscopic filter paper (Whatman).
Images were taken using a FEI Tecnai G2 20 transmission electron microscope with a Gatan ultrascan 1000 ccd camera. Acceleration voltage was 80 kV. Sizes of CNTs were measured from the TEM images.
For energy dispersive X-ray spectroscopy (EDX), the specimens were diluted in double distilled water, sonicated, and placed on pioloform-coated nickel grids. Scanning transmission EM (STEM) micrographs were made with an FEI Tecnai 20 electron microscope at 20,000 × magnification and 120 kV acceleration voltage with a high angle annular dark field detector. EDX spectra were made with a Standard SUTW detector (EDAX) in rectangular selections within the field of view of these micrographs and each spectrum was recorded for 2 min. Data on the elemental composition were gained using Peak ID and Quantify functions of TEM imaging and analysis (TIA) software, FEI company.
Images were taken using a FEI Tecnai G2 20 transmission electron microscope with a Gatan ultrascan 1000 ccd camera. Acceleration voltage was 80 kV. Sizes of CNTs were measured from the TEM images.
For energy dispersive X-ray spectroscopy (EDX), the specimens were diluted in double distilled water, sonicated, and placed on pioloform-coated nickel grids. Scanning transmission EM (STEM) micrographs were made with an FEI Tecnai 20 electron microscope at 20,000 × magnification and 120 kV acceleration voltage with a high angle annular dark field detector. EDX spectra were made with a Standard SUTW detector (EDAX) in rectangular selections within the field of view of these micrographs and each spectrum was recorded for 2 min. Data on the elemental composition were gained using Peak ID and Quantify functions of TEM imaging and analysis (TIA) software, FEI company.
6
Ultrastructural Analysis of Infected Dictyostelium
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A confluent 6-cm dish of infected Dictyostelium was fixed for one hour with 2% glutaraldehyde in Hl5c and stained for 20 min with imidazole (0.1 M)-buffered 2% osmium tetroxide, as previously described ([61 (link)]; [12 (link)]). Afterwards cells were collected using a cell scraper and resuspended in 1 ml of phosphate-buffered saline (PBS) in an Eppendorf tube. After two washes with PBS, the samples were sent to the EM platform of the Faculty of Medicine, University of Geneva for further processing. There, samples were postfixed, embedded in Epon resin and further processed as previously described [62 (link)]. Images were taken with a Tecnai 20 electron microscope (FEI).
7
TEM Sample Preparation for D. discoideum
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Sample preparation for TEM was performed as described in [68 (link)]. Briefly, D. discoideum cells were fixed in a 6 cm dish in 2% (w/v) glutaraldehyde in Hl5c for 1 h and stained with 2% (w/v) OsO4 in imidazole buffer 0.1 M for 30 min. Cells were detached with a cell scraper and washed 3 times with PBS. Subsequent sample preparation was performed at the Pôle Facultaire de Microscopie Ultrastructurale (University of Geneva). Samples were incubated in 2% (v/v) of Milloning buffer and rinsed with distilled water. Then, they were incubated in 0.25% (w/v) uranyl acetate overnight and rinsed with distilled water. Samples were dehydrated using increasing concentrations of ethanol, then in propylene oxide for 10 min and finally embedded in 50% Epon-propylene oxide for 1h, followed by incubation overnight in pure Epon. Samples were embedded in 2% agar for subsequent sectioning in an ultramicrotome and placed on TEM grids. Finally, sections were visualized in a Tecnai 20 electron microscope (FEI Company, Eindhoven, The Netherlands).
8
Multimodal Imaging of Particle Morphology
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Imaging of the
particle morphology
was performed with TEM, SEM, and confocal microscopy. TEM was performed
with an FEI TECNAI 20 electron microscope in which samples were prepared
by pipetting a drop of the particle dispersion in ethanol onto a Formvar/Carbon
Films 200 Mesh Copper (100) grid. SEM was performed with an FEI XL30SFEG,
FEI Phenom in which samples were prepared by pipetting a drop of the
particle dispersion onto an aluminum stub (Figure 1 ) or silicon wafer on top of an aluminum
stub and subsequently sputter-coated with a platinum layer of approximately
4 nm. Confocal microscopy was performed with a Leica SP8 confocal
microscope (100× oil immersion lens) with excitation by a 488
nm laser line. The reflection signal was recorded at 488 nm, and the
fluorescence was in the range 500–600 nm. To prepare the samples,
the rods with fluorescent heads were dried from ethanol on a #1.5
coverslip before adding a drop of refractive index matching liquid
(glycerol/water mixture, 85:15 by mass) on top.
particle morphology
was performed with TEM, SEM, and confocal microscopy. TEM was performed
with an FEI TECNAI 20 electron microscope in which samples were prepared
by pipetting a drop of the particle dispersion in ethanol onto a Formvar/Carbon
Films 200 Mesh Copper (100) grid. SEM was performed with an FEI XL30SFEG,
FEI Phenom in which samples were prepared by pipetting a drop of the
particle dispersion onto an aluminum stub (
stub and subsequently sputter-coated with a platinum layer of approximately
4 nm. Confocal microscopy was performed with a Leica SP8 confocal
microscope (100× oil immersion lens) with excitation by a 488
nm laser line. The reflection signal was recorded at 488 nm, and the
fluorescence was in the range 500–600 nm. To prepare the samples,
the rods with fluorescent heads were dried from ethanol on a #1.5
coverslip before adding a drop of refractive index matching liquid
(glycerol/water mixture, 85:15 by mass) on top.
9
Structural Characterization of PI3KC3-C2:MBP-Rubicon Complex
10
Scanning and Transmission Electron Microscopy of Bacterial Isolates
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All isolates were incubated overnight on MHA plates at 36 °C ( ± 1 °C) and re-suspended in 0.1 M phosphate buffer (pH 7.2). Cell pellets were obtained by centrifugation (10,000× g for 10 min at 4 °C) and fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer for 20 min for scanning electron microscopy (SEM) or 24 h for transmission electron microscopy (TEM). For SEM, samples were dehydrated in ascending ethanol series and re-suspended in hexamethyldisilazane (HMDS). After complete evaporation of HMDS samples were sputter-coated with gold (Sputter Coater, SC 502, Polaron, Fisons Instruments®, England) and examined using a scanning electron microscope (JSM 6310, Jeol Ltd.®, Japan). Images were taken at an acceleration voltage of 15 kV51 (link).
TEM analysis was performed as described previously52 (link). Briefly, samples were incubated in 1% OsO4 and embedded in Epon resin. 70 nm ultra-thin sections were imaged with an FEI Tecnai20 electron microscope equipped with a 4 K Eagle-CCD camera. Images were processed with Adobe Photoshop. For measurements of the thickness of the cell walls, ~100 non-dividing cells each were imaged. The ImageJ software package was used for measurements. Cell cycle stages of the three isolates (DR-I1, DR-I3 and DR-I4) were assessed by counting approximately 200 cells each.
TEM analysis was performed as described previously52 (link). Briefly, samples were incubated in 1% OsO4 and embedded in Epon resin. 70 nm ultra-thin sections were imaged with an FEI Tecnai20 electron microscope equipped with a 4 K Eagle-CCD camera. Images were processed with Adobe Photoshop. For measurements of the thickness of the cell walls, ~100 non-dividing cells each were imaged. The ImageJ software package was used for measurements. Cell cycle stages of the three isolates (DR-I1, DR-I3 and DR-I4) were assessed by counting approximately 200 cells each.
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