For visualization of the isolated EVs, samples were vitrified following standard protocols [41 (link)]. Glow-discharged Quantifoil holey carbon film grids (Orthogonal Array of 2 μm Diameter Holes—2 μm Separation, mounted on a 300 M Cu grid, #657-300-CU, Ted Pella) were vitrified in liquid ethane in Vitrobot (FEI) after deposition of 3 μL of sample. Cryotransfer sample holders of the type GATAN Model 626 were used to keep the sample vitrified during electron microscopy analysis. The sample was observed in a JEM-2100F UHR (80–200 kV, JEOL, Ltd., Tokio, Japan) field emission gun (FEG) transmission electron microscope at different magnifications. Micrographs were recorded on TVIPS F216 CMOS camera (2 k × 2 k).
F216 cmos camera
Manufactured by TVIPS
Sourced in Germany
The F216 CMOS camera is a scientific imaging device designed for high-performance microscopy and imaging applications. It features a CMOS sensor that captures digital images with high resolution and low noise, suitable for a variety of research and analysis tasks.
Automatically generated - may contain errors
Lab products found in correlation
Osmium tetroxide, by Polysciences (3 mentions)
Jem 2100f uhr, by JEOL (2 mentions)
Cm12 em, by Philips (2 mentions)
Uranyl acetate, by Electron Microscopy Sciences (2 mentions)
Jem 2100f microscope, by JEOL (1 mentions)
Centurio, by JEOL (1 mentions)
Lambda 1050, by PerkinElmer (1 mentions)
Gemini ultra 55, by Zeiss (1 mentions)
Model 626, by Ametek (1 mentions)
Cm12 electron microscope, by TVIPS (1 mentions)
Uranyl acetate, by Philips (1 mentions)
8 protocols using f216 cmos camera
1
Cryo-TEM Imaging of Isolated Extracellular Vesicles
2
Visualizing Birbeck Granules in 293T Cells
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Pellets of 293T cells with mannan-induced Birbeck granules were fixed with 4% paraformaldehyde for 1 hr at 4°C and stained with 1% osmium tetroxide and subsequently with 1% uranium acetate. After dehydration in ethanol and acetone, the samples were embedded in Quetol 812 resin (Nissin EM, Tokyo, Japan). Sixty-nm-ultrathin sections were cut using a ULTRACUT microtome (Reichert Leica) and mounted onto Formvar-coated copper grid. Images were recorded using a JEM-2100F microscope (JEOL, Tokyo, Japan) at University of Yamanashi operated at 200 keV equipped with an F216 CMOS camera (TVIPS GmbH, Gauting, Germany). Length of Birbeck granules were measured using Fiji software (Schindelin et al., 2012 (link)), and plots were generated using PlotsOfData web tool (Postma and Goedhart, 2019 (link)). For total length of Birbeck granules per 100 µm2, cross sections of cells with nuclei of >5 µm in diameter were selected, and the sum of the lengths of Birbeck granules within the cells were measured. The areas of the cross sections were also measured using Fiji software. 100 µm2 corresponds to the approximate mean of the cross-sectional area of one cell. All the measurements were repeated three times for obtaining technical replicates.
3
Transmission Electron Microscopy Analysis
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Scanning electron micrographs were recorded on a Zeiss Gemini Ultra 55 scanning electron microscope (SEM), which is equipped with a field emission gun, using an in‐lens secondary electron detector.
A JEOL F200 (scanning) transmission electron microscope (STEM/TEM) equipped with a cold field emission gun and operated at 200 kV was employed to record (high resolution) transmission electron micrographs on a TVIPS F216 CMOS camera (2k x 2k). For details on sample preparation for TEM analysis, see the Supporting Information.
A windowless JEOL Centurio energy‐dispersive X‐ray detector (100 mm2, 0.97 srad, energy resolution <133 eV) contained within the transmission electron microscope was used for energy‐dispersive X‐ray (EDX) spectroscopic analysis. For details, see Ref. [31].
UV/Vis spectra of the immobilized films were recorded with a PerkinElmer LAMBDA 1050 UV/Vis/NIR spectrophotometer equipped with a 150 mm integrating sphere. Diffuse reflectance (R) data were converted into the Kubelka‐Munk function, F(R), which is represented in the spectra as a function of wavelength.
A JEOL F200 (scanning) transmission electron microscope (STEM/TEM) equipped with a cold field emission gun and operated at 200 kV was employed to record (high resolution) transmission electron micrographs on a TVIPS F216 CMOS camera (2k x 2k). For details on sample preparation for TEM analysis, see the Supporting Information.
A windowless JEOL Centurio energy‐dispersive X‐ray detector (100 mm2, 0.97 srad, energy resolution <133 eV) contained within the transmission electron microscope was used for energy‐dispersive X‐ray (EDX) spectroscopic analysis. For details, see Ref. [31].
UV/Vis spectra of the immobilized films were recorded with a PerkinElmer LAMBDA 1050 UV/Vis/NIR spectrophotometer equipped with a 150 mm integrating sphere. Diffuse reflectance (R) data were converted into the Kubelka‐Munk function, F(R), which is represented in the spectra as a function of wavelength.
4
Cryo-EM Imaging of Extracellular Vesicles
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EVs were vitrified following standard protocols [51 (link)]. Glow-discharged Quantifoil holey carbon film grids (Orthogonal Array of 2μm Diameter Holes - 2μm Separation, mounted on a 300M Cu grid, #657-300-CU, Ted Pella) were vitrified in liquid ethane in Vitrobot (FEI) after deposition of 3 μL of sample. Cryo-transfer sample holders of the type GATAN Model 626 were used to keep the sample vitrified during electron microscopy analysis. The sample was observed in a JEM-2100F UHR (80-200kV, JEOL, Ltd.) field emission gun (FEG) transmission electron microscope at different magnifications. Micrographs were recorded on a state of the art TVIPS F216 CMOS camera (2k x 2k).
5
Ultrastructural Analysis of iAT2 ALI Cultures
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iAT2 ALI cultures on Transwell inserts fixed in 2% glutaraldehyde + 1% paraformaldehyde in 0.1M cacodylate buffer at pH 7.4 for 1 hour at RT. They were then washed in cacodylate buffer + 2% sucrose and postfixed in 1.5% osmium tetroxide (Polysciences) overnight at 4°C. The membrane was excised from the insert, block stained in 1.5% uranyl acetate (EMS) for 1 hour at RT. The samples were dehydrated quickly through acetone on ice, from 70% to 80% to 90%. The samples were then incubated 2 times in 100% acetone at RT for 10 minutes each, and in propylene oxide at RT for 15 minutes each. Finally, the samples were changed into EMbed 812 (EMS), left for 2 hours at RT, changed into fresh EMbed 812, and left overnight at RT, after which they were embedded in fresh EMbed 812 and polymerized overnight at 60°C. Embedded samples were thin sectioned (70 nm), and grids were stained in 4% aqueous uranyl acetate for 10 minutes at RT, followed by lead citrate for 10 minutes at RT. Electron microscopy was performed on a Philips CM12 EM operated at 100 kV, and images were recorded on a TVIPS F216 CMOS camera with a pixel size of 2.89 nm per pixel.
6
Vitrification and Cryo-TEM Imaging of Nanoparticles
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Liposomal, polymeric nanoparticle and extracellular vesicle solutions were vitrified following standard protocols described elsewhere [20 (link)]. Quantifoil holey carbon film grids (Orthogonal Array of 2 µm Diameter Holes—2 µm Separation, mounted on a 300 M Cu grid, #657-300-CU, Ted Pella) were vitrified in liquid ethane in Vitrobot (FEI) after a negative glow-discharged treatment of the grids and deposition of 3 µL of each sample. Cryo-transfer sample holders of type GATAN Model 626 kept the sample vitrified during electron microscopy analysis. Every sample was observed on a JEM-2100F UHR (80-200kV, JEOL Ltd. Akishima, Tokio, Japan) field emission gun (FEG) transmission electron microscope at different magnifications (8000× and 30,000×). Low-dose micrographs were recorded on a state of the art TVIPS F216 CMOS camera (2k × 2k).
7
TEM Imaging of SARS-CoV-2 in iAT2 Cultures
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Transmission electron microscopy (TEM) was performed according to methods detailed in (Huang et al., 2020 (link)). Briefly, iAT2 ALI cultures seeded on Transwell inserts were infected with SARS-CoV-2 at a MOI of 5 or mock-infected. At 24 hpi, cells were fixed in 10% formalin for 6 h, post-fixed in 1.5% osmium tetroxide (Polysciences), stained in 1.5% uranyl acetate (Electron Microscopy Sciences, EMS) and embedded samples were thin sectioned (70 nm). Grids were stained in 4% aqueous uranyl acetate followed by lead citrate and imaged on a Philips CM12 Electron Microscope operated at 100kV. Images were recorded on a TVIPS F216 CMOS camera with a pixel size of 0.85-3.80 nm per pixel.
8
SARS-CoV-2 Infection of iAT2 ALI Cultures
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iAT2 ALI cultures on Transwell inserts were infected with SARS-CoV-2 at an MOI of 5 or mock-infected. At 1 dpi, cells were fixed and inactivated in 10% formalin for 6 h at 4°C and removed from the BSL-4 laboratory. The cells were washed with PBS and then post-fixed in 1.5% osmium tetroxide (Polysciences) overnight at 4°C. The membrane was excised from the insert, block stained in 1.5% uranyl acetate (Electron Microscopy Sciences, EMS) for 1 h at room temperature (RT). The samples were dehydrated quickly through acetone on ice, from 70% to 80% to 90%. The samples were then incubated 2 times in 100% acetone at RT for 10 min each, and in propylene oxide at RT for 15 min each. Finally, the samples were changed into EMbed 812 (EMS), left for 2 h at RT, changed into fresh EMbed 812 and left overnight at RT, after which they were embedded in fresh EMbed 812 and polymerized overnight at 60°C. Embedded samples were thin sectioned (70 nm) and grids were stained in 4% aqueous uranyl acetate for 10 min at RT followed by lead citrate for 10 min at RT. Electron microscopy was performed on a Philips CM12 EM operated at 100kV, and images were recorded on a TVIPS F216 CMOS camera with a pixel size of 0.85-3.80 nm per pixel.
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