The exosome samples were diluted with an appropriate volume of PBS. Diluted exosome samples (10 μL) were applied dropwise onto copper grids for 1 minute, followed by staining with 2% uranyl acetate (10 μL) applied dropwise for 1 minute. Any excess was removed by blotting with filter paper following each step. The grids were air dried for 15 minutes and imaged at a voltage of 120 kV on a FEI Tecnai G2 spirit TEM (Thermo-Fischer, Waltham, MA, USA).
Fei tecnai g2 spirit tem
Manufactured by Thermo Fisher Scientific
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The FEI Tecnai G2 Spirit TEM is a transmission electron microscope (TEM) designed for high-resolution imaging and analysis of nano-scale structures. It provides sub-Angstrom resolution and enables the study of materials at the atomic level. The Tecnai G2 Spirit TEM is equipped with advanced features such as a field emission gun (FEG) electron source and specialized detectors to support a wide range of analytical techniques.
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8 protocols using fei tecnai g2 spirit tem
1
Exosome Negative Staining Visualization
2
TEM Imaging of Diluted Exosomes
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Exosome samples were diluted using PBS at an appropriate volume. Next, the diluted exosomes (10 μL) were added to the copper mesh for 1 min with excess liquid absorbed using filter paper, followed by a 1min staining with 2% uranyl acetate (10 μL) with excess staining solution absorbed using filter paper. Next, the copper mesh underwent a 15-minute air-drying and was imaged on an FEI Tecnai G2 Spirit TEM (Thermo Fisher Scientific Inc., Waltham, MA, USA) at a voltage of 120 kV.
3
Ultrastructural Analysis of Induced Cardiomyocytes
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The induced‐hPSC‐derived cardiomyocytes were directly scraped off from the dish and then fixed with 2% glutaraldehyde overnight at 4°C.26 These samples were postfixed with 0.25% osmium/0.25% K4Fe(CN)6, 1% tannic acid, followed with 50 mmol/L uranyl acetate. Then specimens were washed 3 times and dehydrated with a series of ethanol. Finally, the cell samples were embedded in araldite 502 resin (Polysciences Inc, Warrington, PA), and polymerization proceeded at 65°C for several days. The ultrathin sections (≈60 nm) obtained by ultramicrotome (Leica EM UC7; Leica, Wetzlar, Germany)) were mounted in EM‐grids, stained with lead citrate, and then observed by FEI Tecnai G2 Spirit TEM (FEI, Hillsboro, OR).
4
Ultrastructural Analysis of Testes
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Fresh testes were fixed in 2.5% (Vol/Vol) glutaraldehyde in 0.1 M phosphate buffer (PB), pH 7.4, for 2 h at 4 °C, washed with PB, postfixed in 2% OsO4 for 1.5 h, dehydrated in a graded ethanol series before being transferred to acetone, and embedded in Poly/Bed 812. Ultrathin sections were taken with a Leica EM UC7 ultramicrotome (Leica, Inc.), doubly stained with uranyl acetate and Reynold’s lead citrate, and then imaged on a FEI Tecnai G2 Spirit TEM (FEI Company) at 120-kV accelerating voltage.
5
Ultrastructural Analysis of Efemp1 Mutant Mouse Eyes
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Eye globes were collected from 16-month-old female Efemp1+/+ and Efemp1ki/ki mice, fixed in Karnovsky’s fixative (Electron Microscopy Sciences, Hatfield, PA), embedded and sectioned for TEM at either Schepens/MEEI Morphology Core or Novartis Fort Worth electron microscopy facilities. Eyes were collected from 18-month-old female Efemp1+/+:Cfb+/+, Efemp1ki/k: Cfb+/+, Efemp1+/+:Cfb−/− and Efemp1ki/ki:Cfb−/− mice, and processed at the electron microscopy facility of the UMass Medical School. TEM analysis for eyes of 12-month-old male and female EFEMP1ki/ki mice from FB compound study was similar as described previously (11 (link),22 (link)). Eyes from vehicle and compound-treated female and male groups (N = 14–16 per group) were processed at the Schepens/MEEI Morphology Core. Ultrathin sections (70 nm) were collected onto single slot formvar–carbon coated grids. The grids were imaged at a direct magnification of 18 500 using an FEI Tecnai G2 Spirit TEM (FEI, Hillsboro, Oregon) at 80 kV. The microscope was interfaced with an AMT XR41 digital CCD camera (Advanced Microscopy Techniques, Woburn, Massachusetts) for digital TIFF file image acquisition.
6
TEM Observation of ZGSO:0.5%Cr Particles
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Observations of ZGSO:0.5%Cr3+ particles were carried out on a FEI® Tecnai Spirit G2 TEM (ThermoFisher Scientific Inc., Hillsboro, OH, USA) working with an acceleration voltage of 120 kV. For the analysis, one drop of particle suspension is deposited on a carbon film-coated copper grid.
7
Negative Staining of Extracellular Vesicles
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Samples were prepared based on a previously published protocol [17 (link)]. Briefly, 5 μL of the EV sample in filtered PBS was placed on carbon-coated grids for 4 min (Ted-Pella B 300M, Mason Technology, Dublin, Ireland). Grids were washed for 15 s with 0.2 μm filtered PBS at room temperature and were contrasted with 2% uranyl acetate (3 min at room temperature), washed once, and examined by FEI TECNAI Spirit G2 TEM (Thermo Fisher Scientific, Waltham, MA, USA).
8
Transmission Electron Microscopy of Biological Samples
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Samples were prepared adapting a previously published protocol, with some modification [17 (link)]. Briefly, Ted-Pella B 300M carbon-coated grids (Ted-Pella, Redding, CA, USA) were cleaned and hydrophilized using plasma glow discharge for 15 s (Gatan SOLARUS Advanced Plasma Cleaning System, Gatan, Inc., Pleasanton, CA, USA) prior to use. Five μL of sample in 0.2-µm-filtered PBS was placed on carbon-coated grids for 5 min. Carbon grids were washed once (15 s) at room temperature (RT) with 0.2 µm filtered PBS and were contrasted with 2% uranyl acetate (3 min, RT), washed once, and examined by FEI TECNAI Spirit G2 TEM (Thermo Fisher Scientific, Waltham, MA, USA) operated at 100 kV. TEM images were acquired at 30,000× and 68,000× (Figure S1 ).
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