mEVs were isolated as described above. After 13,500 rcf centrifugation, the pellet was re-suspended in 1.5 mL 0.2 µm filtered PBS and centrifuged for 1 h at 100,000 rcf at 4 °C in a polypropylene ultracentrifuge tube. The sample was post-processed as described earlier [48 (link)]. In brief, the sample was fixed with 4% paraformaldehyde and postfixed in 1% osmium tetroxide (Taab, Aldermaston, UK). The pellet was dehydrated in graded ethanol, block stained with 1% uranyl acetate in 50% ethanol, and embedded in Taab 812 (Taab, Aldermaston, UK). After an overnight polymerization at 60 °C, the sample was sectioned to ultrathin sections using a Leica UCT6 ultramicrotome (Leica Microsystems, Wetzlar, Germany, UK) and examined with a Hitachi 7100 transmission electron microscope (Hitachi Ltd., Tokio, Japan). Images were taken by Veleta 2 k × 2 k MegaPixel side-mounted TEM CCD camera (Olympus, Tokio, Japan). Brightness and contrast were adjusted using Adobe Photoshop 7.0 (Adobe Systems Incorporated, San Jose, CA, USA).
Veleta 2 k 2 k megapixel side mounted tem ccd camera
Manufactured by Olympus
Sourced in Japan
The Veleta 2 k × 2 k MegaPixel side-mounted TEM CCD camera is a high-resolution imaging device designed for transmission electron microscopy (TEM) applications. It features a 2048 × 2048 pixel sensor that provides detailed, high-quality images. The camera is side-mounted, allowing for convenient integration with TEM systems.
Automatically generated - may contain errors
3 protocols using veleta 2 k 2 k megapixel side mounted tem ccd camera
1
Isolation and Ultrastructural Analysis of Extracellular Vesicles
2
Transmission Electron Microscopy of EVs
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The EV pellet was fixed within the ultracentrifugation tube with 4% PFA for 60 min. The sample was then post-fixed in 1% OsO4 for 15 min, rinsed with distilled water, block-stained with 1% uranyl acetate in 50% ethanol for 20 min, dehydrated in graded ethanol and embedded in Taab 812 (Aldermaston, T031). After polymerization at 60 °C O/N, 50–60 nm ultrathin sections were cut with a Leica UCT ultramicrotome (Leica Microsystems, UK). TEM images were taken with a Hitachi 7100 TEM instrument (Hitachi Ltd, Japan) equipped with a Veleta 2 k × 2 k MegaPixel side-mounted TEM CCD camera (Olympus, Tokio, Japan).
3
Ultrastructural Analysis of Extracellular Vesicles
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EV-containing pellets were processed as described in our previous papers [18 (link),22 (link)]. Briefly, pelleted EVs were fixed with 4% paraformaldehyde at room temperature for 1 h, rinsed by PBS and postfixed in 1% osmium tetroxide (OsO4) for 20 min. After rinsing with distilled water, pellets were dehydrated by a series of increasing ethanol concentrations, including block staining with 1% uranyl-acetate in 50% ethanol for 30 min, finally embedded in Taab 812 (Taab; Aldermaston, UK). Following polymerization at 60°C for 12 h, 50–60 nm ultrathin sections were cut using a Leica UCT ultramicrotome (Leica Microsystems, UK) and examined using a Hitachi 7100 transmission electron microscope (Hitachi Ltd., Japan).
Electron micrographs were made by Veleta 2k × 2k MegaPixel side-mounted TEM CCD camera (Olympus). Contrast/brightness of electron micrographs was edited by Adobe Photoshop CS4 (Adobe Systems Incorporated, CA, USA). Analysis of micrographs was performed in ImageJ [38 (link)] by two skilled persons independently. Data originate from 400 EVs pro samples mean diameter was calculated from measured area.
Electron micrographs were made by Veleta 2k × 2k MegaPixel side-mounted TEM CCD camera (Olympus). Contrast/brightness of electron micrographs was edited by Adobe Photoshop CS4 (Adobe Systems Incorporated, CA, USA). Analysis of micrographs was performed in ImageJ [38 (link)] by two skilled persons independently. Data originate from 400 EVs pro samples mean diameter was calculated from measured area.
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