Oneview cmos camera

Quality and purity of EV-containing samples (isolated by different methods) were evaluated by TEM. In brief, 10 μL of EV sample was applied to freshly glow discharged carbon Formvar 300 mesh copper grids (Agar Scientific, London, UK) for 2 min. The grid was then blotted dry with filter paper and stained with 2% uranyl acetate for 10 s. The water droplet was then removed and the grid was air dried for 15 min. Grids were imaged using a FEI Tecnai 12 TEM at 120 kV using a Gatan OneView CMOS camera (Gatan, Pleasanton, CA).

Sample fixation and preparation were performed by the Electron Microscopy Facility team at the Sir Willian Dunn School of Pathology (University of Oxford). Leaf discs of 2 mm diameter were collected from leaf 5 of 40 DAS maize or 25 DAS S. viridis plants and placed into 3% glutaraldehyde in 0.06 m Sorenson's phosphate buffer pH 7.2. Samples were transferred to a Leica AMW microwave tissue processor for fixation, osmification (with 1% OsO₄), dehydration with an acetone series, and resin infiltration (LRW Hard). The samples were then removed from the microwave and transferred to fresh resin for 4 days, renewing the resin twice a day. The embedded samples were transferred into gelatin capsules and filled with resin for polymerization at 60°C for about 65 h. Sections of 150 nm were cut using a Leica UC7 ultramicrotome, collected onto formvar‐coated copper slot grids and post‐stained with lead citrate. Images were acquired on a Thermo Fischer Tecnai T12 TEM with a Gatan OneView CMOS camera.
Thylakoid occupancy of chloroplasts was quantified using ImageJ. The thylakoid area was measured by adjusting the image threshold to create a binary mask on which only the thylakoid membrane was apparent. This area value was divided by the chloroplast planar area to give the thylakoid occupancy level.

EV size distribution and concentration was ascertained in fractions 2 and 3 by Nanoparticle Tracking Analysis (NTA) using a NanoSight NS500 (Malvern Panalytical, UK) and NTA 2.3 software. Where necessary, samples were diluted in PBS to achieve a concentration of 2 × 10⁸–2 × 10⁹ particles per mL. The camera level was set to 14 and detection threshold 5. Three recordings of 30–60 s were obtained for each sample and estimations of size distribution were averaged across recordings. EV marker and contaminating proteins were selected according to the 2018 International Society for Extracellular Vesicles position paper [15 (link)]. Transmission electron microscopy of a pooled sample of extracted control CSF EVs. 10 µL of extracted CSF EVs was applied to freshly glow‐discharged carbon‐coated 200 mesh copper grids for 2 min, blotted with filter paper, and stained with 2% uranyl acetate for 10 s, blotted and air dried. Grids were imaged in a FEI Tecnai 12 transmission electron microscope at 120 kV using a Gatan OneView CMOS camera.