Su5000 fe sem

At day 7, medium was aspirated from wells of test scaffolds and controls followed by their move to a new 12-well TCP plate. Wells were washed twice with 0.01 M PBS and thrice with deionised water followed by chemical fixing using Karnovsky’s reagent (Polysciences, Inc., Warrington, PA, USA) (5% glutaraldehyde/3.2% PFA for 8 min at room temperature and then washed twice with 0.01 M PBS. Gradual dehydration was performed using an alcohol series of increasing ethanol concentration, 25, 50, 75, 90, and 100% ethanol, for 8 min each at room temperature, followed by volume ratio mix of 100% ethanol/100% hexamethyldisilizane (HMDS) for 8 min at room temperature. Wells were chemically dried overnight at room temperature using 2–3 drops of 100% HMDS. All reagents were from Sigma-Aldrich.
To maximise image quality, scaffolds were coated with a thin layer of gold-palladium (18 nm) using Emitech K500X sputtering system (Quorum Technologies, Lewes, UK) at 25 mA for 150 s. Field emission scanning electron microscopy, FESEM (SU5000, Hitachi, Tokyo, Japan), was used to study surface topography at 5 KV voltage from three random locations per cell-seeded scaffold type.

Scanning electron microscopy (SEM) with backscatter electron imaging (BEI) and energy-dispersive X-ray spectroscopy (EDS) were used to study the surface structure of the tested substrates, element mapping, and qualitative mineral identification. A Hitachi SU5000 FE-SEM (Schottky FEG) provided the SEM analyses, and the EDS was performed by a Dual Bruker XFlash system and a high-resolution automated electron backscatter diffraction (HR EBDS) system. Carbon coating of substrates was carried out to improve imaging quality, increase chemical analysis precision, and better topographic examination while avoiding surface charging and potential thermal damages. For each substrate (representing different Ω, T, and t), three random locations were analyzed, and a mosaic map of nine ordinary SEM images (3 × 3) was acquired at each locality. Each mosaic map covers a 3786 × 2781 µm region, with each pixel representing 1 µm² surface area.

After time-lapse imaging, the cell culture medium in the dish was removed, and the platform was washed twice with 1% PBS for 5 min each time. Then, the cells were fixed with 4% paraformaldehyde for 15 min. After cell fixation, the platform was washed with 1% and 0.25% PBS for 5 min each, followed by rinsing twice in DI water for 10 min each time. Subsequently, cells were dehydrated for 5 min each time using a series of increasing ethanol concentrations (30%, 50%, 70%, 80%, 95%, and 100%). Cells were dried using a critical point dryer, in which CO₂ was the transitional medium. A thin layer of gold was then sputter-coated on the platform using a thin film coater (Q150 coater, Quorum Technologies Ltd., UK). High-resolution images of the fixed cells were captured using a field emission scanning electron microscope (SEM) (SU5000 FE-SEM, Hitachi, Japan) with a 10 kV electron beam. These images were used to analyze the number and length of filopodia and long protrusions of cells. Typical filopodia were narrow with widths of 200–400 nm and lengths of 4–30 μm. Long protrusions had widths >400 nm, and protrusion length was defined as the distance between the edge of the cell membrane and the protrusion tip, which could be 5–50 μm long.

Cells on platforms were washed twice with PBS and fixed for 15 min with 4% paraformaldehyde. The cells were then treated with ethanol at concentrations of 30%, 50%, 70%, 80%, 90%, 95%, and 100% for 5 min each, and then dried in a critical point dryer (EM CPD300, Leica, Germany) for 4 h. To prevent charging during imaging in the scanning electron microscope (SEM, SU5000 FE-SEM, Hitachi, Japan), a thin layer of Au was coated onto the dried samples using a thin film coater (Q150 coater, Quorum Technologies, Lewes, UK). The coated samples were then mounted onto a 51 mm stub using conductive tape. Samples without and with cells were captured using a secondary electrons detector with a 10 kV accelerating voltage to achieve high-resolution images.

Scanning electron microscopy (SEM) was performed as described previously (Bhar et al., 2021 (link)). Briefly, bacterial pellets were fixed with Trump's fixative, washed with 0.1M sodium cacodylate, pH 7.24 and then fixed with OsO4. The sample is dehydrated in 25%–100% graded ethanol series, dried by Tousimis Autosamdri‐815 and mounted on 12 mm Carbon Conductive Adhesive Tab and aluminum stub with sputter gold/palladium coating before being imaged by Hitachi SU‐5000 FE‐SEM. Negative staining was performed using Transmission electron microscopy (TEM) as described previously (Bhar et al., 2021 (link)). Briefly, purified bacterial OMVs pellet, stool derived bEVs or concentrated MNV‐1 were resuspended in fixating solution and 10 μl droplet of the homogenate was poured onto 400‐mesh carbon coated Formvar nickel grid. The excess solution was removed, and the grid was fixed. The grid was stained using floatation on 10 ml of 1% aqueous uranyl acetate and observed with FEI Tecnai G2 Spirit Twin TEM. Finally, images were obtained using Digital Micrograph software and a Gatan Ultrascan 2kX camera.