Jsm 7500f field emission sem

To visualize the extracellular secretion of the DS-decorated extracellular vesicles, ADSCs were seeded onto a 0.1% gelatin-coated High Grid-500 glass-bottom μ-Dish (ibidi, Gräfelfing, Germany) at a density of 5 × 10⁴ cells with DMEM containing 10% FBS, 1% AA, and 20 μM Ac₄ManNAz for 48 hours. Subsequently, the cells were washed twice with DPBS and treated with serum-free medium containing Cy5.5-labeled DBCO-DS (10 μM) for 2 hours. After washing twice with DPBS, the cells were incubated with serum-free medium for 24 hours and fixed with a 4% paraformaldehyde solution. Then, the cells were observed under a confocal laser microscope. For SEM imaging, the cells were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) at 25°C for 1 hour and then maintained at 4°C for 12 hours. The cells were washed with 0.1 M cacodylate buffer, incubated with 1% osmium tetroxide at 25°C for 1 hour, and then washed with 0.1 M cacodylate buffer. Thereafter, the cells were dehydrated with serial concentrations of ethanol (50, 75, 95, and 100%), and the glass bottoms were detached from the dishes; this was followed by drying at 25°C for 24 hours. The dried coverslips were then mounted onto an aluminum stub with a carbon adhesive tab and sputter-coated with gold for 60 s. The samples were imaged using a JSM-7500F Field Emission SEM (JEOL, Tokyo, Japan).

Silver nitrate (Daejung Chemicals and Metals, Siheung, Korea); C343 and DCM (Sigma-Aldrich, St Louis, MO, USA); DNBP (Tokyo Chemical Industry, Tokyo, Japan); and all other chemicals were used as received without further purification. Homogeneous silver colloidal nanoparticles with diameters of 59–219 nm were synthesized via a kinetically controlled seeded-growth method.^{40 (link)} The SCFs were prepared by attaching the silver nanoparticles to glass slides that were pre-coated with poly(diallyldimethylammonium chloride).^{17 (link)} Half of the SCFs were removed using a dilute nitric acid solution for the evaluation of the fluorescence enhancement. Then, 1.0% (w/v) polystyrene (PS; avg. m.w. 208 000; Wako Pure Chemical, Osaka, Japan) solutions in tetrahydrofuran (Samchun Chemicals, Seoul, Korea) containing fluorophores (C343, DCM, or DNBP in the concentration of 2 × 10⁻⁴ M) were spin-coated on the SCFs. The thicknesses of the spin-coated PS films were determined as 180 nm from the interference patterns in the UV-vis absorption spectrum (see Fig. S2†).^{42,43 (link)} The steady-state extinction spectra of the SCFs were measured using a Mega-900 UV-vis spectrophotometer (Scinco, Seoul, Korea), and scanning electron microscope (SEM) images of the SCFs were obtained using a JSM-7500F field emission SEM (Jeol, Tokyo, Japan).