Jsm 5510 sem

The nanocrystals were prepared using a wet media milling technique with a ball mill, and Tween 80 was used as a stabilizer to prevent aggregation. The stabilizer was dissolved in water at 1% w/w, and the QUE powder was added to the solution and stirred mechanically to ensure adequate wetting. The resulting suspension was introduced into a milling chamber containing zirconium oxide beads, and the milling procedure was conducted at a rotational speed of 300 rpm for four cycles, each consisting of a 5 min milling period followed by a 5 min interval. The nanocrystals were dried and stored in a low-temperature, low-humidity environment until required for subsequent applications. The particle size, zeta potential, and polydispersity index were measured using a Horiba Particle Size Analyzer SZ-100 instrument, and the particle sizes and morphologies of the samples were observed using a scanning electron microscope (JSM–5510-SEM, Make: Jeol, Model: JSM-5510, Tokyo, Japan) [30 (link)].

For each experiment at least 15 gemmae were observed for each line. Plants were imaged using a Leica DFC310 FX camera connected to a Leica M165 FC stereomicroscope. Confocal laser scanning microscopy was carried out with the Zeiss LSM510 Meta microscope using the Zeiss Plan-Neofluar 25x/0.8 water immersion lens with Argon/2 laser excitation at 488 nm in order to observe fluorescence emitted by YFP and chlorophyll at 505–550 nm and 645–710 nm, respectively. Fluorescence images were constructed by making maximum intensity projections from a Z-stack containing the epidermal cell layer of gemmae. For scanning electron microscopy (SEM) gemmae collected from gemma cups were immediately fixed in dry methanol, critical point dried using a Tousimis Autosamdri-815, mounted on aluminium stubs and coated with a gold/palladium mixture using a Quorum technologies SC7640 sputter coater. The samples were then imaged with a JEOL JSM-5510 SEM. All processing of confocal microscopy images was carried out using Fiji (Schindelin et al., 2012 (link)). Other images were adjusted using Adobe Photoshop CS4.

Images were obtained using a Leica M165FC stereomicroscope, Leica M series Plan APO 1.0× objective and Leica DFC310 FX camera. For confocal scanning laser microscopy (CSLM), plants were stained with 15 µM propidium iodide for 15 min, then submerged in water. Images were acquired with a Leica SP5 confocal microscope using a Leica HCX APO 40×/0.80 W U-V-I dipping lens with sequential scans. YFP fluorescence was detected using excitation at 514 nm with an argon laser and emission was measured between 524 and 568 nm using an Acousto-Optic Tunable Filter. PI was excited at 543 nm using a helium-neon laser and emission measured between 568 and 659 nm. Images were processed using FIJI to create brightest-point 3d projections (Schindelin et al., 2012 (link)).
For scanning electron microscopy, samples were fixed in dry methanol, critical point dried using a Tousimis Autosamdri-815, mounted on aluminium stubs and coated with a gold/palladium mixture using a Quorum Technologies SC7640 sputter coater. Samples were imaged immediately with a JEOL JSM-5510 SEM.