Orius sc200

Transmission electron microscopy was used for observation of viral particles in starting material and also after DNase treatment in experiment to evaluate different DNase concentrations. 10–20 μL of viral (AAVrh.10hCLN2) suspension was applied on freshly glow-discharged copper grids (400 mesh, formvar-carbon coated) for 5 min, washed and stained with 1 droplet of 1% (w/v) water solution of uranyl acetate. Grids were prepared in duplicates. The grids were observed with transmission electron microscope Philips CM 100 (FEI, Netherlands), operating at 80 kV. At least 10 grid squares were examined thoroughly and representative micrographs (camera ORIUS SC 200, Gatan, Inc.) were taken at different magnifications.

EVs were purified from coACCM and AF using the ExoQuick TC-ULTRA kit (System Bio, Palo Alto, CA, USA) or AF EVs were purified by ultracentrifugation with a Beckman XL-80 Ultracentrifuge (Beckman Coulter, Brea, CA, USA) and a SW41TI rotor by 107,000× g spin for 90 min at 4 °C. The resulting supernatant was collected as the EV-depleted fraction, and the pellet was washed twice with cold PBS, then resuspended in cold PBS and immediately stored at −80 °C or on ice then further analyzed. Qualitative and quantitative analysis of EVs was performed using nanoparticle tracking analysis (NTA) with the ZetaView particle analyzer model PMX-120 (Particle Metrix, Inning am Ammersee, Germany), transmission electron microscopy (see below), the Bradford method as described previously [72 (link)] (BioRad, Hercules, CA, USA), Western blotting (see below), and LC-MS/MS (see below).
For TEM either total AF, purified EVs from AF, or EV-depleted AF were applied to electron microscopic 200 mesh copper grids and negatively stained with 2% aqueous uranyl acetate. They were examined and measured in a JEOL JEM-1400 electron microscope at 80 kV. Images were acquired on bottom-mounted CCD camera Orius SC200 (Gatan, Pleasanton, CA, USA), and were used to measure the size, relative numbers, and shape of EVs.

The ultrastructure of the bacterial cells was examined using transmission electron microscopy. Overnight cultures were mixed with CgLAO (50 μg/mL) for 2 h and compared with untreated cells. The cells were fixed in 3% glutaraldehyde and 1% paraformaldehyde in 0.1 M phosphate buffer, post-fixed in 2% osmium tetroxide, and embedded in epoxy resin. Ultrathin sections were cut and examined under transmission electron microscopy (CM 100; Philips, Amsterdam, Netherlands), operating at 80 kV. Micrographs were recorded with a CCD camera (Orius SC 200; Gatan Inc., United States).

The morphology and mesostructure of the MSPs were characterized using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). SEM imaging was conducted using a field-emission electron microscope (Ultra Plus; Carl Zeiss, Jena, Germany) with an accelerating voltage of 1 kV. Samples for SEM imaging were deposited onto a gold membrane and sputter-coated prior to analysis. For TEM, ~4 µL of the liposome suspension was applied to the Formvar-coated 400-mesh copper TEM grid for 30 s. Before applying the sample, the grids were coated with an ~4 nm thick carbon layer and glow discharged (ACE200 vacuum coater; Leica, Munich, Germany). Samples were stained with 1% aqueous uranyl acetate and examined using a transmission electron microscope (CM100; Philips, Amsterdam, The Netherlands). Images were recorded with a CCD camera (Orius SC 200; Gatan, Pleasanton, CA, USA) using the Digital Micrograph software. X-ray diffraction (XRD) analysis (AXS D4 Endeavor; Bruker, Billerica, MA, USA) was carried out with Cu Kα radiation (λ = 0.154 nm) and a Sol-X detector. A scanning step of 0.02° and a counting time of 6 s per step were used for the measurements. The hydrodynamic size distribution, polydispersity index, and zeta-potential were measured using dynamic light scattering (ZetaSizer Nano ZSP; Malvern Instruments, Malvern, UK).