Ultrascan ccd camera

Direct observation of weld formation
at elevated temperatures was achieved through in situ transmission
electron microscopy (TEM) using a DENS solutions Wildfire TEM holder.
AgNS were dropcast onto a DENS solutions Nano-Chip, which consists
of a microelectromechanical system (MEMS) based chip design with electron
transparent SiN windows. The temperature of the chip is controlled
by a 4-point probe. The TEM was subsequently performed on a FEI Titan
80–300 Thermo Fisher Scientific, fitted with a Schottky field
emission gun. The operating voltage was set to 300 kV, and images
were recorded using a Gatan UltraScan CCD camera. The temperature
ramp rate was set to 15 °C/min and heated from RT to 200 °C.
Heating was paused for 2 min at 120 °C and at every 10 °C
increment after that. Images were captured every 20 s.

Isolated exosomes were fixed with 2% glutaraldehyde overnight at 4°C and then diluted 10‐fold with PBS for electron microscopic observation. The sample was plated onto the glow discharged carbon‐coated grids (Harrick Plasma), which were immediately negatively stained using 1% uranyl acetate solution (Jeong et al., 2018). The samples on grids were analyzed with a Tecnai 10 transmission electron microscope (FEI, Instrumentation was used in the Kangwon Center for Systems Imaging) operated at 100 kV. Images were collected with a 2k × 2k UltraScan CCD camera (Gatan).

EM grids were prepared in a specially designated biosafety lab (BSL2). The purified M complex or NTNHE was stained in 2% uranyl acetate aqueous solution. Electron microscopy was carried out in a JEOL 2010 F TEM operated at 200 kV. Electron micrographs were recorded at a magnification of 50,000× in a 4 K by 4 K Gatan Ultrascan CCD camera. For the M-complex, we picked 10769 particles, computationally sorted the raw particle images into 100 classes in
EMAN2. Many well-defined 2D class averages were obtained. We rejected raw particle images that did not produce good class averages. After such rejection, 6657 particle images remained in the final data for 3D reconstruction. For NTNHE, we picked 10039 particles, only kept 3371 particles after reference free 2D classification. Initial model calculation and 3D refinement was performed in EMAN2, and the estimated final resolution was ~18 Å. The 3D surface rendering was prepared by UCSF Chimera.

The immunogold staining procedure was carried out to test for the presence of exosomal membrane proteins in hybrid NPs using antibodies against CD63 and tyrosine-protein kinase Met (C-Met). For immunogold staining, fixed hybrids NPs and exosomes were adsorbed onto 300 mesh Formvar/Carbon grid and blocked with 0.1% BSA for 30 min. The grids were then washed and incubated overnight at 4 °C with an antibody against CD63 (polyclonal anti-CD63 antibody, SC-15363, Santa Cruz Biotechnology, San Jose, CA, USA) and C-Met (polyclonal anti-c-Met antibody, AF276, R&D Systems, Minneapolis, MN, USA). All the grids were rinsed and floated on 10 nm gold conjugated secondary antibody (EM Goat anti-Rabbit or Rabbit anti-goat IgG: 10 nm Gold, BBI Solutions) for 1 h at room temperature. The grids were washed, fixed in 2% glutaraldehyde, and contrasted with 1% uranyl acetate solution. The grids were imaged with Tecnai T-12 TEM (FEI, Hillsboro, OR, USA) and Gatan Ultrascan CCD camera (Gatan, Pleasanton, CA, USA).