Talos l120c

Embryos were whole-mount fixed with Karnovsky’s fixative solution containing 2% glutaraldehyde and 2% paraformaldehyde overnight at 4 °C. After washing thrice with 0.05 M sodium cacodylate, they were postfixed with 1% aqueous osmium tetroxide. Then, embryos were stained en bloc using 0.5% aqueous uranyl acetate and were dehydrated through a graded series (30–100%) of ethanol, 20 min for each step. Embryos were incubated with progressively concentrated propylene oxide dissolved in ethanol and infiltrated with increasing concentrations of Spurr’s resin. The samples were embedded in Spurr’s resin, baked in an oven at 65 °C overnight, and sectioned using an ultramicrotome (EM UC7, Leica, Wetzlar, Germany). The sections were observed using a TEM (Talos L120C, FEI, Czech Republic) at the NICEM transmission electron microscope laboratory (Seoul National University, Seoul, Republic of Korea).

Assemblies were characterized in solution by dynamic light scattering (DLS). Samples were measured using an UNcle (UNchained Labs) according to the manufacturer’s directions. Briefly, 8.8 μL of sample was loaded in triplicate into the capillary cassette. For each replicate, 10 acquisitions 10 seconds in length were collected. Assemblies were further characterized by negative-stain electron microscopy. Samples were diluted to between 0.1 and 0.5 mg/mL total protein depending on the assembly stoichiometry, applied to a glow-discharged thick carbon film 400 mesh copper grid (Electron Microscopy Sciences), and stained with 2% uranyl formate. Care was taken to ensure the stain thickness was sufficient to support the larger assemblies. Micrographs were collected on a Talos L120C (FEI) at up to 48,000× magnification. Individual micrographs were processed with ImageJ.

ARS synthesis and content of acetyl groups in the ARS were confirmed by 600 MHz ¹H-nuclear magnetic resonance (NMR) spectroscopy (AVANCE 600, Bruker, Germany). The morphologies of ARS NPs were observed by transmission electron microscopy (TEM) (Talos L120C, FEI, Czech). The sizes of ARS NPs were measured by a dynamic light scattering (DLS) spectrophotometer (DLS-7000, Otsuka Electronics, Osaka, Japan). The zeta potentials of ARS NPs were measured by electron light scattering (ELS) spectrophotometer (DLS-7000, Otsuka Electronics, Osaka, Japan).

An aliquot (4 μL) of BMC-NPs were applied to glow-discharged (Glow discharge system, PELCO EasiGlow™, TED PELLA, Redding, CA) carbon-coated grids (Quantifoil, R2/2, 200 mesh, EMS, Hatfield, PA, USA). After the grids were then blotted for 90 s at 4 with 100% humidity, the samples were plunge-frozen for vitrification (Vitrobot mark IV, FEI, Hillsboro, Oregon). Images were collected on TEM microscope (Talos L120C, FEI, Hillsboro, Oregon) at 120 kV.

The shapes of the LOS vesicles with different edge activators were examined using transmission electron microscopy (TEM; Talos L 120C; FEI, Hillsboro, OR, USA). Approximately 4 μL of each formulation was loaded onto a 200-mesh copper grid (Quantifoil Micro Tools GmbH, Jena, Germany), and manual blotting was performed at 4 °C for 1.5 s without extra staining process. Subsequently, the plunge-freezing method was employed for fixation by applying liquid ethane to the samples. The fixed samples were examined using TEM at an accelerated voltage of 120 kV [24 (link)].

The P. gingivalis cells with different concentrations of coumarin were cultured at 37°C anaerobically for 4 h. The cell pellets were washed with PBS and fixed with 2.5% glutaraldehyde at 4°C. After exposing to 2% osmium tetroxide for 2 h, the cells were dehydrated in a series of ethanol (30%, 50%, 70%, 85%, 95%, 100% and 100%) and dried in acetone solutions three times (50%, 100% and 100%) for 15 min each. Finally, the samples were embedded in resin blocks, cut into ultrathin (70-nm) sections, and stained with uranyl acetate and lead citrate. Images of morphological changes in P. gingivalis cells were observed by transmission electron microscopy (TEM, FEI Talos L120C).