Em420

Imaging was performed using a Philips EM420 as described before [31 (link)].

Transmission electron microscopy (TEM, Philips EM 420, 120 kV, brightfield mode) was used to determine particle size, particle size distribution, and shape of the AgNPs tested. High-resolution Field Emission-Scanning Electron Microscopy (FEI Quanta 450 FEG) was employed for imaging E. coli cell surface morphology and visualizing potential NP interactions with the E. coli surfaces. Samples were drop-casted on to the double-sided adhesive carbon tape attached to the aluminum stub for standard reflective-mode microscopy and on to copper grid with formvar coating for transmissive-mode SEM (STEM), and allowed to dry at room temperature while any excess suspension was absorbed along the edge using a blotting paper. A low vacuum imaging regime (40–80 Pa) of water vapor was applied during the reflective-mode imaging in order to neutralize potential sample charging. E. coli surfaces were then scanned by electron beam using a low accelerating voltage of 3–5 kV for reflective imaging, and 30 kV for transmissive imaging, with a working distance of around 10 mm. Upon EM analysis, energy dispersive spectroscopy (EDS) analysis was performed on the AgNPs and E. coli surfaces for assessing potential nano-bio interactions using AZtec Energy Advanced with X-Max SDD 50 sq mm, 127 eV resolution detector (Oxford Instruments). EDS information was collected in three modes: Point mode, ID mode and Mapping mode.
UV–Vis Spectroscopy (Hach DR6000) was used to measure the localized surface plasmon resonance, λ_max (maximum wavelength at which the plasmonic peak was observed), of the AgNPs. Hydrodynamic diameters (HDDs) of the AgNPs were determined using the dynamic light scattering (DLS; Zetasizer Nano ZS90, Malvern Panalytical) and Smoluchowski equation was used to estimate zeta (ζ) potential of the AgNPs based on electrophoretic mobility of the NPs using the ζ potential software.

In this study two aSNPs with similar sizes from different companies were used to corroborate a material-specific but not a fabrication-specific response of the cells on aSNP treatment. The Ludox TM-40 silica particles were purchased from Sigma-Aldrich (St. Louis, MO, USA) and the NexSil20 aqueous silica dispersion was obtained from Nyacol Nano Technologies, Inc. (Ashland, MA, USA). All nanoparticles were characterized with respect to shape, size and size distribution in the dry state as well as in solution. Transmission Electron Microscopy (TEM) imaging was performed using a Philips EM420, operating at 120 kV, on carbon-coated copper grids. The apparent hydrodynamic diameter (D_h)_z was determined by Dynamic Light Scattering (DLS), both in a ready-to-use instrument (Malvern Zetasizer Nano ZS, single angle measurements at 173°, He-Ne-Laser with λ = 633 nm) and a fully equipped setup with a Coherent Verdi V2 diode-pumped solid-state laser (λ = 532 nm), an ALV-SP125 goniometer with single photon detector SO-SIPD and an ALV-5000 Multiple-Tau digital correlator. Angle-dependent measurements were carried out between 30° and 150° and the data were evaluated by exponential fitting and q = 0 extrapolation; μ₂ values as an estimate for the polydispersity of the sample were determined at 90° (assuming a Gaussian distribution, a μ₂ value of <0,04 approximately corresponds to a standard deviation of <28%) [13 (link)].
Additionally to the DLS measurements, the zeta potential of the particles was measured with the Zetasizer (measuring angle for zeta potential: 17°) to study the aggregation behaviour of the nanoparticle dispersions.

For electron microscopy, small tissue pieces from gray matter of BA10 (left hemisphere) and from underlying white matter were obtained perpendicular to cortical surface. The tissue pieces were fixed by immersion with mixture of 2,5% glutaraldehyde and 4% paraformaldehyde in 0,1 M phosphate buffer for 1 week, then postfixed in 1% osmium tetroxide for 1 hour, stained with uranyl acetate for 1 hour, dehydrated in ethanol series and embedded in Araldit epoxy resin. Sections were cut using Reichert ultramicrotome, and semithin 1 μm sections stained with toluidine blue were used for orientation in cortical layers. Small pyramids were trimmed on layer V (layer of big pyramidal cells) and on adjacent white matter. Ultrathin sections were cut, put on formvar-coated, slot-type grids, counterstained with lead citrate and viewed with the electron microscope Philips EM 420.