Formvar coated copper grids

The hydrocolloid of nano-Ag (AgNPs) obtained from Nano-Tech (Warsaw, Poland) was produced by an electric non-explosive patented method (patent number US2009020364 A1) from high-purity metals (99.9999%) and high-purity demineralized water [7 ]. The physical and chemical properties of AgNPs were characterized by Chwalibog et al. [8 (link)]. The shape and size of NPs were inspected with a Jeol JEM-1220 transmission electron microscope (TEM) at 80 KeV (JEOL, Tokyo, Japan), with a Morada 11 megapixel camera (Olympus Soft Imaging Solutions GmbH, Münster, Germany) (Figure 1). Samples of Ag for TEM were prepared by placing droplets of hydrocolloids onto formvar-coated copper grids (Agar Scientific Ltd, Stansted, UK). Nanoparticles of Ag were mostly spherical and polydispersed. The stability of the colloidal dispersions of the nanoparticles (zeta potential) was measured by the electrophoretic light-scattering method with a Zetasizer Nano ZS, model ZEN3500 (Malvern Instruments, Worcestershire, UK). The zeta potential of Ag nanoparticles was −36.4 mV, and the average diameter of particles was 70 nm (Figure 1). AgNPs were dissolved in ultra-pure water (Milli-Q water system, Millipore Corp., Billerica, MA, USA).Figure 1

Size distribution and TEM image of silver nanoparticles.

The size and shape of nanoparticles were inspected using transmission electron microscopy (TEM: JEM-2000EX; JEOL, Tokyo, Japan) at 80 keV. Images were captured with a Morada 11 megapixel camera (Olympus Soft Imaging Solutions GmbH, Münster, Germany). Droplets of sample solutions were placed onto Formvar-coated copper grids (Agar Scientific, Stansted, UK), and immediately after air-drying, the grids were inserted into the TEM (Fig. 1a–c). The macroscopic structure of the nanoparticle powders was visualized using a Nikon D7000 digital camera with a Nikon AF-S Micro-Nikkor 105mm f/2.8G IF-ED VR lens (Nikon, Tokyo, Japan) (Fig. 1d–f).Fig. 1

Nanoparticles visualized using transmission electron microscopy (a–c) and a digital camera (d–f). Images of diamond (a) and (d), graphite (b) and (e), and graphene oxide (c) and (f)

The DN (Fig. 1a) and GN (b) had a spherical shape. The DN powder was cinnamon brown (d), while the GN powder was the darkest and mostly fine-grained (e). The shape of GO was an irregular single layer (c), and the powder was dark brown (f).

The shape and size of the nanoparticles were inspected using the transmission electron microscope (TEM), JEM-1220 (JEOL, Tokyo, Japan) at 80 kV with a Morada 11-megapixel camera (Olympus Soft Imaging Solutions, Münster, Germany). An aliquot of 5 μL of 50 μg/mL ND sample was placed onto formvar-coated copper grids (Agar Scientific Ltd., Stansted, UK) and air dried prior to observation.

Transmission electron microscopy images (TEM) were performed to visualize the morphology of nanomaterials. Droplets of nanoparticles’ solutions at a concentration of 50 mg/L were placed onto Formvar-coated copper grids (Agar Scientific, Stansted, UK), and after air-drying, the grids were inspected by TEM (JEM-2000EX; JEOL, Tokyo, Japan) at 80 keV. The images were captured with a Morada 11-megapixel camera (Olympus Soft Imaging Solutions GmbH, Münster, Germany). In order to evaluate the stability of the nanoparticles’ hydrocolloids, zeta potential, and average hydrodynamic diameter measurements were performed. Measurements were conducted with a Zetasizer Nano-ZS90 analyzer (Malvern, Worcestershire, UK). Zeta potential was assessed for all nanostructures’ concentrations used in the experiments (3.13, 6.25, 50, 100 mg/L) after 120 s of stabilization at 25 °C using the micro-electrophoretic technique with the Smoluchowski approximation. The average hydrodynamic diameter of the nanostructures (using 6.25 mg/L concentration) was measured using a dynamic light scattering technique after 120 s of stabilization at 25 °C. Each measurement was repeated three times.

The shape of the GFM was inspected by a digital camera, scanning electron microscope (SEM), and transmission electron microscope (TEM). The macroscopic structure of GFM powder was visualized using the digital camera Nikon D7000 with the lens Nikon AF-S Micro-Nikkor 105 mm f/2.8G IF-ED VR (Nikon, Tokyo, Japan). SEM analysis of the GFM was performed by means of an FEI Quanta 200 electron microscope (FEI Co., Hillsboro, OR, USA). All imaging was performed in triplicate. Samples of GFM aqueous suspensions (25 μg/mL) for TEM observations were prepared by placing droplets of the suspension onto formvar-coated copper grids (Agar Scientific Ltd., Stansted, UK). Immediately after the droplets had air-dried, the grids were inserted into the TEM for observation with the JEM-2000EX TEM at 80 keV (JEOL, Tokyo, Japan), and images were captured with a Morada 11 megapixel camera (Olympus Soft Imaging Solutions GmbH, Münster, Germany).
Samples for TEM visualization of the interaction of the GFM with each bacterium were prepared by mixing suspensions (200 μL of 25 μg/mL) of pG, GO, and rGO with bacterial cell suspensions (200 μL containing ≈ 5 × 10⁸ cfu/mL in 0.85% NaCl). Control samples of bacteria were treated with ultrapure water. The samples were gently mixed for 15 min at room temperature, and then droplets of the samples were placed onto formvar-coated copper grids and observed by TEM.

Transcranial perfusion was carried out on six male mice (n=3 per group), with 4% paraformaldehyde followed by PFC dissection. Following post-fixation in osmium tetroxide and dehydration in ascending ethanol series followed by propylene oxide, the samples were embedded in Araldite resin (Agar Scientific, Essex, UK). For each specimen, semi-thin (0.5 μm) and thin (70–90 nm) sections were obtained from polymerized blocks using a Reichert-Jung Ultracut E ultramicrotome (Leica-Microsystems, Wetzlar, Germany). Semi-thin sections were stained with toluidine blue and examined using a light microscope. Thin sections from selected areas of the trimmed blocks were made and collected on formvar-coated copper grids (Agar Scientific). Thin sections were double contrasted with 2% uranyl acetate and Reynolds lead citrate stain, and examined using a Jeol 2000FXII transmission electron microscope (JEOL, Peabody, MA, USA), operated at 80 kV. Electron micrographs were obtained of areas of interest with a Megaview-III digital camera and AnalySIS software (EMSIS, Münster, Germany). A minimum of 50 myelinated axons were measured per animal.