Em ace600 coater

The morphology of the nanofibrous layers was studied using a Zeiss ULTRA PLUS scanning electron microscope (ZEISS, Oberkochen, Germany). The samples were coated with a very thin layer of Au and Pd in a Leica EM ACE600 coater (Leica, Wetzlar, Germany). Images were acquired using an SE detector; the working distance ranged from 4 mm to 6 mm, and the accelerating voltage was 3.5 kV. Randomly selected images representative of the entire implant area covered in a nanofibrous layer were used to measure the diameters of 80 individual nanofibers in the ImageJ software and the results were represented with a histogram and defined with mean +/- SD. The deposited nanofibrous layers were evaluated first immediately after the spinning process and again after their cross-linking and the subsequent lyophilization in order to determine how these processes affect the morphology of deposited nanofibers. Furthermore, the ability of the deposited material to retain its fibrous structure was assessed.

Glass coverslips (12 × 24 mm, thickness 1.5) were washed extensively with ethanol, air-died and then baked for 3 h at 200°C. For the coating procedure, coverslips were transferred with forceps into a custom-built holder, coated on both sides for 20-30 min in 0.01% poly-L-lysine (Sigma-Aldrich; #P8920) in distilled water, washed subsequently for 20 min in water, air-dried and stored at room temperature until use.
Sapphire discs (Leica; #16770158) were washed and sonicated in water and then again in pure ethanol. Individual discs were subsequently dried with dry nitrogen gas and placed into a custom-built metal holder. In preparation for the deposition of a thin (approximately 4 nm) carbon-coordinate system, each disc was covered with a SEMF2 finder grid (SPI; #2305C-XA) and the metal holder containing the grids was placed into an EM ACE600 coater (Leica). Carbon-coated discs were baked for 12h at 120°C, and then stored at room temperature until use. On the culture day, discs were briefly sterilized by UV-exposure in a tissue culture hood and then coated for 20 min with a small drop of 0.01% poly-L-lysine (Sigma-Aldrich; #P8920) in distilled water, followed by three washes with water. Each sapphire disc was then placed onto a separate glass coverslip.

Gold 200 mesh quantifoil 1.2/1.3 grids were covered with a fresh layer of 2 nm carbon using a Leica EM ACE600 coater. The grids were glow discharged at 15 mA for 30 s using Pelco easiGlow followed by the application of 4 μL 1 mg/mL PEI (MAX Linear Mw 40 k from Polysciences) dissolved in 25 mM HEPES pH 7.9. After 2 min, PEI was removed using filter paper immediately followed by two washes with water. The grids were dried at room temperature for 15 mins. Graphene oxide (Sigma) at 0.4 mg/mL was centrifuged at 1000 g for 1 min and applied to the grids for 2 mins. Excess Graphene oxide was blotted away followed by two water washes. The grids were dried once again for 15 mins at room temperature before use. 2.5 μL of 0.15 mg/mL α1β1γ2S_EM receptor sample was applied to the grids with blot-force of 1 for 2 s using FEI Vitrobot in 100% humidity.
Grids were loaded into a Titan Krios microscope operated at 300 kV. Images were acquired on a Falcon three direct-detector using counting mode at a nominal magnification of 120,000, corresponding to a pixel size of 0.649 Å, and at a defocus range between −1.2 to −2.5 µm. Each micrograph was recorded over 200 frames at a dose rate of ~0.6 e−/pixel/s and a total exposure time of 40 s, resulting in a total dose of ~37 e−/Ų.

The morphology of the scaffolds was analyzed by analytic scanning electron microscopy (SEM) using a JEOL JSM-6010LV (Tokyo, Japan). Prior analysis all samples were sputter-coated with gold using a Leica EM ACE600 coater (Leica Microsystems, Wien, Austria). Elemental composition was performed by energy dispersive spectroscopy (EDS, Bruker XFlash model with detector 5030) installed in the SEM. Three independent areas were selected in the scaffolds.

The macro-/micro-structures of the TGs were examined by SEM using a JEOL JSM-6010LV instrument (Tokyo, Japan; 10 kV and 16 mm working distance). Prior to analysis, the samples were sputter-coated with gold (Leica EM ACE600 coater; Leica Microsystems, Wien, Austria) for 60 s at 30 mA, 0.1 mbar and 50 mm working distance. The elemental composition of the scaffolds was determined by EDS using single-point analysis in three independent regions of the CTGs and PTGs (Pegasus X4M, Edax, Mahwah, NJ, USA; 10 kV and 11 mm working distance).

Scanning electron microscopy (SEM) cross-sections of thin pristine m-PBI (6 μm thickness) and the corresponding composite PP-PBI membrane were prepared using a liquid nitrogen breaking method. First, the specimen (0.5 × 2.0 cm) was wetted in IPA and then immersed in liquid nitrogen for approximately 20 s with the help of a pair of tweezers. Next, the sample was broken into two pieces and the cross-sections were placed face up in a slotted specimen stub (12 mm diameter, Agar Scientific, Stansted, UK). Lastly, the SEM samples were sputter-coated with a 10 nm layer of chromium using a LEICA EM ACE600 coater (Leica Microsystems, Heerbrugg, Switzerland).
SEM images of the thin m-PBI layer (6 μm thickness) and of the prepared composite PP-PBI membrane cross-section samples before and after cycling tests in the VRFB were obtained using a Hitachi Regulus 8230 series high-resolution scanning electron microscope (Tokyo, Japan), equipped with an energy dispersed X-ray analysis (EDX) detector. Experimental conditions of 6.0 kV accelerating voltage and 2 to 3 μA current were used for both electron imaging and EDX analysis. Secondary electron (SE) images were recorded with an in lens detector at a working distance (WD) of 8 to 9 mm depending on the sample. The built-in software “Hitachi Regulus” was used for SEM imaging and the software “Oxford-Aztec 3.3” was used for EDX analysis.