Merlin feg sem

First, 200 µM NKB(H₃W) and NKB(H₃F) fibrils were prepared in 10 mM Tris at pH 7.4, without ThT. Then, 10 µL of each sample was aliquoted onto a 300-mesh copper formvar TEM grid and left to incubate for 10 min. The excess sample was subsequently wicked off, and the grid was negatively stained with 0.5% (w/v) uranyl acetate replacement stain (ProSciTech Pty Ltd., Townsville, Australia) for 20 s. Excess stain was wicked off, and the grid was cleaned in water for 20 s and left to dry. Images were taken with a Zeiss Merlin FEGSEM at a 30 kV acceleration voltage.

The Zeiss Merlin FEG-SEM instrument (Jena, Germany) was used. Powder samples were mounted on aluminum stubs using double-adhesive carbon tape and further sputtered with Au/Pd to reduce charging effects. A sputter coater (Polaron, Ashford, UK) was used. The sputtering settings were 4 × 10⁻² mbar and 35 mA and for a sputtering time of 30 s.

SEM images were recorded in a Zeiss Merlin FEG-SEM using secondary electron contrast at a magnification of × 2000 with an image size of 2048 × 1536 pixels, equal to 56.7 × 42.5 μm² (pixel size = 27.7 nm). The SEM was operated at an acceleration voltage of 5 kV, a probe current of 300 pA, and a working distance of 5 mm. All images were acquired with the same image contrast and brightness settings in the SEM. During SEM image acquisition, lath-bainite regions were oversampled and are therefore overrepresented in the data.

Transmission electron microscopy of CNF was carried out using high-resolution transmission electron microscopy, HR-TEM (JEM-2100 transmission electron microscope, JEOL, Tokyo, Japan). Scanning electron microscopy of BC was carried out using high-resolution scanning electron microscope (Zeiss Merlin FEG-SEM, Zeiss, Oberkochen, Germany) while scanning electron microscopy of CNF was carried out using an FEI Quanta 200 scanning electron microscope (FEI Company, Eindhoven, The Netherlands). Water in the wet BC pellicles was first exchanged with isopropyl alcohol then freeze-dried before SEM examination. Atomic force microscopy (AFM) of the isolated nanofibers was carried out using a Veeco MultiMode scanning probe microscope (Veeco Instruments Inc., Plainview, NY, USA) equipped with a Nanoscope V controller. A droplet of the aqueous fiber suspension was dried onto a mica surface prior to AFM examination and images were collected using a tapping mode etched silicon tip with a nominal spring constant of 5 N/m and a nominal frequency of 270 kHz.

Overnight cultures of E. coli, P. aeruginosa, and S. aureus incubated on AR-Ti, NaOH-, and KOH-etched samples were fixed for 1 h with 4% paraformaldehyde, 1.25% glutaraldehyde, and 4% sucrose in PBS. Following fixation, they were washed in PBS, followed by dehydration in an ascending ethanol order from 50% (v/v) to absolute ethanol and further dried using hexamethyldisilazane (HMDS; Sigma-Aldrich, St. Louis, MI, USA). After the dehydration process, the discs were mounted on aluminium stubs using double-sided carbon tape, sputter-coated with 2 nm platinum, and examined using a Zeiss Merlin FEG-SEM (Zeiss, Jena, Germany).