Conductive carbon tape

Energy-dispersive X-ray (EDX) analysis was performed using a detector
(SDD Ultim max 100, Oxford, UK) at 20 kV. Samples were prepared using
the following procedure: A small amount of powder electrocatalyst
sample (1–3 mg) was put on a 13 mm polished metal disk and
covered with a metal disk of the same size. Samples were pelleted
with a manual press until a pellet with a thickness of about 50 μm
was obtained. Standard scanning electron microscope (SEM) pin mounts
(Agar Scientific) covered with conductive carbon tape (Agar Scientific)
were used to hold the pelleted samples.

Biofilms were grown as described above but on 12 mm round poly-L-lysin-coated glass coverslip (Corning). Coverslips were washed 1× with PBS and fixed for 24 hr at room temperature with 2% (v/v) formaldehyde, 0.5% (v/v) glutaraldehyde, and 0.15% (w/v) Ruthenium Red in 0.1 M phosphate buffer (pH 7.4). Coverslips were then rinsed two times with phosphate buffer and post-fixed for 2 hr at 4°C with 1% osmium tetroxide and 1.5% (w/v), potassium ferricyanide (K3[Fe(CN)6]) in 0.065 M phosphate buffer (pH 7.4). Coverslips were rinsed once in distilled water followed by a stepwise dehydration with ethanol (i.e., 50%, 70%, 80%, 95%, 2 × 100%). Samples were then treated stepwise with hexamethyldisilizane (i.e., 50% HMDS/ethanol, 2 × 100% HMDS) and air-dried overnight. The next day samples were mounted on 12 mm aluminum stubs for SEM using carbon adhesive discs (Agar Scientific), and additional conductive carbon tape (Agar Scientific) was placed over part of the sample to establish a conductive path to reduce charging effects. To further improve conductivity, the surface of the samples was coated with a 6 nm layer of Au using a Quorum Q150R S sputter coater. Samples were imaged with a Scios FIB-SEM (Thermo Scientific) under high-vacuum conditions at an acceleration voltage of 20 kV and a current of 0.40 nA.