Electrochemical CLSM

The cell was filled with sterile-filtered biofilm supernatant water (0.2 µm PES membrane filter, VWR). Stained biofilms were placed close to the working electrode.

Electrochemical CLSM measurements were carried out using an upright Leica TCS SPE equipped with a 20x/NA 0.5 water immersion dipping lens (HCX APO L U-V-I UV, Leica Microsystems, Wetzlar, Germany). Measurements were taken at regions (area of 75.625 µm²) close to the working electrode. The pinhole was set to 2 AU to increase the depth of field in order to counteract the influence of potential z-movement of the sample within the time of analysis.

A time series experiment was setup where the applied potential was changed in 15 s intervals and an image was recorded at the end of each 15 s interval. Each potential step was repeated 3 times during the time series experiment. Each cycle was divided in two parts. First, the reduction potential was gradually lowered in an environmentally relevant range from + 0.2 to − 0.5 V. In order to repeat this cycle, an over potential was applied in three increments (from + 0.7 to + 1.1 V) to ensure re-oxidation of the redox-active compounds in the sample.

As a control, the fluorescence intensity of the biofilms was recorded in the same way at open circuit potential (OCP).