Cfi plan fluor 40 1

To track single bacterial cells interacting with flat, pro-and anti-attachment TUs, an inverted TE2000 microscope (Nikon, Japan) equipped with a Hamamatsu Orca Flash 4.0v2 sCMOS camera and an OKOLab cage incubation system to maintain temperature (37 °C) and humidity constant (95%) was used. Differential interference contrast (DIC) brightfield imaging was carried out using a single channel white MonoLED (Cairns) light source and a 40× objective (Nikon, CFI Plan Fluor 40×/1.3). Experiments were conducted using a custom designed single well holder in static growth conditions (Carabelli et al 2020) . Coverslips were sealed with a silicone and halogen free high-vacuum grease (Apiezon M). To obtain single bacterial cell tracking data, previously developed bespoke MATLAB scripts were employed. To measure bacterial population density above selected topographies, time series z-stacks (40 µm range -2 µm steps) were acquired every min for 4 h from selected TUs. To avoid sample disturbance an optical relay was used to recreate the sampling area virtually, facilitating rapid scanning in the z-dimension using a remote piezo-driven objective (Hook et al, 2019)