Overnight cultures of P. aeruginosa grown in LB were used to spin down 1 mL of cells in a fresh Eppendorf tube at 1000 × g for 1 min. The pellet was re-suspended in 1 mL phosphate buffer saline (PBS), spun down again at 1000 × g and re-suspended in 1 mL of minimal medium (M9 succinate). The OD600 was taken and the cells normalised to 0.5 OD600 in 1 mL of selected medium. Nanosensors were suspended at 1.5 mg mL−1 in selected medium. Polyacrylamide nanosensor suspensions were filter sterilised using 0.22 µm PES filters and 500 µL of medium with nanosensors were combined with 175 µL medium and 75 µL normalised cells to obtain a final concentration of 1 mg mL−1 nanosensors and a starting OD600 of 0.05. For approaches without nanosensors, another 175 µL medium was added instead of medium with nanosensors. To each well of the eight-well chamber (Ibidi, glass bottom), 300 µL of the suspension was added before the chamber was placed in a box wrapped in aluminium foil and placed in a static incubator at 37 °C. After an incubation of 48 h, the media was carefully removed and replaced by fresh medium containing 2.5 µg mL−1 CellMask™ Deep Red plasma membrane stain (Thermo Fisher Scientific).

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