No 1.5 coverslip

The cells were seeded on 35 mm petri dishes with glass bottom microwells (no. 1.5 coverslip, MatTek, Ashland, MA, USA) 2 days prior to imaging. Nanoparticle dispersions were prepared by adding both the 40 and 100 nm particles to cDMEM to obtain concentrations of 100 and 20 µg/mL or 6.25 and 80 µg/mL for the 40 and 100 nm particles, respectively. The medium in the petri dishes was removed and replaced with nanoparticle dispersion 24 h before imaging. Immediately prior to imaging, the particle dispersion was removed from the dishes and replaced with Live Cell Imaging Solution (Invitrogen, Waltham, MA, USA). The dishes were placed on a CellDiscoverer 7 (Zeiss, Oberkochen, Germany) microscope with 37 °C heating and 5% CO₂. Images at a lower magnification were obtained using a 50× plan apochromatic water immersion objective (used with autocorrection rings) in phase gradient contrast and epifluorescence mode in combination with an Axiocam 506 camera (Zeiss, Oberkochen, Germany). A 470 nm LED with the AF488 filter setting was used to image the 40 nm yellow/green particles, while the 100 nm red particles were imaged using a 590 nm LED and the AF568 filter. High resolution images were taken in confocal mode with the LSM900 and AiryScan 2 detector at 50× magnification with the same filter settings, but instead exciting the two particles with a 488 and 566 nm laser, respectively.

After cultivation of 24 h-old P. aeruginosa biofilms in 50 mm glass bottom dishes (No. 1.5 coverslip) (MatTek Corporation, Ashland, OR, USA), the supernatant was removed and biofilms were incubated with 1.87 × 10¹⁰ GQD mL⁻¹ for 15 min at room temperature. Biofilms were irradiated with laser pulses at a laser fluence of 2.00 J cm⁻². As VNB efficiently scatter light, the generation of VNB inside biofilms could be detected by dark-field microscopy. Because of the short nature of VNB generation (lifetime < 1 µs), the camera (EMCCD camera, Cascade II: 512, Photometrics, Tucson, AZ, USA) was synchronized with the pulsed laser by an electronic pulse generator (BNC575, Berkeley Nucleonics Corporation, San Rafael, CA, USA). Dark-field pictures were taken before, during VNB formation and immediately after illumination, in order to elucidate any conformational changes in the biofilm structure. After loading the biofilms with GQD and irradiation with pulsed laser light, as described above, 100 µL supernatant was removed and 100 µL tobramycin (at 16 µg mL⁻¹) or control solution (0.9% NaCl (w/v)) was added for 24 h at 37 °C. Then, the sessile cells were washed with physiologic saline and harvested by 2 rounds of 5 min vortexing and 5 min sonication followed by plate counting (n = 3 × 3).