543 nm laser

Details of the imaging system have been described elsewhere^{8 (link)}. Briefly, images were acquired with a spinning disk confocal microscope (Yokogawa) a 543 nm laser (OEM DPSS), and an Andor iXon 897 EMCCD camera. For single-cell resolution imaging, a 60× water objective with a numerical aperture of 1.2 plus a 1.5× post-magnification lens was used. To avoid evaporation, immersion oil with a refractive index of 1.3300 ± 0.0002 (Cargille) was used instead of water. The time difference between each image acquisition was 10 minutes, and the total imaging time was 8 hours. Only the bottom 5 μm of the biofilm was imaged (with a z step size of 0.2 μm) to avoid excessive photobleaching and phototoxicity. For coarse-grained imaging, a 20× multi-immersion objective was used without post-magnification. In this case, the time difference between each image acquisition was 30 minutes, and entire biofilms were imaged (with a z step size of 1 μm). At the end of the coarse-grained time course, the biofilm clusters were imaged again with high magnification to determine cell lengths. All image acquisitions were automated using Nikon Element software. All cells harbored mKO fluorescent proteins expressed from the chromosome. Experimental images in Fig. 4 were false-colored to differentiate between different growth conditions.