X cite120 120 watt high pressure metal halide arc lamp

Time-lapse microscopy was done using fully-automated Olympus IX81 inverted microscopes (Olympus, Tokyo, Japan). Imaging was done using a 100X NA1.3 oil objective (Olympus) and either a F-View II CCD camera (for cib, Olympus Soft Imaging Solutions, Münster, Germany) or an ORCA-flash 4.0 v2 sCMOS camera (all other data, Hamamatsu, Hamamatsu, Japan). Fluorescent imaging was done using a X-Cite120 120 Watt high pressure metal halide arc lamp (Lumen Dynamics, Mississauga, Canada) and Chroma 49000 series fluorescent filter sets (N49002 for GFP and N49008 for RFP, Chroma, Bellows Falls, Vermont). Focus was maintained using the Olympus Z-drift compensation system and the entire setup was controlled with either the Olympus CellM or CellSens software. The sample was maintained at 37°C by a microscope incubator (Life imaging services, Basel, Switzerland). Images were taken every 3 (rpsM, elongation rate), 5 (cib) or 7.5 (recA, trpL, pheA, metA) minutes for several hours. We quantified the homogeneity of the illumination field and found that light intensities varied by less than 11% within the microcolony. Any potential negative effects of light exposure (bleaching, photo toxicity, etc.) are thus not expected to contribute to the observed spatial patterns of gene expression as they would affect all cells equally.