Next, sterile LB 1/10 medium was injected into the system at where It is the pixel intensity at time t, I0 is the pixel intensity at t=0 h, and the argument (x,y) represents the horizontal and vertical pixel indices. To avoid inconsistencies associated with light flicker or changes to ambient light levels in the laboratory, the pixel intensities in all images were normalized to average background light levels obtained between the two microchannels where no biofilms existed. Following the creation of OD x-y maps, the average OD value at a given time slice was obtained from ODt=∑x,y ODt(x, y)/n, where n is the number of pixels in the region of interest. This procedure has the effect of creating images on a black background with the pixel brightness corresponding to the quantity of biofilm present. The 3D biofilm structure was estimated using a previously validated calibration method to convert time-varying OD maps (ODt(x,y)) into biofilm topographical maps (hb,t(x,y)) at each pixel. Because the optical density to biofilm height calibration is applied individually to every experiment, it produces unique and self-consistent results for each experiment, irrespective of strain, growth conditions or even microchannel geometry. This approach was been shown to yield more reliable values for hb than direct measurements using confocal laser scanning microscope, especially for thin, earlygrowth-stage biofilms, or residual biofilm layers after sloughing, as in the present case. In this work, we used an improved multipoint calibration approach instead of the previous two-point method. See Electronic Supplementary Information for more details (Section S1).