Images were acquired using LAS AF software (Leica) and exported to TIF format. All image analysis procedures were performed using Matlab (MathWorks). First, a flat-field correction was applied to compensate for field curvature. For this purpose, at least 10 empty field images were taken using the same microscope and camera configurations used later in the corresponding experiment. Images were then averaged, and a correcting matrix was generated. This correcting matrix was applied on a second set of empty field images to check that it produced a flat field (fig. S1B). This correcting matrix was then used on the experimental images to correct for field curvature. Cell segmentation was generated from phase images using MicrobeTracker (40). Algorithm parameters were fine-tuned to obtain the best segmentation mask possible. Manual curation was nevertheless necessary, so each frame was manually corrected to guarantee proper segmentation masks. From these segmentation masks, we extracted cell length, area, and fluorescence intensities in all relevant channels. Cell size was used as a proxy to check the uniformity of growth conditions. For this purpose, cell size histograms were generated for all frames. Frames that produced cell size histograms 1 SD outside the norm were discarded. Fluorescence shift with respect to phase images was checked by manually curating a set of segmentation masks and then comparing its resulting values to the original ones obtained without shift correction. Fluorescence values were background subtracted using MicrobeTracker algorithm (40). Fluorescence intensities were generated by normalizing fluorescence values by their corresponding cell area and exposure time. All fluorescence intensities are thus reported in arbitrary units/(ms × μm2).

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