Cell cultures were pregrown overnight, starting from a 1:1000 dilution from a master glycerol stock. Cells were grown at 37°C in M9 medium (Fluka-Sigma M9 salts supplemented with 100 μM CaCl2, 20 mM MgSO4, 0.2% casamino acids, and 0.5% glucose or glycerol). Appropriate concentrations of arabinose/glucose/glycerol/Tc were added according to the experimental requirements. Overnight saturated cultures were diluted 1:1000 in the same medium and incubated at 37°C for 2 to 3 hours until they reached OD600 = 0.1 to 0.2. Thus, cells were grown for approximately 16 generations in the same growth medium before their analysis by fluorescence microscopy. A total of 2 μl of cells were placed onto agarose pads containing the same growth medium used for preimaging growth. Agarose pads were generated by stacking an adhesive frame (Frame-Seal Incubation Chamber, Bio-Rad) onto a microscope slide. In the cavity formed by the adhesive frame, a volume of 200 μl of hot M9 medium + 1.5% agarose was added. Another microscope slide was placed over the frame immediately after pouring M9 + agarose to obtain a uniform and leveled gel. M9 medium used to generate agar pads was supplemented with the appropriate arabinose/glucose/glycerol/Tc concentrations. Pads were allowed to cool down for 30 min at room temperature, and then, using a sterile scalpel, a square of about 0.25 cm2 of the agarose gel was cut. The rest of the agarose pad was removed, and 2 μl of the appropriate culture was placed on top of the pad. The culture droplet was allowed to dry for approximately 5 min, and then, a coverslip was placed over the frame, sealing it carefully. Sealed pads were transferred to the slide holder of a Leica AF6500 inverted epifluorescence microscope, inside an environmental chamber that was kept at 37°C along the course of the experiment. Cells were allowed to adapt to the pad and temperature for 30 min before we started to image. Images were acquired with ×630 magnification using HCX PL S-APO 63× 1.3 oil objective. We acquired images in phase contrast and in the green and red fluorescence channels. Filters used for fluorescence images were 562/40-nm excitation and 641/75-nm emission for mKate2 and 482/18-nm excitation and 520/28-nm emission for GFP. A Leica EL6000 external light engine, equipped with a mercury vapor lamp (HXP Short Arc Lamp, Osram), was used for fluorescence excitation. All images were obtained using the excitation lamp at maximum power. Images were acquired using a 12-bit Andor iXon885 high-speed camera, without binning. For red fluorescence images, we used two exposure times (500 ms and 2 s) in all images taken. For the green fluorescence channel, variable exposure times were applied (10 ms, 100 ms, and 1 s). To avoid fluorescence bleaching from previous exposures, snapshots were taken at least four fields of view away from each other. Linearity of the fluorescence emission with exposure time was checked using a set of predefined fluorescence beads (Rainbow Fluorescent Particle Slide, Spherotech) (fig. S1B).

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