Spectra x

Microfluidics experiments were performed on a Nikon Ti Eclipse inverted fluorescence microscope with a perfect focus system, oil immersion objective 100× NA1.4, motorised stage, sCMOS camera (Hamamatsu Flash 4) and LED excitation source (Lumencore Spectra X). The temperature chamber (Okolabs) allowed performing the experiments at 37°C. We recorded time‐lapse movies with a frame rate of 1/3 min using NIS‐Element software (Nikon) across three spectral channels (LED excitation wavelengths λ: 555, 508, 440 nm) to image mKate2, MutL‐mYpet and CFP reporters, respectively. One image is acquired from each channel consecutively using exposure times of 100 ms (λ = 555 nm), 300 ms (λ = 508 nm) and 75 ms (λ = 440 nm); LED intensity for all channels was 50% maximal output. One single triband dichroic and three separate emission filters were used to separate excitation and emission light. Up to 48 fields of view can be imaged across the microfluidic device within the 3‐min time‐lapse window. On average, 20 channels with bacteria were present per field of view such that about ~1,000 mother cell traces were recorded per experiment.

Widefieled image acquisition was done on a Zeiss AxioObserver Z1 widefield microscope, equipped with a Lumencor SpectraX illumination system and a Hamamatsu Orca Flash 4.0 V2, sCMOS, cooled fluorescence camera (16 bit, 2048 × 2048 pixel (4 MP), pixel size 6.5 µm). A 63×, 1.4-NA, i-plan apochromat oil-immersion objective was used. For optimal representation in figures, images were adjusted for brightness and exported as RGB TIF files using Fiji^{53 (link)}.
Confocal images were acquired with a Leica SP8 inverse confocal laser scanning microscope with a 63×, 1.4-NA Plan-Apochromat oil-immersion objective. The sequential scanning mode was used and the number of overexposed pixels was kept at a minimum. Fields were recorded at a resolution of 512 × 512 pixels, 8 bit depth or 1024 × 1024 pixels, 8 bit. For optimal representation in figures, maximum intensity projections were calculated and images were adjusted for brightness and exported as RGB TIF files using Fiji^{53 (link)}.

Cells were transferred onto Concanavalin A-treated MatTek dishes (MatTek Corp., Ashland, MA) and visualized at room temperature Microscopy was performed using an inverted epi-fluorescence microscope (Nikon Ti) equipped with a Spectra X LED light source and a Hamamatsu Flash 4.0 sCMOS camera using a 100x Plan-Apo objective NA 1.4 and the NIS Elements software. Representative images were processed using ImageJ software. Brightness and contrast were adjusted to the same values for images belonging to the same experiment and were chosen to cover the whole range of signal intensities. Image processing for PB analysis was performed using Diatrack 3.05 particle tracking software (Vallotton and Olivier, 2013 (link)) as described below.