Plan apochromat 100x oil ph3

To image membrane and DNA compartmentalisation of the cells, FM1-43FX dye (Invitrogen) and DAPI (Stratech Scientific) were used, respectively. Overnight cultures were adjusted to initial OD₆₀₀ = 0.01 in LB. The cells were incubated at 37°C until OD₆₀₀ = ~0.2. A volume of 500 μl of the culture was stained with 5 μg ml^-1 of FM1-43FX at room temperature for 10 min. The cells were adjusted to 33 mM sodium phosphate pH 7.4 and fixed by addition of 2.4% formaldehyde and 0.04% glutaraldehyde. Fixed cells were inoculated on agarose pads, which were prepared with 1.5% agarose in PBS and set in Gene Frames (Thermo Scientific). Cells were imaged using a Zeiss AxioObserver equipped with a Plan-Apochromat 100x/Oil Ph3 objective and illumination from HXP 120V for phase contrast images. For FM1-43FX images Zeiss filter set 38 (Ex: 470/40 nm, beamsplitter 495 nm, Em: 525/50 nm) was used. DAPI images were captured using the Zeiss filter set 96 (Ex: 390/40 nm, beamsplitter 420, Em: 450/40 nm). For phenotype analysis in S2 Table we used MicrobeJ plugin for Fiji 600 [95 (link)].

Cultures were grown at 37°C to OD₆₀₀0.4–0.5. Cells were harvested by centrifugation at 7000 x g for 1 min before being applied to agarose pads, which were prepared with 1.5% agarose in PBS and set in Gene Frames (Thermo Scientific). Cells were immediately imaged using a Zeiss AxioObserver equipped with a Plan-Apochromat 100x/Oil Ph3 objective and illumination from HXP 120V for phase contrast images. Fluorescence images were captured using the Zeiss filter set 45, with excitation at 560/40 nm and emission recorded with a bandpass filter at 630/75 nm. For localisation analysis and generation of demographs, the MicrobeJ plugin for Fiji was used and >500 cells were used as input for analysis (Ducret et al., 2016 (link)).