125 μl gene frame

For TLFM, appropriate dilutions of cell cultures were transferred to agarose pads containing the appropriate medium on a microscopy slide and covered with a cover glass attached to a 125-μl Gene Frame (Thermo Fisher Scientific) to hold the cover glass on the microscopy slide. TLFM was performed on a Ti-Eclipse inverted microscope (Nikon, Champigny-sur-Marne, France) equipped with a ×60 Plan Apo λ oil objective, a TI-CT-E motorized condenser, and a Nikon DS-Qi2 camera. Green fluorescent protein (GFP) was imaged using a quad-edge dichroic (395/470/550/640 nm) and a fluorescein isothiocyanate (FITC) single emission filter. A SpecraX LED illuminator (Lumencor, Beaverton, OR, USA) was used as the light source, using the 470/24 excitation filter. Temperature was controlled with an cage incubator (Okolab, Ottaviano, Italy).
Images were acquired using NIS-Elements software (Nikon), and the resulting pictures were further handled with the open source software ImageJ. The average cellular fluorescence of cells was determined using the open source software Ilastik (55 (link)), which was trained to robustly identify and segment bacterial cells and exclude debris and out-of-focus cells. Background fluorescence was subtracted using NIS-Elements software.

Appropriate dilutions of cultures or spore suspensions were placed on agarose pads (MOPS medium supplemented with 1.5% LSL-LE 8200 agarose [Lonza, Basel, Switzerland] and 30 mM l-valine) on a microscopy slide and covered with a cover glass attached to a 125 μL Gene Frame (Thermo Fisher Scientific, Waltham, MA, USA). The process of making agarose pads has been previously described in more detail by De Jong et al. (55 (link)). Automated TLFM monitoring was performed on a widefield Ti-Eclipse inverted microscope (Nikon, Champigny-sur-Marne, France) equipped with a 60× Plan Apo λ oil objective, a TI-CT-E motorized condenser, and a Nikon DS-Qi2 camera. GFP was imaged using a quad-edge dichroic (395/470/550/640 nm) and a FITC single emission filter. A SpectraX LED illuminator (Lumencor, Beaverton, OR, USA) was used as light source, using the 470/24 excitation filter. Temperature was controlled at 37°C with an Okolab cage incubator (Okolab, Ottaviano, Italy). While phase contrast images were taken every 15 min, GFP was imaged every 30 min in order to avoid bleaching. Images were acquired using NIS-Elements software (Nikon), and the resulting pictures were further handled with the open source software ImageJ. During acquisition of fluorescent images, photobleaching was reduced by lowering the intensity of excitation light and prolonging time intervals between exposures.