Time-lapse Microscopy of Bacterial Cells

Phase contrast and fluorescence microscopy was performed using bacteria spotted on agarose pads, as previously described (Roghanian et al., 2019 (link)). For still images, bacteria were grown to late log phase in CDM containing 100 µM IPTG, diluted in PBS and transferred to agaros pads. Microscopy was performed using Olympus IX53 inverted fluorescence microscope, and the images were analyzed using the software Olympus cellSens Dimensions 1.18. For time-lapse microscopy, bacteria were scraped from o/n chocolate agar plates with 30 µM kanamycin, diluted in PBS and 2 µl were spotted on agarose pads containing CDM with 100 µM IPTG. The samples were transferred to the microscope enclosure and allowed to adjust to 37 °C for 30 min. A Zeiss Axio Observer.Z1 inverted light microscope with a motorized stage was used for this purpose as previously described (Frojd and Flardh, 2019 (link)). Zeiss ZEN 2.3 software was used for analysis of the images. Phase contrast and fluorescence images were recorded every 10 min for up to 6 h at 37 °C in temperature-controlled settings. All images were taken with the 100x objective in phase-contrast and fluorescent channels.