Du885 emccd

Cultures were grown overnight at 37°C in LB and diluted 1:200 into fresh medium (with antibiotics when appropriate). For phase-contrast imaging, cells were spotted onto a 1% (wt/vol) agarose pad with LB. Cells treated with A22 were exposed to the drug for 2 to 3 h before being imaged.
Phase-contrast and epifluorescence images were acquired with a Nikon Ti-E inverted microscope (Nikon Instruments) using a 100X (numerical aperture [NA], 1.40) oil immersion objective and a Neo 5.5 scientific complementary metal oxide semiconductor (sCMOS) camera or a DU885 EMCCD (Andor Technology). The microscope was outfitted with an active-control environmental chamber for temperature regulation (Haison Technology, Taipei, Taiwan). Images were acquired using μManager v. 1.4 (52 (link)). Cell contours were automatically segmented using Morphometrics (53 (link)), and a local coordinate system was defined based on the meshing algorithm from MicrobeTracker (54 (link)). Some images of cells sampled from the edge of colonies (Fig. 6) had clusters of cells that could not be segmented by Morphometrics. These images were processed with the neural network-based machine-learning framework DeepCell (55 (link)) prior to segmentation with Morphometrics.

Cells were imaged on a Nikon Eclipse Ti-E inverted fluorescence microscope with a 100X (NA 1.40) oil-immersion objective (Nikon Instruments Inc., Melville, NY, USA). Images were collected using an Andor DU885 EMCCD or Neo 5.5 sCMOS camera (Andor Technology, South Windsor, CT, USA). Cells were maintained at 37 °C during imaging with an active-control environmental chamber (HaisonTech, Taipei, Taiwan). Images were collected using µManager v. 1.4⁴⁶ .