Microscopy Imaging of Live Bacterial Cells

Live cells were spotted on 1% agarose pads (prepared with phosphate-buffered saline [PBS]) between a glass slide and a coverslip. When indicated, cells were stained with 5 µg/ml DAPI (4′,6-diamidino-2-phenylindole) (Sigma-Aldrich) and 5 µg/ml FM4-64 (Life Technologies) just before imaging. Image acquisition, analysis, and processing were performed as described previously (45 (link)) using filter sets 31, 49, and 46 (Carl Zeiss) to image FM4-64, DAPI, and GFPmut2-associated fluorescence, respectively. We used a parameter set modified from algorithm 1 of the MicrobeTracker suite (46 (link)) to obtain subpixel cell outlines. Quantitative analysis and plots from the MicrobeTracker data were done on MATLAB (MathWorks, Inc.) using homemade scripts. The values of cell width were obtained by dividing the cell area by the cell length.