Fluorescence Imaging of Bacterial Stress Response

Imaging was performed with a Nikon Eclipse Ti inverted microscope equipped with an Orca R2 (Hamamatsu) camera, a 60X Plan Apo oil objective (NA 1.4, Nikon), an automated stage (Ludl), a Lumencor SOLA fluorescent illumination system, and a custom-designed temperature-controlled Plexiglas enclosure in which the temperature was maintained at approximately 37°C during imaging. Image acquisition was performed using MATLAB scripts interfacing with μManager, as previously described [29 (link)]. Semrock filter cubes for GFP (GFP1828A) and mKate2 (mCherry-B) were used to image mNeonGreen and mNeptune, respectively. mNeonGreen (used for σ^B reporters) was imaged with 2x2 pixel binning at approximately 20% illumination power with 200-ms exposures, and mNeptune (used for cell segmentation) was imaged with 1x1 binning at approximately 24% power with 400-ms exposures). Images were captured at 10-minute intervals.