Na 1.40 oil immersion phase contrast objective

A Nikon Ti-E inverted fluorescence microscope with a 100X NA 1.40 oil immersion phase contrast objective was used for imaging the bacteria. Fluorescence was excited by a 200W Hg lamp through an ND4 or ND4+ND8 (for DAPI time-lapse) neutral density filter. Chroma 41004, 41001, 31000v2 filter-cubes were used to record mCherry, GFP and DAPI images, respectively. Images were captured by an Andor iXon DU897 camera and recorded using NIS-Elements software.

A Nikon Ti-E inverted fluorescence microscope with a 100X NA 1.40 oil immersion phase contrast objective was used for imaging the bacteria. Fluorescence was excited by a 200W Hg lamp through an ND4 or ND8 neutral density filter. Chroma 41004, 41001 and 31000v2 filtercubes were used to record mCherry, GFP and DAPI images, respectively. Images were captured by an Andor iXon DU897 camera and recorded using NIS-Elements software.
Cells were imaged on M9 agar pads for still imaging. For time lapse imaging home-made glass bottom dishes were used. Cells were pipetted to #1.5 glass coverslips on the bottom of the dish and covered with about 1 cm thick slab of M9 agar. No antibiotics were used in M9 agar during imaging. Agar was supplemented with IPTG (10–40 µM) for strains with ZipA-GFP constructs. For DAPI labeling cells were incubated in 0.2 µg/ml DAPI for 1/2 hour before spreading cells on the pads.