Cfi plan apochromat dm 60 lambda oil ph3 1.40 objective

Strains harboring a chromosomal FlhA-mNeonGreen fusion were grown in LB until reaching an OD₆₀₀ of 0.7–0.8. Cells were harvested, washed in PBS, and applied on 1% agarose pads (Sigma). Slides were imaged using a Nikon Eclipse Ti2 inverted microscope equipped with a sCMOS Prime BSI (Photometrics) camera, a Lumencor Spectra III light engine (Lumencor), and a CFI Plan Apochromat DM ×60 Lambda oil Ph3/1.40 objective (Nikon) using a LED-DA/FI/TR/Cy5/Cy7-A Filter Cube (Semrock). Z-stacks were acquired and the FlhA-mNeonGreen foci were counted using MicrobeJ^{31 (link)}.

For live cell microscopy, cultures were spotted on the surface of 1% agarose pads (in PBS) cast on SuperFrost Plus slides (Erpedia). The spotted cultures were allowed to air-dry briefly, then covered with a microscopy cover slip (1.5H, Roth). For fixed cells imaging, in-house flow-chambers were constructed using slides and cover slips treated with 0.1% poly-L-lysine (Sigma-Aldrich) [112 (link)]. The slide and cover slip were assembled in the presence of a double layered parafilm as a spacer. Samples were loaded into the chamber and allowed to adhere to the cover slip at room temperature (RT) followed by fixation with 4% paraformaldehyde for 10 min. The fixed cells were then washed with PBS and mounted in Fluoroshield mounting medium containing DAPI (Sigma-Aldrich). Image acquisition was carried out using a Nikon Eclipse Ti2 inverted microscope equipped with a CFI Plan Apochromat DM 60× Lambda oil Ph3/1.40 objective (Nikon). The filter cube LED-CFP/YFP/mCherry-A (CFP / YFP / mCherry—Full Multiband Triple) (Semrock) was used for imaging rpoS-mCherry fusions and LED-DA/FI/TR/Cy5/Cy7-A (DAPI / FITC / TRITC / Cy5 / Cy7—Full Multiband Penta) (Semrock) was used for P_sicA-eGFP, rflP-mScarlet, DAPI and DiSC₃(5).