Wild-type and ΔmliR cells were harvested by centrifugation (10 min at 8,000 rpm), washed with PBS and diluted 1:100. Aliquots of the suspension (0.1 ml) were stained with 1X CellBrite™ Fix Membrane Dye or Calcofluor White Stain (Biotium). The cells were fixed with 3.7% formaldehyde for 15 min at room temperature with gentle shaking, washed 3 times with PBS and resuspended in PBS to a final OD600 of ∼0.3. The cells incubated with CellBrite™ Fix Membrane Dye were then stained with 250 ng/ml of DAPI (4′, 6′-diamidino-2-phenylindole). The stained cells were immobilized on agarose pads, and visualized by fluorescence microscopy as described below.
Images were captured using a confocal microscope (Carl Zeiss LSM 880) with an objective C Plan-Apochromat 63x/1.4 Oil DIC M27 and Airyscan detector in superresolution mode. For CellBrite stained cells a 543 nm laser and BP 420-480 nm plus BP 495–620 nm filter set were used. Images from DAPI and Calcofluor White stained cells were acquired using excitation laser at 405 nm and BP 420–480 nm plus BP 495–620 nm filter set. Airyscan images were processed and analyzed with the ZEN 2.3 SP1 FP3 Black software. Image sizes were 32,89 × 32,89 μm (932 × 932 pixels).
Images were captured using a confocal microscope (Carl Zeiss LSM 880) with an objective C Plan-Apochromat 63x/1.4 Oil DIC M27 and Airyscan detector in superresolution mode. For CellBrite stained cells a 543 nm laser and BP 420-480 nm plus BP 495–620 nm filter set were used. Images from DAPI and Calcofluor White stained cells were acquired using excitation laser at 405 nm and BP 420–480 nm plus BP 495–620 nm filter set. Airyscan images were processed and analyzed with the ZEN 2.3 SP1 FP3 Black software. Image sizes were 32,89 × 32,89 μm (932 × 932 pixels).