PEP Confocal Microscopy Raw Images

For confocal imaging, 4ml cultures of the relevant strains were grown in TAP liquid medium for 5 days. 24 hours before imaging, cells were pelleted via centrifugation at 1500 x g for 15 minutes at RT and resuspended in an equal volume of High-Salt-Minimal (HSM) media (Sueoka et al., 1967). On the day of imaging, cells were pelleted again, and the cell pellet washed with 1X Phosphate-Buffered Saline (PBS).

Cells were then fixed via incubation for 15 minutes in 3.7% PFA, followed by a further washing with 1X PBS. Prior to observation, cells were subjected to 15 minutes of staining with 0.5µg/ml DAPI (ThermoScientific) in the dark. All imaging was conducted using a LSM980 Axio Observer Confocal Microscope (Zeiss) with a 63X oil lens, and a total of 30 images collected per strain.</p><p dir="ltr"><br></p><p dir="ltr">456 = PEPS3-Venus-3xFLAG</p><p dir="ltr">457 = PEPS9-Venus-3xFLAG</p><p dir="ltr">647 = PEPS12-Venus-3xFLAG</p>