Microscopy and Cell Cycle Analysis

Images for colony morphology were acquired with G:BOX imaging system (Syngene). Florescence microscopy was performed essentially as described previously [44 (link)]. Images were acquired on a DeltaVision Microscopy System (Applied Precision, LLC) using an inverted microscope (TE200; Nikon), a charge-coupled device camera (CoolSNAP HQ; Roper Scientific) and either a 40× objective with a 0.75 NA or a 20× with a 0.50 NA (Nikon). Figures were prepared for publication using Adobe Photoshop and Adobe Illustrator. No further manipulations other than adjustments in brightness and contrast were made. To determine DNA contents, cells were grown to exponential phase, fixed with 70% ethanol, and treated with RNAase. The cells were then stained with propidium iodide overnight at 4°C and fluorescent intensity was quantified with CellProfiler image analysis software [45 (link)]. For time-lapse experiments, cells were grown to early exponential phase in liquid YPD medium and were placed on YPD agarose pads. 10 z sections spaced 1 μm apart without binning were acquired every 2 min. All images were projected and processed with the softWoRx program (Applied Precision, LLC).