Live-cell imaging of an Atg9 reservoir becoming the PAS (overexpressed Atg9-mChe). Atg9-mCherry GFP-Atg8 (MMY120) cells were grown to a log phase and transferred to SD-N medium for 20 min before being imaged. Images were collected by time-lapse fluorescence microscopy using a DeltaVision RT system (Applied Precision) equipped with a CoolSNAP HQ camera (Photometrics). Frames were taken every 5 s for 4 min and 5 s. At each time point, images were generated by collecting a stack of three pictures with focal planes 0.20 µm apart and by successively deconvolving and analyzing them with the softWoRx software (Applied Precision).
