Softworx package

A DeltaVision microscope system (GE Healthcare) was used to acquire live-cell fluorescence images as described previously (Li et al., 2015 (link)). Briefly, an agarose pad with 2% raffinose was prepared on a concave slide, and an aliquot of yeast cells was placed on the agarose. The slide was then sealed with LVP (Li et al., 2015 (link)), and images were acquired at each of the indicated time points at 30°C. Images were acquired with a 63× (NA = 1.40) objective lens on an inverted microscope (IX-71; Olympus) equipped with a CoolSNAP HQ2 CCD camera (Photometrics). Pixel size was set at 0.10700 µm. For images attained during time course experiments, 10 optical sections with a 0.5-µm thickness were acquired at each time point. To reduce phototoxicity, a neutral density filter was used to diminish the intensity of the excitation light to ∼50% or less of the equipment output. For GFP, the excitation spectrum was set at 470/40 nm, and emission spectrum at 525/50 nm; for mApple, excitation was at 572/35, and emission at 632/60 nm. Acquired images were deconvolved using the SoftWorx package (GE Healthcare). Projected images are used for display (Fig. 2, D and G; and Figs. 6 I, S1, and S4).