Deltavision personal system

Cells were grown in SCD or YMM medium containing ammonium sulfate as the sole nitrogen source, and then shifted to nitrogen-free YMM for 30 minutes, after which the indicated amino acids were added. Cells were collected by centrifugation (600 x g, 2 min) and subjected to microscopy. The cells were observed on a Leica AF6500 fluorescence imaging system (Leica Microsystems) mounted on a DMI6000B microscope (HCX PL APO 100/1.40–0.70 oil-immersion objective lens, xenon lamp (Leica Microsystems)) under the control of the LAS-AF software (Leica Microsystems). For time-lapse imaging, the cells were grown in YMM medium containing ammonium sulfate as the sole nitrogen source, and then shifted to nitrogen-free YMM on a glass bottom dish (Matsunami Glass) mounted with 2 mg/ml of concanavalin A (Sigma). Cells were then subjected to time-lapse imaging after addition of glutamine (final concentration of 0.5 mg/ml). Images were recorded using a DeltaVision Personal system (Applied Precision) mounted on a IX71 microscope (UPlanSApo 100x/1.40 oil-immersion objective lens, LED lamp (OLYMPUS)). ImageJ software (National Institutes of Health) was used to process and produce merged images.

Yeast cells were fixed with 70% ethanol, washed with phosphate-buffered saline (PBS), and incubated in PBS containing 1 µg/ml 4′6,-diamidino-2-phenylindole (DAPI) to visualize DNA. Images were acquired using the DeltaVision Personal system (Applied Precision, Issaquah, WA, USA). A Z series in 0.4-µm steps was acquired for DAPI images, and ImageJ (National Institutes of Health, Bethesda, MD, USA) was used to generate projected images. Adobe Photoshop (Adobe Systems, Inc., San Jose, CA, USA) was used to process and produce merged images.