Yeast strains containing the pRS316-GFP-Atg8 plasmid were collected from rich medium (SD medium lacking the appropriate nutrients or SGal medium for yeast strains containing the yeast ORF collection plasmids) or starvation medium at 30 or 37°, and washed once with water before imaging. Cells were visualized with a Zeiss AxioImager M2 florescence microscopy system using a 63 × oil lens. Images were captured and deconvolved using Volocity 6.3 (PerkinElmer) software. The fluorescence microscopy experiments were repeated three times.