2.4. Fluorescence Staining, Microscopy, and Image Processing

We cultivated cells of strains K7, K7GE01, K7GE21, K7GE31, and K7GE41 until the early log phase (<5 × 10⁶) and fixed them with medium containing 3.7% (w/v) formaldehyde (Wako, Osaka, Japan). We then triple-stained cells with fluorescein isothiocyanate-conjugated concanavalin A (Sigma, St. Louis, MO, USA) for the cell wall, rhodamine-phalloidin (Invitrogen, Carlsbad, CA, USA) for the actin cytoskeleton, and 4′,6-diamidino-2-phenylindole (Sigma) for nuclear DNA, as described previously [19]. We acquired fluorescence microscopy images of the cells using a microscope (Axio Imager) equipped with a special lens (6100 EC Plan-Neofluar; Carl Zeiss, Oberkochen, Germany), a cooled-charge-coupled device camera (CoolSNAP HQ; Roper Scientific Photometrics, Tucson, AZ, USA), and appropriate software (AxioVision; Carl Zeiss). We analyzed the micrographs of the cells with image processing software designed for diploid cells (CalMorph, ver. 1.3) [20]. We obtained the morphological data of 501 traits from the single-cell data. Descriptions of each trait have been presented previously [19]. The CalMorph user manual is available at the S. cerevisiae Morphological Database (https://www.yeast.ib.k.u-tokyo.ac.jp/CalMorph/).