Yeast Cell Imaging and Tracking

Yeast cultures were grown at 30°C in synthetic complete media with 2% glucose (SC) to OD 0.2-0.6. Cells were pipetted onto 0.8 mm coverglass and covered with a 2% agarose pad made with SC. Before imaging, the cells were allowed to proliferate on the slide for 60 min. Imaging was performed at 30°C using a Zeiss Observer Z1 microscope with a 63X/1.4NA oil immersion objective and Orca-r2 C10600-10B camera (Hamatsu Photonics). Temperature of the agarose pad was held at 30°C using Tempcontrol 37-2 digital from PeCon. Images were taken every 3 min and imaging sessions were 8 h long. Up to 12 positions were imaged using an automated stage and Axiovision software. Definite Focus was used to keep the cells in focus during time-lapse microscopy. NLS-NES-GFP expressing strains were exposed for 15 ms using a Colibri 470 LED module. Clb2- and Cdc6-yeCitrine strains were exposed for 250 ms using Colibri 505 LED module. Spc42-mCherry was imaged using Colibri 540-580 LED module for 750 ms. All Colibri modules were used at 25% power. Image segmentation, cell tracking and quantification of nuclear fluorescence signals was performed using MATLAB (The MathWorks, Inc.) as described in Doncic et al. (2013) (link). For every strain, data are from at least two repeats with different transformants.