Visualizing Sporulation Dynamics in Yeast

Cells were induced to sporulate using the resuspension method in Sterlini-Mandelstam (SM) medium (60 (link)). At different time points, 150 µL of cell cultures was harvested and resuspended in 10 µL PBS containing 5 µg mL⁻¹ FM4-64 membrane dye and/or 2 µg mL⁻¹ DAPI to visualize DNA, as needed. Finally, 3 µL of cell suspension was placed on a glass bottom culture dish (MatTek) and covered with 1% agar pad made with SM medium. Cells were viewed using a DeltaVision core microscope system equipped with an environmental chamber at 22 °C. Cell images were captured with a Photometrics cool snap HQ2 camera. Eight planes were acquired every 0.2 µm and the data were submitted to deconvolution using SoftWorx software (61 (link)).