Fluorescence Imaging of Fission Yeast Telomeres

A DeltaVision fluorescence microscopy system (Applied Precision), which is based on an Olympus wide-field IX71 fluorescence microscope equipped with an oil-immersion objective lens (Plan Apo 60×; NA = 1.4; Olympus) and a CoolSNAP HQ2 CCD camera (Photometrics), in a temperature-control room, was used for imaging of the fission yeast cells41 (link). Cells were grown on YES agar at 30 °C or 26 °C for the cdc10-129^ts strain. To induce meiosis, the cells were transferred to an ME plate at 26 °C. After the formation of zygotes, the cells were suspended in EMM-N medium. Then, the cells were mounted on a 24 mm × 60 mm cover glass coated with 0.2% (w/v) lectin. Images were collected of 15 focal planes at 0.5 μm intervals. To measure the telomere-ade8 or ade8-ade1 distance, horsetail nuclei were observed in zygotes. The distance was measured only when the nucleus was moving straight in either direction (not making a turn). Time-lapse observation was performed as described12 (link). Images were deconvolved with SoftWorx software (Applied Precision). The distance was measured using Priism software (University of California, San Francisco)42 (link). Each trace was initiated at the center of the GFP focus.