Deltavision fluorescence microscopy system

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 CoolSNAP HQ2 CCD camera (Photometrics), was used to image the yeast cells. For time-lapse observation, living cells were mounted on 35-mm glass-bottomed culture dishes (MatTek) coated with 0.2 mg/mL soybean lectin (Sigma) and observed at 26 °C unless otherwise specified. A set of images of 15 focal planes at 0.5 μm intervals was taken at each time point. Images were deconvolved using the DeltaVision SoftWorx software (Applied Precision).

To assess the phagocytic activity, NEL-MG at a density of 2 × 10⁵ cells/mL were seeded on a 12 mm coverslip in 24-well cell culture dishes. NEL-MG were treated with 2 μL of red fluorescent latex beads (2 μm, Sigma-Aldrich, St. Louis, MO, USA), HiLyte™ Fluor 488-labeled amyloid β peptide 25–35 (2 μL), or pHrodo-conjugated synaptosomes for 2 h at 37 °C. HiLyte™ Fluor 488-labeled amyloid β peptide 25–35 (Anaspec, AS-633308) was prepared according to the manufacturer’s protocol. Phagocytic activity was then stopped by adding 2 mL of ice-cold PBS. The cells were washed twice with ice-cold PBS, fixed, stained with a microglial marker (IBA-1), and counterstained with DAPI. The cells were analyzed using confocal microscopy (TCS SP5, Leica) and a DeltaVision fluorescence microscopy system (Applied Precision).

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.