Dmi8 spinning disk microscope

Time-lapse images are single confocal slices captured with Leica DMi8 spinning disk microscope and 40×/0.75 Plan Apo objective, as described in Fixed Cell Imaging, at 3 m intervals over 18–24 h. Image sequences were adjusted for brightness and contrast using Fiji. For nuc:cyto mean intensity over time cell traces, nucleus and cytoplasm were manually segmented in Fiji for each time point. For live precision/recall analysis, true positives were manually determined by rapid loss of GFP-Nuc from the nucleus into the cytoplasm co-incident with rapid increase of RFP-Cyto in the nucleus.

RPE‐1 cells expressing mNG‐MCAK under a doxycycline‐inducible promoter were transfected with siRNAs, seeded onto Ibidi 8 Well Glass Bottom µ‐slides and induced by adding doxycycline. For pulse‐chase experiment, 24 h post‐induction and 48 h post‐transfection, cells were washed 2× with PBS and incubated in medium without FBS and doxycycline. Cells were subsequently imaged with a Leica DMi8 spinning disk microscope at 40× or 63× magnification (HC PL APO 40×/1.3 Oil or HC PL APO 63×/1.40–0.60 Oil) equipped with a Hamamatsu Orca Flash 4.0 LT. Imaging was performed at 37°C and 5% humidified CO₂. Telophase cells were chosen for time‐lapse imaging and 13 stacks at a distance of 0.5 µm were taken every 20 min. If not enough telophase cells were present, cells in prometaphase and metaphase were imaged and quantification started at telophase.