Csu 10 spinning disk

Live imaging at 37°C was performed with a confocal microscope (DM4000; Leica), equipped with a 100× 1.4 NA oil immersion objective (Leica), an XY Piezo-Z stage (Applied Scientific Instrumentation), a CSU10 spinning disk (Yokogawa), an electron multiplier charge-coupled device camera (ImageEM; Hamamatsu Photonics), and a laser merge module (LMM5; Spectral Applied Research) equipped with 488- and 593-nm lasers, as previously described (Zhang et al., 2017 (link)). To minimize photobleaching in monopolar spindle assays, images were acquired with a 1-min time interval, with 1-µm spacing for GFP and mCherry z-stacks covering 15 µm total. To improve precision for the bipolar spindle assays, the time interval was 30 s with 0.5-µm z-spacing covering 3 µm total, so not all kinetochore pairs are visualized. For fixed cell imaging, the same microscope was used with a four-line laser module (405 nm, 488 nm, 561 nm, and 639 nm; Vortran Stradus VersaLase 4).