Csu x1m 5000

Both live and fixed images were collected with a Yokogawa CSU-X1M 5000 spinning disk confocal on a Zeiss Axio Observer inverted motorized microscope with a Zeiss 63× Plan Apo 1.4 numerical aperture (NA) lens. Images were acquired with a Hamamatsu OCRA R2 charge-coupled device camera controlled with Zen software (Zeiss, Thornwood, NY). For time lapse, images were collected every 2 s for 1–3 min. Laser power for 488 nm was 30%, with exposure time 1000–1500 ms. Laser power of 561 nm was 25%, with exposure time 850–1500 ms. For imaging of immunostaining, laser power for 488 nm was set to 25%, with 95-ms exposure; for 561 nm, laser power was 12% with exposure time 800 ms. For two-color colocalizations in Figure 6, the TACC3 channel was translated in the x-axis, after calculating the frame-to-frame velocity of the growing MT plus end, in order to account for the 1-s time delay between channels, for each examined MT (using the Translate function of ImageJ [National Institutes of Health, Bethesda, MD]). To further confirm correct translation, time-lapse colocalizations were examined with both combinations of imaging—red channel first, green channel second; then green channel first, red channel second.