Description

Time lapse of Aurora-B GFP and mCherry microtubules at the metaphase-to-anaphase transition. The laser illumination was set near to but not achieving complete total internal reflection TIRF to allow visualization of microtubules at the cortex as well as the more interior central spindle. Aurora B foci are seen at the tips of microtubules at multiple sites at the cortex as well as a bright central region (the central spindle).This video corresponds to the image in Fig. S4 and video 6 from J Cell Bio 186:727-738, 2009. Total internal reflection microscopy was performed using a microscope (Perfect-focus TE2000; Nikon) with a 100×, 1.45 NA objective (Nikon) and illumination from either a 488-nm argon laser (100 mW) or a 491-nm solid-state laser (100 mW) and a 561-nm solid-state laser (50 mW). For dual-color TIRF microscopy, we used a triplepass dichroic filter (z491/561/633rpc) and changed the emission filter (ET525/50 or ET595/50; both from Chroma Technology Corp.) with a filter wheel placed before the camera. In some cases, an excitation notch filter was used in the filter cube (NF01-405/488/561/635; Semrock, Inc.). Images were typically captured every 2 -3 s with a 50 -200 ms exposure with an EM charge-coupled device camera (iXon; Andor Technology). The microscope was controlled and images were acquired using open source MicroManager software (http://www.micro-manager.org).