TIRF time lapse of myosin-GFP (left), mCherry-tubulin (middle), and overlay (myosin-GFP in green and mCherry-tubulin in red) in wild-type S2 cells from metaphase to anaphase. This cell has many astral microtubules touching the cortex (within the TIRF illumination field) in the bottom left and very few in the top right. Despite this asymmetry (perhaps due to a tilting of the spindle), myosin localization occurs in a normal, symmetrical manner, and myosin clears from both poles. Video corresponds to video 4 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).