Description

Time lapse of mCherry-tubulin by wide-field epifluorescence at the coverslip interface (left), mCherry-tubulin by TIRF (middle), and myosin-GFP by TIRF (right). The acquisition of Cherry-tubulin by TIRF was at 2s intervals, whereas the mCherry-tubulin by epifluorescence and myosin-GFP TIRF were at 36 and 10 s intervals, respectively. Video corresponds to Fig 3A and video 2 from J Cell Bio 186:727-738, 2009. Note that this video is the wild type control for another video in the series that shows the same biological process in the presence of kinesin-6 RNAi. 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).