Ronald D. Vale, James A. Spudich, Eric R. Griffis (2011) CIL:11993, Drosophila melanogaster, epithelial cell. CIL. Dataset

TIRF time lapse of myosin-GFP in a cell with a symmetric monopolar spindle from metaphase to anaphase (cell was depleted of Klp61F [kinesin-5] and BubR1 by RNAi). At anaphase, myosin-GFP concentrates at the cell perimeter and becomes depleted from the central regions of the adhered cortex. Slowly, myosin breaks symmetry and ultimately relocalizes to the middle. This video corresponds to the image in Fig. 6 and video 10 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 50-200ms 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).

本数据集展示了细胞中肌球蛋白-GFP在具有对称单极纺锤体的细胞中从有丝分裂中期至分裂期的时序记录（通过RNA干扰耗竭了Klp61F [肌球蛋白-5]和BubR1）。在分裂期，肌球蛋白-GFP在细胞周边区域富集，并从附着细胞质的中央区域逐渐耗竭。随后，肌球蛋白逐渐打破对称性，最终重新定位至细胞中央。本视频与J Cell Bio 186:727-738, 2009中图6和视频10所展示的图像相对应。

采用全内反射显微镜技术，使用Perfect-focus TE2000（尼康）显微镜、100×、1.45 NA物镜（尼康）以及488-nm氩激光（100 mW）、491-nm固态激光（100 mW）和561-nm固态激光（50 mW）进行照明。对于双色全内反射显微镜成像，我们使用了三通分光镜（z491/561/633rpc）并利用置于相机前的滤光轮更换发射滤光片（ET525/50或ET595/50；均来自Chroma Technology Corp.）。在某些情况下，滤光盒中使用了激发截止滤光片（NF01-405/488/561/635；Semrock, Inc.）。图像通常每2-3秒捕获一次，曝光时间为50-200ms，使用EM电荷耦合器件相机（iXon；Andor Technology）。显微镜的控制和图像采集使用开源的MicroManager软件（http://www.micro-manager.org）进行。