Visualization of F-actin network movement in motile keratocytes with FSM. F-actin flows slowly from the leading edge to the cell body in motile cells. Video is paired phase-contrast and AF546-phalloidin FSM time-lapse images. 30x real time (frames were collected at 2-s intervals and are displayed at 15 frames/s). Phalloidin was conjugated to AlexaFluor546 for live cell FSM imaging. Cells were imaged in culture media (Leibovitz's L-15 medium without phenol red supplemented with 14.2 mM Hepes, pH 7.4, 10% FBS, and 1% antibiotic-antimycotic). Images were acquired using an inverted microscope (Diaphot-300; Nikon) with a 60× NA 1.4 oil plan-Apo objective (Nikon) every 2 with the 60× oil objective. All time-lapse images were collected with a cooled back-thinned CCD camera (MicroMax 512BFT; Princeton Instruments) with a 2× optovar attached using MetaMorph software version 6 (Molecular Devices). Video corresponds to Fig. 1 and video 2 in JCB 178:1207-1221, 2007.
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