Fluorescence recovery after photobleaching

FRAP experiments were performed on a laser scanning confocal microscope (SP5; Leica Microsystems) with a 63× oil objective and 3× optical zoom using the FRAP wizard with LASAF software package. A square ROI (5 µm×5 µm) was manually selected within the F-actin cortex of either animal cap epithelium or mesoderm for bleaching (Kim and Davidson, 2011). To maximize frame rate, 256×256-pixel images were acquired at 700 Hz (∼0.37 s/frame). Bleaching was achieved by zooming in to the square ROI (‘zoom-in’ feature on software) and exposing the 488 nm argon laser for two time steps (∼0.74 s). The laser power was set to 25% and the ND filter setting to 100% for bleaching. Five frames were collected before bleaching and 30 frames after at the same frame rate. After acquisition, intensity profiles were analyzed using custom Matlab software to calculate half-life and immobile fraction.