FLS growth and segmentation of actin bundles and tip complex assembly. (A) Images from HILO and confocal or wide-field illumination on the same fields of view. Individual FLSs are segmented based on the actin fluorescence from the z-stack. Red arrows indicate straight tip-to-end distance. A mask on the actin channel is used as an overlay on any other channel, measuring intensities inside the tip complex area defined at the membrane with background correction (at the base slice), and along the shaft. Output includes protein intensity information and shape parameters of the bundle. (B) Single example of bundle growth in time, together with its 3D reconstruction (blue mesh). Actin structures are shown by setting each voxel’s transparency according to its measured intensity (darker indicates higher intensity). The 3D reconstruction overlay uses the output of FLS Ace rendered using Blender software. Black scale tripods indicate 1 μm along each axis. (C) Time-lapse montages of six example FLS tip complexes showing the intensity increase of actin, TOCA-1, and another tip complex protein (scale bars = 5 µm). Protein concentrations labeled/unlabeled were Actin 210/14,000; TOCA-1 10/3; VASP 20/16; N-WASP 20/1; GBD 2/0; Fascin 300/416; Diaph3: 20/10; and Ena: 40/40 in nanomolars.