Fig. 3: Optical flow as a tool to study actin dynamics with top 15% optical flow vectors show distinct regions of activity throughout differentiation.

Actin dynamics within hNPC can be captured using optical flow algorithm (A–F). To visualize optical flow’s use, a single analysis time point of two subsequent frames from a 7-minute movie (with 0.06255 Hz acquisition) showing intracellular actin is labeled (A, B). Overlay of two subsequent frames analyzed with time gradient shown (A). Optical flow vectors (green) overlaid on the second of the two subsequent frames from (A, B). Probability distribution of optical flow vectors of detected actin speeds between subsequent frames (C). The magnitude calculated by optical flow algorithm is overlaid on the masked cell region (D). Orientation of optical flow vectors displayed on the overlaid cell region; color shown is indicative of directionality of optical flow vectors detected as displayed by circular map showing directionality in radians (E). Kymograph that captures the probability (as indicated by color) of directional actin flow (as indicated on the y-axis) over time (along the x-axis) (F). Max projection of actin image time-series showing top 15% magnitude flow vectors over all frames from 5-min movies at specific differentiation time points (G–I). Colorscale indicates the proportion of frame pixels that were found to be in the top 15% magnitude throughout the entire film. Examples of undifferentiated cells (G), naive differentiated cells (H), and mature differentiated cells (I). All scale bars represent 50 microns.