Figure 4. Bisecting cell ROI into front and back halves.

A. A cell ROI is fitted into an ellipse and its shape parameters are measured. The centroids (X_C, Y_C) of the cell ROI at subsequent time frames (T_j-1 and T_j) are used to calculate the cell displacement. The ROI is split into two halves along the minor axis (perpendicular to the major axis). θ = angle perpendicular to the major axis, φ = angle (radians) subtended by the minor axis. To determine the back and front half of the bisected ROI, dist1 and dist2 are calculated. dist1 < dist2. dist1 corresponds to the back half and dist2 corresponds to the front half of the bisected ROI. (xh1, yh1): half-centers of front half; (xh2, yh2): half-centers of back half. B. The cell ROIs across time frames (F0–F10) were fitted into an ellipse. The ellipse was bisected along the minor axis (perpendicular to the major axis) to generate two halves of the moving cell.