ILEE and human eye have different tendency to judge the topological structure of cytoskeleton, especially between two bright bundles. A cropped portion of the demonstration image from Fig. 3 a and its derived images are generated and demonstrated to explain the discrepancy related to the determination of the topological structure between ILEE and hand-drawn binary ground truth (i.e., human eye evaluation). When ILEE binary image is compared with ground truth, they mostly match with each other with only minor and non-influential differences. When both binary images were skeletonized based on their topological structure, a numerous branches were detected from the ground truth, but not from ILEE. This phenomenon is very obvious in the highlighted (i.e., circled) area, where two bright actin bundles are very close to each other. This is intriguing, as where the human eye identified many branches and intervals, ILEE did not. These structures have a strong impact on the topological structure. Therefore, the topology-sensitive indices, linear density, segment density, and branching activity display contrast and stable inconformity between ground truth and ILEE (Fig. 4 d). However, since the hand-drawn ground truth image is not a rigorously defined ground truth, we surmise that inconformity cannot be interpreted to any inaccuracy of ILEE. At the same time, the skeletonization algorithm may be replaced/upgraded as an entire module, for further improved performance.