ILEE pipeline and the demonstration of cytoskeletal indices. (a) ILEE is an adaptive local thresholding approach that applies to both 2D and 3D data structures, with an output of 12 cytoskeletal indices. Please note that total connected element, total branch, and total node (marked “*”) are non-normalized indices for development purposes of advanced users and cannot be directly used for biological interpretation. (b) Schematic diagram of the ILEE algorithm. ILEE requires a sample image with enhanced edge gradients, a computed gradient threshold, and an implicit Laplacian smoothing coefficient, K₂, to generate a binary image and skeletonized image for index computation. Z-axis maximum projection (red box) is only conducted in the 2D mode. (c) Visualized demonstration of ILEE performance. The raw data, binary image generated by ILEE, and demonstration of computed features by both 2D and 3D modes are shown. TDT, total distance transformation map, used to compute bundling indices; skeleton NCE, non-connected element (filament pieces) of the skeleton image with each element by different colors, used to estimate connectivity indices; branching, the skeleton image with each branch in different colors and each node by a black cross; anisotropy, local anisotropy level, shown as length of red lines, and direction of the first eigenvector, shown as the direction of red lines. Scale bars are marked as red solid line.