Determination of global gradient threshold. In order to determine the global gradient threshold using estimated mean and standard deviation (STD) of coarse background gradient as an input of ILEE, we constructed a non-linear estimation model g_thres= μ_G.cbg+ k(σ_cbg) σ_G.cbg. The global gradient threshold of 30 samples with 800*800*25 resolution randomly collected from our actin image database were evaluated manually (similar to MGT), and the mean and STD of gradient values in coarse background are calculated following the approach described in the Methods and Fig. S5. For the 2D mode, images were first projected to 800*800 resolution. Then, the coefficient k of each sample was calculated and corelated with STD of the coarse background. (a) The correlation of STD of coarse background and k. Green, standard mode, where each sample have the similar weight of regression; Red, conservative mode (default of this paper), where a weight equal to k² is applied. (b) The g_thres-σ_cbg relationship restored from (a). (c and d) Similar to (a and b), but the reference global gradient threshold were manually evaluated using a 3D visualizing interface.