K-FOCUS is a robust object-based colocalization tool in live-cell imaging. (A) Example images demonstrating the accumulation of Halo-ATG9A in lysosomes, captured immediately after or 24 h following labeling with JFX650 in U2OS cells (scale bar = 10 µm). (B) A comparison between different colocalization analysis methods for the data shown in A, including threshold-based Pearson and Manders coefficients, as well as wavelet spot detection–based SODA and K-FOCUS. The box indicates the interquartile range, the whiskers the 10–90% confidence interval, the square indicates the average, and the horizontal line is the median; statistical significance was assessed by a two-tailed t-test. (C) Example images of U2OS cells expressing GFP-LC3B and Halo-ATG13 under both control and EBSS conditions (scale bar = 5 µm). (D) A comparison between different colocalization analysis methods for the data shown in C, including threshold-based Pearson and Manders coefficients, as well as wavelet spot detection–based SODA and K-FOCUS analyses. The box indicates the interquartile range, the whiskers the 10–90% confidence interval, the square indicates the average, and the horizontal line is the median; statistical significance was assessed by a two-tailed t-test.