Model-free quantification and visualization of colocalization in fluorescence images

The spatial association between fluorescently tagged biomolecules in situ provides valuable insight into their biological relationship. Within the limits of diffraction, such association can be measured using either Pearson's Correlation Coefficient (PCC) or Spearman's Rank Coefficient (SRC), which are designed to measure linear and monotonic correlations, respectively. However, the relationship between real biological signals is often more complex than these measures assume, rendering their results difficult to interpret. Here, we have adapted methods from the field of information theory to measure the association between two probes' concentrations based on their statistical dependence. Our approach is mathematically more general than PCC or SRC, making no assumptions about the type of relationship between the probes. We show that when applied to biological images, our measures provide more intuitive results that are also more robust to outliers and the presence of multiple relationships than PCC or SRC. We also devise a display technique to highlight regions in the input images where the probes' association is higher versus lower. We expect that our methods will allow biologists to more accurately and robustly quantify and visualize the association between two probes in a pair of fluorescence images. © 2018 International Society for Advancement of Cytometry.