Demonstrating a relationship between two bio-molecules is a common biological problem that can be tackled using fluorescence microscopy. The ability of microscopists to demonstrate relationships in an objective fashion was enhanced by the introduction of Pearson's correlation coefficient (PCC), a standard statistical analysis designed to measure the strength of a linear relationship between two variables, in this case fluorescent intensities from two images.

When PCC is calculated for all of the pixels in an image pair, regardless as to whether they contain data from positively labelled structures, or background intensities, these calculations generate "global" values for PCC. In this tutorial, one of our Application Specialists discusses some of the ambiguities with using global PCC, and describes how using a thresholded PCC can bring clarity to colocalization studies in fluorescent microscopy.