Fluorescence colocalization microscopy analysis can be improved by combining object‐recognition with pixel‐intensity‐correlation

The question whether two proteins interact with each other or whether a protein localizes to a certain region of the cell is often addressed with fluorescence microscopy and analysis of a potential colocalization of fluorescence markers. Since a mere visual estimation does not allow quantification of the degree of colocalization, different statistical methods of pixel‐intensity correlation are commonly used to score it. We observed that these correlation coefficients are prone to false positive results and tend to show high values even for molecules that reside in different organelles. Our aim was to improve this type of analysis and we developed a novel method combining object‐recognition based colocalization analysis with pixel‐intensity correlation to calculate an object‐corrected Pearson coefficient. We designed a macro for the Fiji‐version of the software ImageJ and tested the performance systematically with various organelle markers revealing an improved robustness of our approach over classical methods. In order to prove that colocalization does not necessarily mean a physical interaction, we performed FRET (fluorescence resonance energy transfer) microscopy. This confirmed that non‐interacting molecules can exhibit a nearly complete colocalization, but that they do not show any significant FRET signal in contrast to proteins that are bound to each other.