title: Fluorescence colocalization microscopy analysis can be improved by combining object-recognition with pixel-intensity-correlation.
abstract: 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.
authors.affiliation: Dept. of Vascular Biology and Thrombosis Research, Center for Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Moser
authors.affiliation: Dept. of Vascular Biology and Thrombosis Research, Center for Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Hochreiter
authors.affiliation: Inst. of Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.
authors.forename: Ruth
authors.initials: R
authors.lastname: Herbst
authors.affiliation: Dept. of Vascular Biology and Thrombosis Research, Center for Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria.
authors.forename: Johannes A
authors.initials: JA
authors.lastname: Schmid
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002451:Cell Compartmentation; D031541:Fluorescence Resonance Energy Transfer; D057809:HEK293 Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods*; D057054:Molecular Imaging / Q000379:methods; D012984:Software
chemical_list: D001426:Bacterial Proteins; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria
keywords: Colocalization; Fluorescence; Image processing; ImageJ; Microscopy