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.

Mechanical Regulation of Microvascular Angiogenesis

“…Gels were imaged at 40X to a depth of 25 µm, which is the approximate depth of a single vessel. A region of interest (ROI) was drawn around vascular structures, and signal overlap between αSMA and isolectin B4 were quantified for the ROI using Manders coefficients as determined by the Fiji plugin coloc2 (62).…”

A green tea polyphenol Epigallocatechin-3-gallate modulates Tau Post-translational modifications and cytoskeletal network

“…4B, white arrows). We performed the co-localization analysis for AGEs+actin and AGEs+tubulin for glycation by coloc2 plugin in Fiji on the background subtracted images (55). It was observed that Pearson's Co-Correlation Co-efficient (PCC) for actin+AGEs was increased in presence of MG suggesting actin glycation.…”

“…To ensure the glycation of cytoskeleton, co-localization analysis was carried out for AGEs (red) and actin (green) and AGEs (red) tubulin (green) fluorescence. The analysis was carried out on the background subtracted images the reported algorithm (55). The Pearson's coefficient of correlation was determined by the coloc-2 plugin of Fiji software.…”

Editorial: Methods and advances for systems and synthetic biology, nanobiotech and medicine

“…Treatment options for these and other diseases often rely on detailed analysis of cellular signaling. Moser et al [7] provide now a method to improve fluorescence colocalization microscopy analysis, which can be used to show whether two proteins interact with each other.…”