Quantitative Colocalization Analysis in Fluorescence Microscopy

Spatial organization of macromolecules is fundamental to cellular function, with colocalization providing key insights into molecular interactions and biological processes. However, quantification remains challenging due to diverse localization patterns and irregular sample geometries. Here, we present a protocol for analyzing colocalization between two fluorescent probes using coAnalyzer, a MATLAB-based software package. coAnalyzer features a user-friendly graphical interface and supports region of interest (ROI) selection, image merging, line scanning, signal isolation, scatterplot generation, and quantitative analysis. coAnalyzer enables colocalization analysis of any two fluorophores, regardless of the proteins or dyes involved. Its broad applicability across a wide range of organisms and sample types demonstrates the robustness, flexibility, and versatility of the platform. Key features • Provides both quantitative and semi-quantitative colocalization analysis using multiple coefficients. • Features a user-friendly graphical user interface (GUI) that provides step-by-step visualization of the analysis process. • Supports 1D (line scan) and 2D analysis. • Analyzes large datasets and generates histograms for effective data presentation.