Quantitative Colocalization Analysis in Fluorescence Microscopy
- 1Department of Biology, Texas A&M University, College Station, TX, USA
Abstract
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.
Related
- MUFASA: a continuous-time stochastic framework for realistic fluorescence microscopy simulation
- High-precision correlative fluorescence and electron cryo microscopy using two independent alignment markers
- Localization accuracy in single-molecule fluorescence microscopy
- Multiphoton tomographic fluorescence lifetime imaging microscopy -TomoFLIM
- Quantitative fluorescence lifetime imaging of protein microenvironments by genetically encoded compact benzodiazole probes
BibTeX
@article{myers2026quantitative,
title = {{Quantitative Colocalization Analysis in Fluorescence Microscopy}},
author = {Charles James Myers and Beiyan Nan},
journal = {Bio-protocol},
year = {2026},
month = oct,
doi = {10.21769/bioprotoc.5832},
url = {https://doi.org/10.21769/bioprotoc.5832},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Quantitative Colocalization Analysis in Fluorescence Microscopy
AU - Charles James Myers
AU - Beiyan Nan
PY - 2026
DA - 2026/10/20
JO - Bio-protocol
DO - 10.21769/bioprotoc.5832
UR - https://doi.org/10.21769/bioprotoc.5832
AB - 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.
SN - 2331-8325
ER -