ColocalizationAnalysis: an ImageJ/Fiji Macro

This macro analyzes overlap of multiple target channels within defined regions of interest in multi-channel images. ROIs can be drawn during the analysis session or loaded from a pre-saved overlay, making the workflow more dynamic. The color channels are intensity thresholded and converted to binary masks for relative area quantification. Multiple channel masks can be overlayed to quantify channel intersection area fractions. Within the individual thresholded area masks as well as the channel intersection masks, total area and pixel intensities are measured.

In addition, pixel intensity histograms are provided from each channel mask and their intersection mask. The final values represent area fractions of thresholded areas within each channel, and area fractions of channel intersections, including mean intensities and intensity histograms for the thresholded areas. Moreover, it is possible to enable co-occurrence and intensity correlation analysis. This includes several conventional quantifiers and metrics of colocalization.

The script is robust with respect to diverse fields and applications, and the image processing pipeline can be adjusted accordingly. In addition, all of the session information of the macro is documented, and an instruction outlining additional features and details is provided.

Contents of the archive:

1. ColocalizationAnalysis_.ijm (Macro)

2. Introduction_to_Colocalization.pdf (A concise description of colocalization, and a case study)

3. ColocalizationAnalysis_UserManual.pdf (User Guide)

4. CodeDesign.pdf (Outline of the internal architecture of the script)

5. README.md

6. License.txt