Tutorial for the xsColoc colocalization analysis plug-in of ImageJ

Introduction to the xsColoc colocalization analysis plugin for ImageJ/Fiji. Access the plugin via Plugins > Tracking > xsColoc with a multi-channel 8 or 16-bit image open.

Set initial parameters for particle recognition including background filter size, dynamics, sensitivity, and contrast. Apply these settings to both channel 1 and channel 2 separately, adjusting noise filtering options as needed.

Click 'Go' to detect particles in both channels. Use the merge and show particles functions to visually inspect the detected particles and verify colocalization by toggling between channels.

Use ImageJ's region of interest tools to specify the image area containing particles. This step ensures proper random simulation by excluding background areas from the colocalization testing.

Configure reference and test channels, set maximum distance threshold, and specify duplicate removal settings. Click 'Test colocalization' to run the analysis including 100 randomization simulations.

The plugin saves three files: a TIFF image, an MCC descriptor file containing detection and ROI parameters, and a data table with particle measurements and colocalization results.

Review the results table containing reference channel particles, nearest neighbor distances, and 100 simulated colocalization columns. Interpret minus-one values as unmatched particles and analyze the visualization of detected colocalizations.