Multi-Channel Nuclear Analysis: An ImageJ/FIJI Plugin for Automated Nuclear Segmentation and Multi-Channel Fluorescence Analysis

Quantitative analysis of fluorescence microscopy images is essential for studying expression levels, subcellular localization and co-occurrence of proteins and other biomolecules. While several automated tools exist for specific applications, there remains a need for user-friendly, customizable tools that can analyze multi-channel fluorescence images with nuclear segmentation capabilities. Here we present Multi-Channel Nuclear Analysis, an open-source ImageJ/FIJI plugin that combines the robust nuclear segmentation capabilities of StarDist with versatile multi-channel analysis features. The tool provides a graphical user interface for configuring analysis parameters, processes multiple images in batch mode, and generates both individual and consolidated measurement tables to facilitate downstream analysis. A companion tool for merging separate channel files into multi-channel images extends compatibility to diverse microscopy systems. Together, these tools enable researchers without extensive programming experience to perform comprehensive quantitative analysis of nuclear-centered multi-channel fluorescence images.