# ColocZStats: a z-stack signal colocalization extension tool for 3D slicer

## TL;DR

ColocZStats is a new tool for 3D Slicer that enables users to analyze colocalization of signals in 3D confocal microscopy images within user-defined regions.

## Contribution

The tool introduces ROI-based colocalization analysis in 3D Slicer, filling a gap in existing visualization platforms.

## Key findings

- Current 3D visualization tools lack robust ROI-based colocalization analysis features.
- ColocZStats provides a user-friendly interface for thresholding and ROI selection in 3D image stacks.
- The extension generates colocalization metrics and reports in diagrams and spreadsheets.

## Abstract

Confocal microscopy has evolved to be a widely adopted imaging technique in molecular biology and is frequently utilized to achieve accurate subcellular localization of proteins. Applying colocalization analysis on image z-stacks obtained from confocal fluorescence microscopes is a dependable method of revealing the relationship between different molecules. In addition, despite the established advantages and growing adoption of 3D visualization software in various microscopy research domains, there have been few systems that can support colocalization analysis within a user-specified region of interest (ROI). In this context, several broadly employed biological image visualization platforms are meticulously explored in this study to understand the current landscape. It has been observed that while these applications can generate three-dimensional (3D) reconstructions for z-stacks, and in some cases transfer them into an immersive virtual reality (VR) scene, there is still little support for performing quantitative colocalization analysis on such images based on a user-defined ROI and thresholding levels. To address these issues, an extension called ColocZStats (pronounced Coloc-Zee-Stats) has been developed for 3D Slicer, a widely used free and open-source software package for image analysis and scientific visualization. With a custom-designed user-friendly interface, ColocZStats allows investigators to conduct intensity thresholding and ROI selection on imported 3D image stacks. It can deliver several essential colocalization metrics for structures of interest and produce reports in the form of diagrams and spreadsheets.