Biological phenomena usually evolves over time and recent advances in high-throughput microscopy have made possible to collect multiple 3D images over time, generating (or 4D) datasets. To extract useful information there is the need to extract spatial and temporal data on the particles that are in the images, but particle tracking and feature extraction need some kind of assistance.

This manuscript introduces our new freely downloadable toolbox, the Visual4DTracker. It is a MATLAB package implementing several useful functionalities to navigate, analyse and proof-read the track of each particle detected in any stack. Furthermore, it allows users to proof-read and to evaluate the traces with respect to a given gold standard. The Visual4DTracker toolbox permits the users to visualize and save all the generated results through a user-friendly graphical user interface. This tool has been successfully used in three applicative examples. The first processes synthetic data to show all the software functionalities. The second shows how to process a 4D image stack showing the time-lapse growth of Drosophila cells in an embryo. The third example presents the quantitative analysis of insulin granules in living beta-cells, showing that such particles have two main dynamics that coexist inside the cells.

Visual4DTracker is a software package for MATLAB to visualize, handle and manually track stacks of microscopy images containing objects such cells, granules, etc.. With its unique set of functions, it remarkably permits the user to analyze and proof-read 4D data in a friendly 3D fashion. The tool is freely available at https://drive.google.com/drive/folders/19AEn0TqP-2B8Z10kOavEAopTUxsKUV73?usp=sharing