ThirdPeak is a flexible tool designed for the robust analysis of two- and three-dimensional tracking data.

Biological processes, though often imaged and visualized in two dimensions, inherently occur in at least three-dimensional space. As time-resolved volumetric imaging becomes increasingly accessible, there emerges a necessity for tools that empower non-specialists to process and interpret intricate datasets. We introduce ThirdPeak, an open-source tool tailored for the comprehensive analysis of two- and three-dimensional track data across various scales. Its versatile import and export options ensure seamless integration into established workflows, while the intuitive user interface allows for swift visualization and analysis of the data. When applied to live-cell diffusion data, this software demonstrates the benefits of integrating both 2D and 3D analysis, yielding valuable insights into the understanding of biological processes.