High-Throughput Focal Adhesion Analysis Software for Automated Detection, Tracking, and Classification of Cell-Matrix Adhesions.

Focal adhesions are dynamic multi-protein complexes that mechanically link the actin cytoskeleton to the extracellular matrix through integrin receptors. A single migrating cell can contain >1000 adhesion structures simultaneously, exhibiting highly heterogeneous fates: only ∼3.5% of nascent adhesions mature into focal contacts or focal adhesions, while the majority turn over within minutes. Resolving this heterogeneity requires automated detection, tracking, and classification of individual adhesion trajectories at scale. Here we provide a step-by-step Basic Protocol for the Focal Adhesion Package, a MATLAB-based pipeline that processes time-lapse fluorescence images through 11 sequential steps, from sub-pixel stage drift correction and cell masking through point source detection, particle tracking, focal adhesion segmentation, feature extraction, and machine learning-based classification into 9 functional groups. The pipeline optionally integrates traction force microscopy and multi-channel fluorescence data to perform event detection of significant signal rise and compute temporal cross-correlations between protein recruitment and force generation at individual adhesions. For a typical 100-frame movie, total runtime is ∼40 min using CPU-parallelized optimization (∼15 min for preprocessing and detection, ∼25 min for feature extraction) on a standard workstation, with GPU acceleration reducing this to ∼27 min. We describe parameter selection criteria for each step and provide supplementary code recipes for programmatic access to outputs. The pipeline can be executed through a graphical user interface or automated using MATLAB scripts. © 2026 Wiley Periodicals LLC. Support Protocol: Software installation and set up Basic Protocol: Running the Focal Adhesion Package Alternate Protocol: Batch processing multiple movies.