napariTFM: An open-source tool for traction force microscopy and monolayer stress microscopy

Cellular force generation and transmission are fundamental processes driving cell migration, division, tissue morphogenesis, and disease progression. Traction Force Microscopy (TFM) and Monolayer Stress Microscopy (MSM) have emerged as essential techniques for quantifying these mechanical processes, but current software solutions are fragmented across multiple platforms with varying degrees of usability and accessibility. Here, we present napariTFM, a comprehensive open-source plugin for the napari image viewer that integrates state-of-the-art algorithms for both TFM and MSM analysis within an intuitive graphical user interface. The software implements TV-L1 optical flow for displacement analysis, Fourier Transform Traction Cytometry (FTTC) for force reconstruction, and finite element methods for stress calculation, supporting both single-frame and time-series analysis of 2D microscopy data. Systematic validation using synthetic datasets with known ground truth values demonstrated excellent accuracy, with correlation coefficients above 0.9 for most situations. Real-time parameter adjustment and immediate visualization capabilities enable interactive optimization of analysis parameters and quality assessment during processing. Finally, we demonstrate the software’s capabilities through analysis of optogenetic contractility experiments in cell doublets. napariTFM addresses critical gaps in the cellular mechanics software ecosystem by combining algorithmic rigor with practical usability, providing the research community with an accessible platform for quantitative studies of cellular force generation and transmission.Author summary: Cell-generated forces are essential in driving and regulating numerous biological processes, including development, wound healing, and disease progression. Traction Force Microscopy (TFM) is a widely used technique to measure these cell-substrate forces. Cells are cultured on elastic gels of known stiffness with embedded fluorescent beads. By comparing images of the gel in deformed (cell-attached) and relaxed states (cell-detached), bead displacements can be measured and converted into traction forces through Fourier-based mathematical methods. For cells in monolayers, Monolayer Stress Microscopy (MSM) extends this analysis to calculate internal tissue stresses, assuming the cell layer behaves as a homogeneous and linear material. Despite TFM’s widespread use, existing software tools remain fragmented and often require programming expertise. napariTFM addresses this gap with a comprehensive, user-friendly platform built on napari, a python-based image viewer. The software combines an intuitive graphical interface for interactive parameter exploration with robust batch processing for large datasets, making quantitative force measurements accessible to researchers without computational backgrounds.