Fig 1.
napariTFM workflow and data structure.
Schematic overview of the napariTFM analysis pipeline showing the flow from raw input data (beads, reference, and optional cell images) through preprocessing, displacement analysis using TV-L1 optical flow, force calculation via FTTC (Fourier Transform Traction Cytometry), and stress analysis using MSM (Monolayer Stress Microscopy). Orange boxes indicate input data, blue boxes show analysis steps and their internal data structures, and green boxes indicate output files generated at each step.
Fig 2.
napariTFM user interface in napari.
Screenshot of the napariTFM plugin integrated within the napari image viewer, showing: (1) the main image display with fluorescent bead data and overlaid displacement vectors, (2) napari’s layer controls, (3) global parameters panel, (4) tabs that access controls for different analysis steps, (5) data input panel, (6) local parameters for the selected step, and (7) action controls (analyze, save data, preview etc.). The interface provides real-time parameter adjustment and visualization of results.
Fig 3.
Validation of displacement analysis and traction force calculation.
(A) Displacement field validation across three scenarios (low, mid, high displacement magnitudes). Top row shows ground truth displacement fields, bottom row shows calculated results using napariTFM’s TV-L1 optical flow algorithm. Vector overlays indicate displacement direction and magnitude, with color maps showing displacement magnitude in pixels. (B) Displacement analysis metrics. Left: correlation coefficients between calculated and ground truth displacement fields. Right: relative error as a function of ground truth displacement magnitude, showing systematic bias at different displacement scales. (C) Traction force validation using FTTC algorithm across three force magnitude scenarios. Top row shows ground truth traction fields, bottom row shows calculated results. Color maps indicate traction force magnitude in kPa, with vector overlays showing force direction. (D) Traction force analysis metrics. From left to right: correlation coefficients between calculated and ground truth traction forces, deviation of traction magnitude (DTM), traction in surrounding regions (DTPS) and deviation of traction angle (DTA).
Fig 4.
Validation of Monolayer Stress Microscopy (MSM) analysis.
(A) Square plate analytical validation using a simple geometric test case with a known analytical solution. Stress tensor components (,
,
) are shown for ground truth (top row) and MSM-calculated (bottom row) fields. (B) Square plate analysis metrics. Bar charts show correlation coefficients between calculated and ground truth stress fields for each stress component, as well as mean relative error. (C) FEM-simulation–based validation using computational data from a finite element model resembling a snapshot of a migrating cell. Stress tensor components are shown for ground truth (top row) and MSM-calculated (bottom row) fields, with layout as in (A). (D) FEM-simulation validation metrics. Correlation coefficients between calculated and ground truth stress fields are shown for each stress component, as well as absolute and relative error with respect to ground truth sress magnitude.
Fig 5.
Validation of napariTFM against experimental data, previously published in eLife [37].
(A) Single-cell analysis showing displacement fields (left column), traction force fields (second column), and stress tensor components (third column) and
(right column). Top row: original analysis from Ruppel et al., eLife 2023. Bottom row: reanalysis using napariTFM. Color maps indicate displacement magnitude in μm, traction force magnitude in Pa, and stress components in mN/m. (B) Population-averaged fields across 29 cells. Top row: averages from original analysis. Bottom row: averages from napariTFM reanalysis. Layout as in (A). (C) Quantitative comparison of analysis outputs. Box plots show distributions of average displacement magnitude (left), average traction force (second from left), average
stress (second from right), and average
stress (right). For each metric, three conditions are shown: Original (original published analysis), napariTFM (reanalysis), and Difference. Horizontal dashed line at zero indicates no difference between methods.