A Step-by-Step Protocol for Quantifying Molecular Forces in Living Cells Using the Vinculin TSMod FRET Tension Sensor.

Genetically encoded FRET-based tension sensors provide a powerful approach for visualizing and quantifying molecular forces within living cells. These probes incorporate an extensible, calibrated elastic linker between a donor-acceptor fluorophore pair, enabling mechanical load changes to be reported as variations in FRET efficiency. This article presents a detailed, stepwise protocol for the expression, imaging, and quantitative analysis of the vinculin tension sensor (VinTS), which reports forces transmitted through vinculin within focal adhesions. The procedure describes optimized conditions for cell culture and guidelines for selecting appropriate imaging modalities, and recommendations for acquiring high-quality ratiometric or spectral FRET datasets. In addition, the workflow includes image processing steps for background subtraction, bleed-through correction, FRET ratio calculation, and spatial mapping of tension across adhesion sites. Representative results illustrate how VinTS can be used to assess cytoskeleton-dependent mechanical forces and to correlate tension distribution with adhesion assembly and maturation. While demonstrated here for vinculin, the methodological framework is readily adaptable to other FRET-based tension sensors and mechanosensitive proteins.