Mechanical Fidelity of Fixed-Cell Traction Force Microscopy

Cellular traction forces contribute to biological processes including cell migration and tissue morphogenesis. Traction force microscopy (TFM) is widely used to quantify these forces in living cells, but it remains unclear how well measurements after fixation reproduce the live-cell state. Here, we systematically evaluated fixation-induced changes by paired comparison of the same cells before and after fixation using different fixation protocols and substrate stiffnesses. Fixation largely preserved the spatial organization and direction of substrate displacements and reconstructed traction fields, whereas displacement, traction, and strain-energy magnitudes showed fixation-dependent changes. Among the protocols tested, glutaraldehyde (GA) provided the closest agreement with live-cell measurements, retaining up to 99% of displacement and traction magnitudes and strain energy. Paraformaldehyde-based protocols caused larger reductions in these quantities while largely preserving their spatial organization. These results show that fixed-cell TFM, particularly with GA fixation under the conditions tested here, can provide measurements close to the live-cell state and offers a practical approach for combining TFM with post-fixation fluorescence imaging in a multi-well workflow.