Traction Force Microscopy

In addition to measuring displacements and strains of the cells, we measured forces applied by the cells to the substrate using traction force microscopy (TFM), which computes the traction (force per area) at the interface between the cells and the compliant substrate [70,71,80–83]. This required markers to be embedded in to the substrate of our cell culture platform. It is crucial to achieve a high contrast image of a single plane of particles at the top surface of the substrate. As cell-induced deformations cause particle displacements throughout the substrate, the exact location of the particles must be known for accurate calculations. Often measurements are assumed to be taken from particles at the top surface of the substrate nearest the cells. Unfortunately, this assumption is often incorrect, resulting in an image that shows beads for various positions through the bulk of the substrate. To address this issue, we make PA substrates with particles located at the cell-substrate interface as described above.

The cells and fluorescent particles were imaged with a widefield florescent microscope (Nikon Ti-E) with a 10× NA 0.3 objective during an experiment. After the experiment, the cells were released from the substrate with trypsin, which eliminated the tractions applied by the cells to the substrate and allowed the substrate to recover to a stress-free state. A final image of the particles was acquired in that stress-free state; this final image was used as a reference to compute cell-induced displacements of the particles using DIC. After displacements were measured, the tractions were computed by applying equations of force equilibrium to the substrate [70,71,80–83]. There are multiple different ways to implement the equations; we used an approach based on the Fourier transform [71] with corrections for a substrate of finite thickness [82,83].