Factors influencing the mechanical properties of soft elastomer substrates for traction force microscopy

Traction force microscopy (TFM) aims at determining the forces exerted by cells on their substrate. Traction quantification relies on the knowledge of the mechanical properties of the substrate, i.e., typically its Young’s modulus. Because of the challenges associated with the mechanical analysis of very soft elastomers, a large range of modulus values are reported in literature for the same materials. In order to provide a reliable characterization, a systematic study was performed, including a large number of micro-scale indentation experiments. The mechanical behavior of four elastomer configurations (Sylgard 184 40:1 and 60:1, and CY9:10 and 10:9), frequently used for TFM studies, was investigated. The good repeatability of the experimental procedure allowed characterizing the modulus variability within each material sample and between batches. The corresponding uncertainty is in the range of 10%, consequently directly affecting the reliability of cell traction force values. Material aging, immersion in a liquid, and illumination were considered as specific factors potentially influencing the stiffness of TFM substrates. The results show that stiffness changes significantly over the first 3 weeks following elastomer production, while immersion in a isoosmolar solution and illumination have generally modest influence on micro-scale stiffness.