Traction Force Microscopy with DNA FluoroCubes

From cell differentiation to morphogenesis and cell migration, a multitude of processes are coordinated by mechanical forces that cells generate. Among diverse techniques to assess the mechanical properties of the cell, traction force microscopy (TFM) has emerged as one of the most popular methods for quantifying cell-generated stresses. Standard TFM procedures rely on fiducial markers in the extracellular environment to measure the deformations that are caused by cellular forces. Typically, fluorescent beads are used as fiducials. However, the replacement of beads with fluorescently labeled DNA structures can have numerous advantages, including a smaller size of the markers and the possibility of customizing the DNA structures, for example to read out orthogonal information or to realize a switchable surface functionalization. Here, we develop a multi-purpose platform for combining such setups with TFM. As fiducials we employ FluoroCubes – nanometer-sized DNA constructs - for TFM. These constructs are grafted to a high refractive index polyethylene siloxane surface for the precise tracking of displacements resulting from cell-generated forces. To ensure a local transmission of traction forces from the adhesion ligands to the substrate, we also graft RGD peptides, which represent the smallest ligands of the extracellular matrix, onto our elastic substrates. To further enhance the spatial resolution of the TFM, FluoroCubes can be supplemented with densely packed fluorescent beads as fiducials. We propose a modification of the Kanade-Lucas-Tomasi (KLT) optical flow tracking (OFT) algorithm for optimal, simultaneous tracking of FluoroCubes and beads. Together, the developed experimental setup and tracking algorithm yield highly resolved maps of traction forces that correlate well with the spatial distribution of kindlin at focal adhesions. ### Competing Interest Statement The authors have declared no competing interest.