Traction Force Microscopy with DNA FluoroCubes.

Mechanical forces at the cell-substrate interface govern processes ranging from migration to differentiation, yet mapping these forces at high spatial resolution remains challenging. Traction force microscopy (TFM) addresses this by quantifying substrate deformations using fiducial markers, typically conventional fluorescent beads. Here, we introduce fluorescently labeled DNA nanostructures (FluoroCubes) as alternative fiducials grafted onto polydimethylsiloxane (PDMS) substrates. Co-anchored with RGD peptides, FluoroCubes remain stably tethered, resist internalization, and enable dense, minimally perturbative labeling. This surface-functionalized platform is compatible with TIRF microscopy and leverages tunable biotin-NeutrAvidin chemistry for precise control of fiducial density. Using a modified multichannel optical flow algorithm, we achieve improved displacement sensitivity and force reconstruction resolution compared with conventional algorithms. FluoroCube-functionalized substrates provide a reproducible, high-resolution method for traction force mapping and offer a versatile foundation for future integration with DNA-based molecular sensors to probe interfacial forces at biointerfaces.