Massively parallel bead-free force spectroscopy with fluorescence.

Single-molecule force spectroscopy (SMFS) has transformed our understanding of biomolecular mechanics. However, current high-throughput implementations rely on beads to apply force, introducing size and surface chemistry variability, requiring per-bead calibration, and are prone to multitether artifacts. Long handles further complicate measurements by convolving target conformational changes with handle stretching. We introduce tether force spectroscopy (TFS), a bead-free SMFS platform in which a single DNA tether serves as both the force applicator and an internal calibrator. In TFS, shear flow acting on identical DNA tethers applies piconewton-scale forces directly to surface-anchored molecules whose conformational dynamics are simultaneously monitored by single-molecule fluorescence. This guarantees single-tether results with uniform, internally calibrated forces and is inherently compatible with single-molecule fluorescence. We achieved high-resolution, high-throughput measurements across hundreds of molecules, enabling both force-extension and rupture experiments without specialized instrumentation. The combination of simplicity and simultaneous force-fluorescence capability makes TFS broadly accessible for correlating structure and function in diverse biomolecular systems.