Cellular Force Nanoscopy with 50 nm Resolution Based on Integrin Molecular Tension Imaging and Localization

Integrin-transmitted cellular forces have rich spatial dynamics and are vital to many cellular functions. To advance the sensitivity and spatial resolution of cellular force imaging, we developed a force-activatable emitter reporting single-molecular tension events and the associated cellular force nanoscopy (CFN). Immobilized on a surface, the emitters are initially dark (>99.8% quenched), providing a low fluorescence background despite the high coating density (>2000/μm²) required for sampling cellular force properly. The emitters fluoresce brightly once switched on by integrin tensions and can be switched off by photobleaching, enabling continuous real-time imaging of integrin molecular tensions in live cells. With multiple cycles of molecular tension imaging and localization, CFN reproduces cellular force images with 50 nm resolution. Applied to both migratory cells and stationary cells, CFN revealed ultranarrow distribution of integrin tensions at the cell leading edge, and showed that force distribution in focal adhesions (FAs) is off-centered and FA size-dependent.