Quantitative Super‐Resolution Imaging of Molecular Tension

DNA-based molecular tension probes have revolutionized the localization of mechanical events in live cells with super-resolution. However, imaging the magnitude of these forces at super-resolution has been challenging. Here, qtPAINT (quantitative tension points accumulation for imaging in nanoscale topography) is introduced as a strategy to image the magnitude of molecular tension with super-resolution accuracy. By leveraging the force-dependent dissociation kinetics of short DNA oligonucleotides on their complementary strands, tension is encoded on individual molecules through their binding kinetics. This method allowed for a quantitative analysis of these kinetics, providing a detailed reconstruction of the force magnitudes acting on each tension probe. The technique integrates a molecular-beacon PAINT imager with a hairpin molecular tension probe, achieving a force quantification range of 9-30 pN and maintaining a spatial resolution of 30-120 nm in low and high-density regions. Additionally, qtPAINT offers a temporal resolution on the order of a minute, enhancing its applicability for studying dynamic cellular processes.