Hydrogel-based molecular tension fluorescence microscopy for investigating receptor-mediated rigidity sensing

Extracellular matrix (ECM) rigidity serves as a crucial mechanical cue impacting diverse biological processes. However, understanding the molecular mechanisms of rigidity sensing has been limited by the spatial resolution and force sensitivity of current cellular force measurement techniques. Here we developed a method to functionalize DNA tension probes on soft hydrogel surfaces in a controllable and reliable manner, enabling molecular tension fluorescence microscopy for rigidity sensing studies. Our findings showed that fibroblasts respond to substrate rigidity by recruiting more force-bearing integrins and modulating integrin sampling frequency of the ECM, rather than simply overloading the existing integrin-ligand bonds, to promote focal adhesion maturation. We also demonstrated that ECM rigidity positively regulates the pN force of T cell receptor-ligand bond and T cell receptor mechanical sampling frequency, promoting T cell activation. Thus, hydrogel-based molecular tension fluorescence microscopy implemented on a standard confocal microscope provides a simple and effective means to explore detailed molecular force information for rigidity-dependent biological processes.