Project Summary Cells are highly dynamic, squeezing, pulling, and tugging on their surroundings and on each other. Each individual interaction involves forces. These forces are felt by specific receptors and molecules. Although small in magnitude (pN), these molecular forces can have profound biological impacts in many aspects of cellular life including the fate of differentiating stem cells, cell division, cancer metastasis, and blood clotting. Therefore, the ability to characterize the interplay between physical forces and biochemical signals is a critical component of understanding signaling pathways in living systems. There are two main techniques used to study molecular mechanobiology: single molecule force spectroscopy (SMFS) and traction force microscopy (TFM) based methods. While powerful, these approaches suffer from several drawbacks. SMFS measures individual receptor forces (pN), but it does so only one molecule at a time. Conversely TFM provides spatial maps of cellular forces, but on the nN scale, orders of magnitude larger than the forces applied by individual cell receptors. To bridge these approaches, we invented molecular tension fluorescence microscopy (MTFM) which uses conventional fluorescence microscopy to map cellular forces with pN resolution by using a calibrated molecular force probe. The goal of this proposal is to transform the capabilities of MTFM allowing orders of magnitude improvement in spatial and temporal resolution as well as the mapping of force orientation. Molecular mechanobiology remains at the fringes of biomedical sciences because of the lack of tools to precisely quantify and link mechanics to cellular biochemistry. Our goal is to transform the field of molecular mechanobiology by developing new imaging technologies to enable the study cellular forces at unprecedented resolution. These technologies, centered around the DNA-based MTFM probes, will provide a broadly applicable platform of technology to investigate molecular mechanics, and the functional outcomes of molecular forces, in diverse biological systems. In Aim 1 we will address the spatial resolution gap, and leverage the DNA-based force probes to develop super-resolution force-PAINT with the goal of dynamic force imaging with 20 nm spatial resolution. In Aim 2 we will probe the dynamics of forces and force fluctuations by harnessing the power of two approaches, FRAP and FCS, to study molecular force dynamics with nsec to msec time resolution. Finally, in Aim 3 we will leverage fluorescence polarization microscopy to measure the 3D orientations of molecular forces. We will use fibroblast focal adhesions, platelet activation and coagulation, and T cell antigen recognition to test and verify our approach. Accomplishment of these goals will provi...