Advancing Visualization and Quantification of Subcellular and Biomolecular Mechanics through Mechanochemical Protocols - Project Summary Exploring cellular mechanics represents a groundbreaking frontier to unravel the complexities of life and disease. Far from being static entities, cells are dynamic, active materials that generate and sustain mechanical forces, serving as critical indicators for various pathologies. Despite remarkable strides in cell mechanics, we stand in a new era that beckons us to surmount many challenges that have long stymied progress. These challenges span from technological obstacles to limited versatile methodologies, each representing a unique puzzle and challenge in biomechanics. Our MIRA program embarks on an unprecedented journey to address biomechanics challenges by leveraging an interdisciplinary approach that melds the strengths of chemistry and mechanical engineering. The PI's expertise in mechanochemistry, mechanical sensing, and fluorescent microscopy has positioned him as a suitable researcher to use mechanochemical protocols to visualize biomechanical mapping into subcellular and molecular levels. We will address the following challenges: First, the cell mechanics field lacks a versatile technique for quantifying multiscale cell mechanics. Existing methods like Atomic Force Microscopy (AFM) and Fluorescence Resonance Energy Transfer (FRET) offer partial solutions but come with their own sets of limitations, such as intricate setups, specialized expertise, and low throughput. Second, high-precision, real-time force measurements at the nanoscale are missing. While AFM provides nanoscale resolution, its limitations for in vivo applications and the viscoelastic nature of cells add layers of complexity that further complicate data interpretation. Third, the obstacle of accurately measuring in vivo dynamic biomechanical regulations is a significant hurdle in cell mechanics. Existing techniques, although precise, are generally unsuitable for in vivo applications due to their invasive nature and complex setups. The dynamic milieu of living systems further complicates the issue, demanding rapid and continuous measurements. These challenges are not mere obstacles; they are opportunities, beckoning us to innovate, explore, and revolutionize biomechanics. As we embark on this exhilarating journey, we are not just solving puzzles but pushing the boundaries of what is possible, opening new avenues for research and clinical applications. This MIRA program will establish a pioneering analytical platform for biomechanics, from building a comprehensive library of Molecular Force Gauges (MFGs), integrating these force gauges into various biological targets for precise biomechanical measurements, to developing a state-of-the-art super-resolution biomechanical microscopy BM-STORM to visualize and quantify cellular and subcellular biomechanics with unprecedented detail. Our research agenda will be based on a three-fold approach. First, we will pioneer a comprehensive library of Molecular Force Gauges (MFGs) designed to offer unprecedented insights into cellular and subcellular mechanics. These MFGs are ingeniously engineered to undergo optical or luminescent transformations in response to mechanical forces, serving as real-time, nanoscale force sensors. Our approach addresses the limitations of existing technologies like flipper probes and FRET, offering a more direct and versatile method for force quantification. Second, we will utilize these MFGs to sense both compressive and tensile forces across various biological targets. This dual-mode operation enriches our understanding of membrane mechanics and offers a robust and reliable metric for tension measurements. Third, we will develop an innovative super-resolution biomechanical microscopy, BM-STORM, to visualize and quantify cellular and subcellular biomechanics with unprecedented detail. This system leverages mechanical force to control the switching between bright and dark states of MFGs, offering a biomechanical counterpart to Stochastic Optical Reconstruction Microscopy
