In this application, we propose to acquire the first commercial optical tweezers-single molecule super-resolution fluorescence microscope and house it in an open-access, shared-use facility at the University of Michigan (U-M) to serve a broad user base at the U-M and beyond. The proposed LUMICKS C-Trap Dymo will be placed into the well-established Single Molecule Analysis in Real-Time (SMART) instrumentation Center to make it available to the entire biomedical research communities of the U-M, Michigan State University, Oakland University, and the Van Andel Institute across the state of Michigan. The C-Trap will advance a significant number of current and future NIH-funded research programs ranging from fundamental mechanistic studies of protein and RNA folding and function to the role of phase separation in disease, from biophysics to cell biology. New users will be trained and guided in their research by the highly experienced SMART Center Manager Dr. Damon Hoff, who earned his PhD on optical tweezers as applied to biomedical problems. The SMART Center has existed since 2010, has a well-practiced intake, training and affordable recharge system, leverages university support as a Core facility of the Center for RNA Biomedicine, and already serves a broad biomedical research community at Michigan. If funded, the C-Trap will complement existing single molecule instrumentation at SMART and expand the Center's reach, enticing researchers at the U-M and other institutions to utilize the combined optical tweezers- fluorescence microscope as well as other resources. PI Walter (U-M) and key collaborator Comstock (Michigan State University) bring complementary expertise to the proposal – while Walter's expertise lies in single molecule fluorescence microscopy, Comstock is an expert in optical tweezers combined with single molecule fluorescence observation. Comstock's expertise in particular will be leveraged to help guide the research on the C-Trap and subsequent data analysis in the most productive scientific directions. An Advisory Committee of complementary biomedical experts will regularly meet to help optimize use of the C-Trap and further ensure scientific rigor and significance, assisting Walter and Comstock in this task. The proposed state-of-the-art C-Trap is equipped with quadruple laser tweezers, a three-wavelength, single-photon-sensitivity laser scanning confocal fluorescence microscope, and stimulated emission depletion (STED) super-resolution fluorescence capability (i.e., a STED nanoscope), thereby accommodating the broadest range of research. In addition to best-in-class force sensitivity and imaging capabilities, the proposed C-Trap offers a most user-friendly software and experimental workflow among optical trapping systems, allowing novice users to quickly perform sophisticated experiments. Successfully establishing the C-Trap in the SMART Center is expected to advance the goals of 7 Major and 10 Minor, robustly NIH-funded Users, spanni...