Developing Molecular Tools to enhance Fluorescent Live Cell Nanoscopy

RESOLFT (Reversible Saturable Optical Fluorescence Transitions) microscopy enables advanced super resolution live-cell imaging but faces challenges related to photobleaching and the limited availability of high-performance reversibly switchable fluorescent proteins (rsFPs). This thesis tackles these challenges through two approaches. Chapter 1 presents a molecular monolayer-platform for studying the binding kinetics of fluorescent probes, employing the ALFA-tag and Nanobody system to investigate the binding and unbinding dynamics of lower-affinity ALFA mutants. Lower-affinity interactions between the fluorophore and target can enable the replacement of photobleached probes, thereby extending imaging durations. While achieving consistent single molecule distributions and precise affinity measurements proved challenging, the chapter outlines strategies to improve the technique for extended imaging. Chapter 2 explores novel red rsFPs, focusing on rsScarlet variants designed for RESOLFT microscopy. Photophysical evaluations identified an rsScarlet mutant as a promising candidate with improved switching performance and increased resistance to fatigue. Structural analysis revealed mutations essential to its functionality, providing valuable insights into protein design. These findings address technical obstacles in live-cell imaging and contribute new molecular tools for studying dynamic cellular processes on the nanometer scale.