Research Projects

The advent of super-resolution imaging techniques has fundamentally transformed fluorescence microscopy by overcoming the diffraction limit, enabling visualization of molecular structures at nm-scale resolution. When combined with molecule-specific labeling, this capability provides a powerful tool to unravel biological complexity and disease pathology. Central to these nanoscopy techniques is the precise optical control over fluorophore emission, often achieved using photoactivatable or caged dyes that switch from a non-fluorescent (off) to a fluorescent state (on) upon light exposure. This light-controlled off-to-on transition, which allows the stochastic activation of sparse subsets of fluorophores over time, is essential for resolving closely spaced molecules at nanometre-scale proximities. This principle lies at the heart of Photoactivated Localization Microscopy (PALM), a powerful single-molecule localization technique that enables super-resolution imaging of biological systems with exceptional spatial resolution and allows real-time tracking of individual molecules with unprecedented precision. Despite its transformative potential, PALM remains constrained by limitations in probe design, which heavily rely on the synthetic incorporation of photolabile protecting groups into the fluorophore structure that function as fluorescence quenchers. Unfortunately, these modifications increase molecular complexity, requiring bottom-up fluorophore redesign to accommodate core structural changes and address associated synthetic challenges. In addition, such structural alterations can negatively impact the photophysical performance of fluorophores and compromise fluorophore stability in biological conditions. More importantly, as the quenching mechanism is not generalizable to diverse libraries of fluorophore families, it requires extensive design efforts to customize quenching mechanisms for each fluorophore scaffold, often placing the red and far-red dyes beyond reach for PALM imaging. To overcome these limitations, we propose a modular and broadly generalizable strategy based on synthetic host–guest recognition that will readily transform any off-the-shelf fluorophore into a PALM-compatible probe without the need for bottom-up redesign of the dye structure. This approach introduces a fundamentally distinct mechanism for fluorescence off-to-on switching, replacing dye-specific quenching strategy with a universal, light-triggered host–guest recognition mechanism that is easily transferrable across fluorophore classes. In our design, fluorescence switching is achieved not by altering the fluorophore itself, but through the optically gated, ultrahigh-affinity binding of a fluorescently labelled cucurbituril host molecule (“imager”) to a photocaged guest ligand (“docking site”) attached to the target biomolecule. Prior to photoactivation, the imager remains unbound and freely diffusing, contributing only background fluorescence (off-state). Upon light-induced uncaging of the guest ligand, the host imager rapidly binds to the target through specific, ultrahigh-affinity host–guest complexation, generating a localized fluorescence signal (on-state) that enables single-molecule localization and facilitates both nanoscopic imaging and tracking of individual biomolecules. Overall, this strategy represents a transformative advance in PALM imaging, offering a plug-and-play platform that eliminates fluorophore-specific design constraints, dramatically simplifies multicolor probe development, and expands high-resolution imaging capabilities using readily available fluorophores. We will benchmark this approach using spatially programmed DNA origami nanostructures and will extend it to 3D volumetric imaging in live cells via antibody, metabolic, SNAP/CLIP-tag, and small-molecule targeting strategies. Finally, we will use this platform for nanoscale mapping of therapeutic antibody–receptor interactions to elucidate their mechanism.