A Photoswitchable HaloTag for Spatiotemporal Control of Fluorescence in Living Cells

Abstract Photosensitive fluorophores, whose emission can be controlled using light, are essential for advanced biological imaging, enabling precise spatiotemporal tracking of molecular features and facilitating super‐resolution microscopy techniques. Although irreversibly photoactivatable fluorophores are well established, reversible reporters that can be reactivated multiple times remain scarce, and only a few have been applied in living cells using generalizable protein labeling methods. To address these limitations, we introduce chemigenetic photoswitchable fluorophores, leveraging the self‐labeling HaloTag protein with fluorogenic rhodamine dye ligands. By incorporating a light‐responsive protein domain into HaloTag, we engineer a tunable, photoswitchable HaloTag (psHaloTag), which can reversibly modulate the fluorescence of a bound dye‐ligand via a light‐induced conformational change. Our best performing psHaloTag variants show excellent performance in living cells, with large, reversible, deep‐red fluorescence turn‐on upon 450 nm illumination across various biomolecular targets and SMLM compatibility. Together, this work establishes the chemigenetic approach as a versatile platform for the design of photoswitchable reporters, tunable through both genetic and synthetic modifications, with promising applications for dynamic imaging.