Toward Far-red Emitting Chemogenetic Labelling for Live Cell Super-Resolution Microscopy using Fluorescence-Activating and Absorption-Shifting Tag

Live cell super-resolution microscopy (SRM) imaging is often limited by the lack of optimal fluorescent labelling approaches. Here, we report two new chemogenetic approaches allowing far-red fluorescence detection to label intracellular protein targets to achieve background free SRM images in fixed and live mammalian cells. These methods are based on Fluorescence-activating and Absorption Shifting Tag (FAST), a small monomeric protein tag that forms reversible fluorescent assembly with various rhodanine fluorophores. We developed a Förster resonance energy transfer based (FRET-FAST) method that allows single molecule localization microscopy (SMLM) imaging of proteins in fixed mammalian cells. Additionally, we applied a far-red emitting variant (frFAST) to conduct fixed and live cell SMLM and stimulated emission depletion (STED) microscopy. Time-lapse SMLM up to 40 minutes revealed cytoskeletal dynamics without significant photobleaching. Additionally, we imaged filopodia in living neuroblastoma cells. Moreover, we succesfully imaged mitochondrial outer membrane, microtubules, cytoskeleton and histone proteins in live cell STED microscopy. It is envisioned that the excellent photostability achieved by the presented far-red emitting FAST labelling techniques provides a new alternative for stochastic and deterministic SRM imaging of living cells, even for longer imaging sessions.