Genetically Targetable and Color‐Switching Fluorescent Probe

Genetically expressed fluorescent proteins (FPs)[1–3] revolutionized the field of cell biology, especially enabling live-cell studies.[4–16] The limitations of FPs however are related to the photophysical properties of their fluorophores, mainly brightness and photostability, which are still are inferior to organic fluorophores.[17–19] Hybrid tagging strategies, such as SNAP-tag[20–22] and HaloTag[23], as well as genetic encoding of fluorescent amino acids[24] are alternatives to FPs that allow genetic targeting of synthetic dyes.[25] Fluorogen activating proteins (FAPs) belong to a recently developed hybrid-tagging strategy that allows target-specific activation of the fluorescence of otherwise nonfluorescent dyes (fluorogens)[26] by hundreds-to-thousands fold. FAPs that bind derivatives of Malachite Green (MG) displayed the greatest fluorogenic activation, up to 20000-fold, primarily because of an extremely low unbound dye fluorescence in water.[26] The high specificity and extremely low background signal from FAP tagging were shown to be very useful in fluorescence microscopy of live cells[27] including super-resolution STED imaging.[28]