Spectrally separated fluorophores allow the observation of multiple targets simultaneously inside living cells, leading to a deeper understanding of the molecular interplay that regulates cell function and fate. Chemogenetic systems combining a tag and a synthetic fluorophore provide certain advantages over fluorescent proteins since there is no requirement for chromophore maturation. Here, we present the engineering of a set of spectrally orthogonal fluorogen-activating tags based on the fluorescence-activating and absorption shifting tag (FAST) that are compatible with two-color, live-cell imaging. The resulting tags, greenFAST and redFAST, demonstrate orthogonality not only in their fluorogen recognition capabilities, but also in their one- and two-photon absorption profiles. This pair of orthogonal tags allowed the creation of a two-color cell cycle sensor capable of detecting very short, early cell cycles in zebrafish development and the development of split complementation systems capable of detecting multiple protein–protein interactions by live-cell fluorescence microscopy.

光谱分离的荧光团允许同时观察活细胞内的多个目标，从而更深入地了解调节细胞功能和命运的分子相互作用。结合标签和合成荧光团的化学遗传学系统提供了优于荧光蛋白的某些优势，因为不需要发色团成熟。在这里，我们提出了一组基于荧光激活和吸收移位标签 (FAST) 的光谱正交荧光激活标签的工程，这些标签与双色活细胞成像兼容。由此产生的标签，greenFAST 和 redFAST，不仅在它们的荧光识别能力方面表现出正交性，而且在它们的单光子和双光子吸收曲线上也表现出正交性。