Fluorescence Lifetime Multiplexing with Environment‐Sensitive Chemigenetic Probes

HaloTag (HT) is a versatile self‐labeling protein that has been widely adopted in fluorescence microscopy. Besides its established use as an intensity‐based marker and sensor, interest in using HT for multiplexed fluorescence lifetime imaging microscopy (FLIM) has recently arisen. Herein, the application of an environment‐sensitive fluorophore was explored for FLIM multiplexing with the free dye and HT mutants. The extended coumarin pyridinium scaffold was selected due to its structural simplicity and the strong sensitivity of its photophysical properties to the environment. It was demonstrated that three‐channel imaging is possible by taking advantage of the propensity of the dye to accumulate in mitochondria and vesicles and the efficient labeling of two distinct HT mutants. Further investigation was conducted on different dehalogenase proteins and their FLIM multiplexing capabilities when paired with an HT mutant. Finally, the polarity of the protein binding pocket was identified as a key feature that affects the lifetime of this kind of fluorescent molecule.