Multicolor conditional labeling is a powerful tool that can simultaneously and selectively visualize multiple targets for bioimaging analysis of complex biological processes and cellular features. We herein report a multifunctional stimuli-responsive Fluorescence-Activating and absorption-Shifting Tag (srFAST) chemogenetic platform for multicolor cell-selective labeling. This platform comprises stimuli-responsive fluorogenic ligands and the organelle-localizable FAST. The physicochemical properties of the srFAST ligands can be tailored by modifying the optical-tunable hydroxyl group with diverse reactive groups, and their chemical decaging process caused by cell-specific stimuli induces a conditionally activatable fluorescent labeling upon binding with the FAST. Thus, the resulting switch-on srFASTs were designed for on-demand labeling of cells of interest by spatiotemporally precise photo-stimulation or unique cellular feature-dependent activation, including specific endogenous metabolites or enzyme profiles. Furthermore, diverse enzyme-activatable srFAST ligands with distinct colors were constructed and simultaneously exploited for multicolor cell-selective labeling, which allow discriminating and orthogonal labeling of three different cell types with the same protein tag. Our method provides a promising strategy for designing a stimuli-responsive chemogenetic labeling platform via facile molecular engineering of the synthetic ligands, which has great potential for conditional multicolor cell-selective labeling and cellular heterogeneity evaluation.
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