Cell wash-free fluorescent probes based on phenothiazine and phenoxazine with high photostability and large stokes shifts for targeted imaging of subcellular organelles

Most organelle-targeting probes require the removal of excess dye to enhance the signal-to-noise ratio before microscopic imaging experiments. However, this washing step may cause cellular damage and interfere with the continuous observation of cellular activities. Here, we report a series of wash-free probes based on small molecule phenothiazine/phenoxazine. Simple modification of phenothiazine/phenoxazine by nitro group significantly enhances the polarity-sensitive characteristic, which are well-suited for wash-free cellular imaging. These probes feature low molecular weight, excellent photostability, and large Stokes shifts (up to 191 nm). By conjugation with targeting groups, a series of probes are developed for specific imaging in different organelles such as lysosomes, mitochondria, the endoplasmic reticulum, lipid droplets, and the plasma membrane, without any washing step. Moreover, by simply replacing S atom in the central core with O atom, the emission of probes shifts from red to green-yellow. Using these two probes, high-contrast, dual-color wash-free imaging can be achieved. Among them, the PXZ-Lipid probe was successfully applied for real-time monitoring of dynamic changes in lipid droplets within living cells. This work establishes a general strategy for designing small-molecule, wash-free fluorescent probes based on phenothiazine/phenoxazine scaffolds, enabling real-time, multi-organelle imaging with minimal cellular disturbance.