A General Strategy for Developing Si-Rhodamine-Based Fluorogenic Dyes for Advanced Bioimaging and Biosensing

Si-rhodamine (SiR) and its derivatives have advanced bioimaging research because of their high photostability and bright near-infrared (NIR) emissions. However, the rational design of SiR derivatives with high fluorogenicity remains challenging. Herein, we report a general strategy for developing high-performance fluorogenic SiR derivatives, termed SFs, by incorporating a molecular rotor at the meso position of the SiR scaffold. This design enables a twisted intramolecular charge transfer (TICT)-based mechanism for fluorescence on/off switching, which results in the achievement of a maximum fluorescence on/off ratio of more than 3000-fold. Chloroalkane (CA)-conjugated SFs exhibit high cellular brightness and outstanding photostability, enabling robust and high-contrast fluorescent labeling of HaloTag in live cells and in vivo. Their suitability for use in stimulated emission depletion microscopy enables advanced fluorescence imaging with subdiffraction resolution in live cells. We demonstrate the ability of SFs to construct chemigenetic Ca 2+ indicators with large dynamic ranges, enabling sensitive detection of intracellular and in vivo Ca 2+ dynamics using both fluorescence intensity and lifetime. This work establishes a general design strategy for NIR fluorogenic dyes, thus opening new avenues for advanced bioimaging and biosensing.