Whole‐cell Lysosome SMLM Imaging as Indicators for Functional Diagnostics with a Low‐Phototoxic Spontaneously Blinking Probe

Lysosomal morphology and pH dynamics are closely linked to lysosome functions, making long‐term single‐molecule localization microscopy (SMLM) imaging of whole‐cell lysosomes a potential indicator for functional diagnostics. However, the phototoxicity of probes in SMLM imaging often compromises the reliability of the observed lysosomal dynamics. Here, we developed Aze‐HMSiR, a spontaneously blinking silicon rhodamine probe with near‐infrared excitation, enabling low phototoxicity, long‐term SMLM imaging of lysosomal morphology, and pH dynamics for up to 50 minutes. This probe enables super‐resolution imaging of key lysosomal characteristics—distribution, size, and lumen pH—critical for understanding lysosomal dynamics and their physiological and pathological roles. By analyzing these parameters, we investigated lysosomal function under acidosis and starvation and evaluated the effects of seven anticancer drugs (paclitaxel, rapamycin, periplocoside, metformin, erastin, polyphyllin and gefitinib). Our results revealed that periplocoside significantly reduced lysosomal size, while other drugs generally induced an increase. Notably, paclitaxel elevated lysosomal pH and led to a sparser lysosomal distribution. These findings underscore the potential of Aze‐HMSiR as a powerful tool for diagnosing lysosomal function and monitoring dynamic changes in response to physiological conditions and pharmacological treatments, establishing it as a robust platform for functional diagnostics and drug screening, particularly in cancer therapy.