A red-emitting, microenvironment-insensitive fluorophore for lysosome-specific imaging in live cells

Lysosomes and the endoplasmic reticulum (ER) are vital for cellular homeostasis, degradation, and signaling, making them key imaging targets. However, existing fluorescent probes suffer from limitations such as pH sensitivity, poor photostability, and cytotoxicity. To overcome these challenges, we developed two red-emitting fluorophores, DM and MM, based on a rigid DCM scaffold with morpholine linkers. DM rapidly localizes to lysosomes within 10 minutes, exhibiting exceptional photostability, pH insensitivity, and resilience in live and fixed cells. MM initially targets the ER before redistributing to lysosomes, enabling studies of inter-organelle dynamics and lysosomal maturation. Both probes, excitable at 561 nm, emit in the red spectral region, reducing autofluorescence and phototoxicity while allowing deep tissue imaging. DM efficiently tracks lysosomal dynamics under normal and stressed conditions, including mitophagy and lysosome-mitochondria interactions. MM's dual-targeting behavior provides insights into ER-lysosome crosstalk, crucial for cellular signaling. Both dyes exhibit negligible cytotoxicity (up to 100 μM), ensuring prolonged imaging without disrupting the cellular function. Their rigid scaffold imparts high stability, making them versatile tools for studying lysosomal and ER-associated processes. DM and MM set a new standard for dynamic organelle imaging, advancing biomedical research on lysosomal biology and disease mechanisms.