Rational design of pH probes with large Stokes shift for tracking lysosome-mitochondria interactions

Tracking the interactions of lysosomes with other organelles by fluorescence imaging is the key to understanding cellular homeostasis. Herein, we focus on the problem of fluorescence crosstalk caused by the small Stokes shift in lysosomes targeted pH probes and introduce rhodamine dyes with large Stokes shift to construct pH probes. In order to accurately construct a probe suitable for lysosomal pH, we established the relationship between the atomic dipole moment-corrected Hirshfeld population (ADCH) charge of rhodamines and pKa values of the corresponding pH probes. The large Stokes shift rhodamine DQF-584 was predicted and verified as the suitable dye to construct lysosomal pH probe. The photostable probe DQF-pH with large Stokes shift of 91 nm could effectively localize in lysosomes and applied in tracking the dynamics of lysosome-mitochondrial interactions.