A ratiometric fluorescent probe for simultaneous detection of pH and viscosity during cell death processes in live cells and in fat tissue of Drosophila melanogaster larvae.

Cell death is essential for maintaining health and development of an organism. Although cell death is not inherently detrimental, processes such as apoptosis, autophagy, and ferroptosis are accompanied by significant alterations in the intracellular microenvironment, including changes in pH and viscosity. However, the simultaneous monitoring of pH and viscosity remains challenging and has not yet been demonstrated in Drosophila melanogaster (Dm) third-instar larvae, a physiologically relevant model of developmentally programmed starvation-induced autophagy. Herein, we report PSM-NAP-OH, a dual-responsive fluorescent probe capable of ratiometric pH sensing and turn-on viscosity response. The probe integrates a dihydroxy-functionalized pentacyclic pyridinium framework for ratiometric pH response and a naphthyl rotor as viscosity-sensitive unit through a restricted intramolecular rotation (RIR). It primarily localizes in mitochondria and enables simultaneous monitoring of pH and viscosity changes during apoptosis, ferroptosis, and autophagy in living cells, as well as during autophagy in Dm third-instar larval tissues. Confocal fluorescence imaging studies revealed distinct pH and viscosity profiles for different cell death pathways. Notably, this work represents the first demonstration of simultaneous visualization of pH and viscosity alterations associated with developmentally programmed starvation-induced autophagy in the fat tissue of Dm larvae. These findings establish pH and viscosity as complementary biomarkers of autophagy and provide a new platform for investigating dynamic microenvironmental changes during development and disease.