Viscosity-sensitive fluorescent probes based on the hemicyanine for the organelle-specific visualization during...

The dynamic monitoring of cell death processes remains a significant challenge due to the scarcity of highly sensitive molecular tools. In this study, two hemicyanine-based probes (5a-5b) with D-π-A structures were developed for organelle-specific viscosity monitoring. Both probes exhibited correlation with the Förster-Hoffmann viscosity-dependent relationship (R² > 0.98) and emitted near-infrared (NIR) fluorescence (652-666 nm). Key advantages of these probes included good photostability (retention over 92 %), minimal cytotoxicity (cell viability over 90 %), and an OFF-ON response to viscosity changes, confirming their suitability for continuous monitoring from physiological to pathological conditions. Moreover, probe 5a selectively targeted mitochondria (Pearson correlation coefficient (Pr) of 0.90) in a manner independent of mitochondrial membrane potential (MMP), while probe 5b localized to lysosomes (Pr = 0.90) independent of pH. In starvation and drug-induced autophagy models, probe 5a exhibited a 1.9-2.3-fold increase in fluorescence intensity. Furthermore, 5a monitored the gradual fusion of mitochondria with lysosomes, achieving a maximum colocalization coefficient of 0.78. Probe 5b demonstrated a 1.8-2.4-fold enhancement in fluorescence intensity during lysosomal autophagy. Additionally, in the ferroptosis process, probes 5a-5b exhibited 3.4-fold and 2.4-fold increase in fluorescence intensity in HeLa cells, showcasing the probes' real-time monitoring capabilities. These findings establish 5a-5b as versatile tools for studying autophagy and ferroptosis via mitochondrial and lysosomal viscosity dynamics, respectively.