Abstract

Mitochondria are cell-dynamic physicochemical compartments whose internal microenvironment (defined by parameters such as local polarity and molecular crowding) undergoes distinct microenvironmental responses under oxidative stress. Here we report MitoDAN, a cationic DAN-based fluorophore designed for preferential mitochondrial accumulation, whose intramolecular charge-transfer (ICT) character confers inherent dual sensitivity to local polarity and molecular crowding. Comprehensive photophysical characterization (including steady-state solvatochromism, Lippert-Mataga analysis, and time-resolved fluorescence measurements) confirms strong ICT-dependent environmental sensitivity and multiexponential excited-state dynamics. Benchmarking in lipid membrane models and macromolecular crowding systems establishes the spectral phasor response of MitoDAN to distinct physicochemical variables. Colocalization and spectral phasor analyses support predominant mitochondrial localization in live U2OS and HeLa cells. HSI and FLIM phasor analyses reveal that sodium arsenite-induced oxidative stress decreases molecular crowding while increasing local polarity. MitoDAN also detects infection-associated crowding changes in -infected HeLa cells, extending its application to biologically induced host-cell remodeling.