A viscosity-responsive benzoindole probe targeting the endoplasmic reticulum for applications in cells and tissues.

Real-time monitoring of dynamic changes in endoplasmic reticulum (ER) viscosity is of paramount importance for elucidating the molecular mechanisms underlying various pathological processes, including autophagy and ferroptosis. To address this need, a viscosity-responsive, ER-targeting fluorescent probe 1a was designed and synthesized, based on a benzoindole hemicyanine scaffold. Probe 1a with red emission operated via the intramolecular charge transfer (ICT) mechanism, exhibiting a robust fluorescence response under high-viscosity conditions, along with a high fluorescence quantum yield (62.05% in glycerol) and excellent photostability (remaining absorption > 95%). Cellular imaging studies confirmed that probe 1a selectively localized to the ER with a high Pearson colocalization coefficient of 0.95 at low concentration (500 nM). Moreover, probe 1a was able to visualize ER viscosity elevation in real-time during autophagy and ferroptosis with higher fluorescence of 1.3-1.8-fold and 1.7-fold, respectively. Notably, probe 1a effectively discriminated between cancer and normal cells, and imaging of human tissue sections revealed significantly enhanced fluorescence intensity in cancer regions. Collectively, these findings established probe 1a as a powerful and reliable molecular tool for tracking ER viscosity dynamics, thereby offering substantial potential for advancing research into ER-associated diseases and facilitating diagnostic applications.