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Real-time monitoring of cell membrane viscosity in a 3D hydrogel cultivation microenvironment using BODIPY-based fluorescent probe

Shi Junli,Chong Hui,Yang Xiaofei,Zhong Guangjie,Wu Shengnan,Gu Zehao,Tao Qi,Wang Dong-An,Xue Huaiguo,Yang Yi,Yao Hang

This study reports a viscosity-responsive fluorescent probe, BODIPY-C, designed to measure viscosity in a 3D live-cell hydrogel microenvironment using a fluorescence lifetime-based method. This method enables real-time monitoring of cell membrane viscosity changes through the fluorescent lifetime signals of the probe. Functionally, the BODIPY-C exhibits microviscosity sensitivity; the non-toxic probe enables long-term tracking with high sensitivity ( R 2  = 0.99). Structurally, the BODIPY-C features polymerizable vinyl groups that facilitate covalent conjugation to hydrogel networks via UV-initiated polymerization. This design synergistically integrates the molecular specificity of BODIPY-C-based viscosity sensing with the mechanobiology of covalent hydrogel networks. Notably, softer 3D hydrogel microenvironments extend the probe’s fluorescence lifetime due to restricted molecular motion from differential elastic collisions between polymer chains. The covalent anchoring of BODIPY-C within hydrogel networks enables in situ monitoring of viscosity dynamics in encapsulated cells, establishing a promising platform for investigating mechanobiological processes. Graphical abstract