Viscosity is a pivotal factor for indicating the dysfunction of the mitochondria. To date, most of the fluorescent probes developed for mitochondrial viscosity have been designed using BODIPY, hemicyanine, or pyridine-based molecular rotors as part of the core structure. Our aim with this research was to extend the range of suitable fluorophores available for the construction of such fluorescent molecular rotors for evaluating the viscosity of mitocondria. Herein, we have developed a green fluorescent protein (GFP)-chromophore-based fluorescent probe (MIT-V) for the detection of mitochondrial viscosity in live cells. MIT-V exhibited a high sensitivity toward viscosity (from 7.9 cP to 438.4 cP). The “off–on” sensing mechanism of MIT-V was ascribed to the restricted rotation of single bonds and excited-state CC double bonds of MIT-V. Cell studies indicated that MIT-V targets the mitochondria and that it was able to monitor real-time changes in the viscosity of live HeLa cell mitochondria. Therefore, we propose that MIT-V can be used as an effective chemosensor for the real-time imaging of mitochondrial viscosity in live cells. Our results clearly demonstrate the utility of such GFP-chromophore-based derivatives for the development of viscosity-sensitive systems.

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The occurrence of endoplasmic reticulum (ER) stress is the main cause of a variety of biological process that are closely related with numerous diseases. The homeostasis of the ER microenvironment...