An Engineered Supramolecular Fluorescent Chemosensor for Multiscale Visualization of Glutamate Dynamics in Living Systems

Glutamate (Glu) plays a critical role in the brain, and the ability to directly measure glutamate activity is essential for understanding its physiological functions and pathological processes. Herein, we engineered a family of Glu sensors (TympG_n) based on host–guest interactions through the indicator displacement method (IDA) strategy. The optimized supramolecular chemosensor TympG₂ exhibited specificity, sensitivity, signal-to-noise ratio, rapid kinetics (∼145 ms), and photostability, enabling it to be suitable for monitoring Glu dynamics in neuronal organelles, brain tissues, and zebrafish. Significantly, we established the first Glu-associated functional network map across 24 deep brain regions using a homemade high-density fiber photometry array. It was discovered that the correlations between adjacent brain regions, especially between cortex, hippocampus and thalamic populations, were significantly reduced in the hypoxic mice compared to normal mice. Overall, TympG₂ enabled spatiotemporally resolved quantification of glutamatergic activity ranging from subcellular compartments to expansive neural networks across various model organisms. This comprehensive approach not only enhances our understanding of Glu dynamics but also aids in identifying potential neurological disruptions associated with altered glutamatergic signals.