GlutaR: A High‐Performance Fluorescent Protein‐Based Sensor for Spatiotemporal Monitoring of Glutamine Dynamics In Vivo

of ~1000 %), and we demonstrate its selectivity for monitoring glutamine fluctuations in multiple cell types. Additionally, using digitonin permeabilization of GlutaR HeLa cells, we generated a calibration curve and performed in situ titration to quantify free glutamine concentrations in subcellular compartments (cytosol, nucleus, mitochondria). Subsequently, we applied GlutaR to investigate how chemical and genetic inhibition of glutamine synthetase (GS) and glutaminase (GLS) differentially alter glutamine levels in subcellular compartments. Finally, we demonstrate GlutaR's ability to monitor dynamic glutamine levels in muscle and liver tissues of diabetic mice in vivo. These findings collectively demonstrate GlutaR as a versatile tool for the spatiotemporal characterization of glutamine metabolism in living cells and tissues.