Glutamine is the most abundant amino acid in serum, used as a key nutrient by cells for protein synthesis, energy production, carbon and nitrogen metabolism, and cellular redox balance. The use of glutamine in the cell is highly compartmentalized, but the dynamics of glutamine metabolism across organelles and individual cells are not fully understood. To illuminate subcellular glutamine dynamics, we developed an intracellular glutamine optical reporter, iGlo. We find iGlo is sensitive and specific for glutamine and can be used to measure glutamine uptake, production, and consumption with high spatiotemporal resolution in multiple cell types. Furthermore, multiplexed imaging of iGlo with a lactate biosensor in single cells reveals temporal crosstalk between glucose and glutamine metabolism to maintain energy homeostasis. Thus, iGlo enables the sensitive and precise study of compartmentalized glutamine dynamics and represents a new and enhanced tool for studying the spatiotemporal dynamics and regulation of metabolism. ### Competing Interest Statement The authors have declared no competing interest. National Institutes of Health, https://ror.org/01cwqze88, T32GM145388, P30CA016042, R35GM138003, DP2GM154012 United States Department of Energy, https://ror.org/01bj3aw27, DE-FC02-02ER63421 UCLA Jonsson Comprehensive Cancer Center, https://ror.org/0599cs764, Strategic Plan Aligned Grant, Graduate Research Award Chan Zuckerberg Initiative (United States), https://ror.org/02qenvm24, MET-000000000151

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