Quantification of extracellular glutamate levels using fluorescence lifetime imaging of the red genetically encoded biosensor R^ncp-iGluSnFR1

Glutamate is the major excitatory neurotransmitters in the mammalian nervous system. Dynamic and appropriate glutamate release is critical for the physiological functions of the nervous system. On the other hand, dysregulated glutamatergic neurotransmission accounts for a variety of disorders ranging from neuropsychiatric to neurodegenerative diseases. Due to the importance of glutamate in both basic and translational neuroscience research, it is urgent to develop tools for quantitative measurement of extracellular glutamate levels with high spatiotemporal resolution. In contrast to the fluorescence intensity-based methods in monitoring dynamic change of glutamate release, here we report a fluorescence lifetime-based quantification method for measuring extracellular glutamate levels. We reveal that R^ncp-iGluSnFR1, an existing genetically encoded glutamate biosensor, exhibits a large fluorescence lifetime response (∼0.6 ns) and a low micromolar affinity (∼5.9 μM) in vitro. The fluorescence decays of R^ncp-iGluSnFR1 comprise two lifetime components (∼0.6–1.0 ns and ∼ 2.3–3.0 ns), both of which were significantly decreased upon glutamate binding. We further used fluorescence lifetime imaging microscopy (FLIM) to quantitatively measure extracellular glutamate levels after expression of R^ncp-iGluSnFR1 at mammalian cell membrane. Intriguingly, the binding affinity of glutamate with R^ncp-iGluSnFR1 at cell membrane reached ∼ 1 μM, which is significantly higher than that in vitro. In addition, the reduction of fluorescence lifetime of R^ncp-iGluSnFR1 in response to glutamate occurs rapidly. Overall the fluorescence lifetime-based method presented here may provide a powerful research tool for studying glutamate dynamics in neurobiology.