期刊
ANTIOXIDANTS & REDOX SIGNALING
卷 25, 期 10, 页码 553-563出版社
MARY ANN LIEBERT, INC
DOI: 10.1089/ars.2015.6593
关键词
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资金
- NIH [DP1 EB016985, R01 NS055031, F32 NS080455]
- David Mahoney Fellowship
Aim: Cytosolic NADH-NAD(+) redox state is central to cellular metabolism and a valuable indicator of glucose and lactate metabolism in living cells. Here we sought to quantitatively determine NADH-NAD(+) redox in live cells and brain tissue using a fluorescence lifetime imaging of the genetically-encoded single-fluorophore biosensor Peredox. Results: We show that Peredox exhibits a substantial change in its fluorescence lifetime over its sensing range of NADH-NAD(+) ratio. This allows changes in cytosolic NADH redox to be visualized in living cells using a two-photon scanning microscope with fluorescence lifetime imaging capabilities (2p-FLIM), using time-correlated single photon counting. Innovation: Because the lifetime readout is absolutely calibrated (in nanoseconds) and is independent of sensor concentration, we demonstrate that quantitative assessment of NADH redox is possible using a single fluorophore biosensor. Conclusion: Imaging of the sensor in mouse hippocampal brain slices reveals that astrocytes are typically much more reduced (with higher NADH: NAD(+) ratio) than neurons under basal conditions, consistent with the hypothesis that astrocytes are more glycolytic than neurons.
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