Related Experiment Video
Updated: Mar 26, 2026
Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence
Published on: February 21, 2012
Cytosolic NADH-NAD(+) Redox Visualized in Brain Slices by Two-Photon Fluorescence Lifetime Biosensor Imaging
Rebecca Mongeon1, Veena Venkatachalam1, Gary Yellen1
1Department of Neurobiology, Harvard Medical School , Boston, Massachusetts.
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. Antioxid. Redox Signal. 25, 553-563.
