title: Cytosolic NADH-NAD(+) Redox Visualized in Brain Slices by Two-Photon Fluorescence Lifetime Biosensor Imaging.
abstract: 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.
  
  
  
  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.
  
  
  
  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.
  
  
  
  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.
authors: Department of Neurobiology, Harvard Medical School , Boston, Massachusetts.
authors: Rebecca
authors: R
authors: Mongeon
authors: Department of Neurobiology, Harvard Medical School , Boston, Massachusetts.
authors: Veena
authors: V
authors: Venkatachalam
authors: Department of Neurobiology, Harvard Medical School , Boston, Massachusetts.
authors: Gary
authors: G
authors: Yellen
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000378:metabolism / Q000648:ultrastructure; D015374:Biosensing Techniques; D003600:Cytosol / Q000378:metabolism* / Q000648:ultrastructure; D005947:Glucose / Q000378:metabolism; D006019:Glycolysis; D006624:Hippocampus / Q000000981:diagnostic imaging / Q000378:metabolism* / Q000648:ultrastructure; D019344:Lactic Acid / Q000378:metabolism; D051379:Mice; D009243:NAD / Q000302:isolation & purification / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism / Q000648:ultrastructure; D010084:Oxidation-Reduction
chemical_list: D009243:NAD; D019344:Lactic Acid; D005947:Glucose