Multiphoton Histology | Plants | Device and Crystal Analysis
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Brain, Nervous System, Tissue, Continued Fundamental
Aspects of our brain imaging projects: |
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| NADH
(beta-nicotinamide adenine dinucleotide) The importance of energy metabolism for brain function: "At times of peak activity, some regions in the CNS use as much energy as any other tissue in the body, including striated muscle." (Adelbert Ames III). It is widely believed that the brain depends almost entirely on oxidative energy metabolism for fulfilling its energy requirements. Some basic bioenergetics: The oxidation of fuel molecules (glucose, ketone-bodies, fatty acids) provides free energy for aerobic organisms. However, electrons from the fuel molecules are not directly transported to the final electron acceptor oxygen. Instead the fuel molecules are being broken down to intermediate metabolites which donate electrons to special carriers, pyridine nucleotides or flavins. These electron carriers then feed the mitochondrial electron-transport chain (ETC) for ATP-synthesis. The reduced
pyridine nucleotide NADH is the principal electron donor for the
respiratory chain in mammalian cells. The oxidation of NADH by the
electron transport chain is coupled to the phosphorylation of ADP
by ATP synthase. The relationship between the rate of ATP production
and the ratio of reduced NADH to oxidized NAD+, known as the redox
ratio, allows the metabolic state of a cell to be measured by its
NADH/NAD+ ratio. When NADH donates
2 electrons to complex I of the respiratory chain, it gets oxidized
to NAD+. Fortunately, NADH is fluorescent whereas NAD+ is not,
hence the NADH/NAD+ ratio can be measured using fluorescence techniques.
Britton Chance first utilized the intrinsic NADH fluorescence in
living cells to measure metabolic states in single cells, tissue
explants and the in vivo brain and heart - this technique
came to be known as redox-fluorimetry. |
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change in the molecular structure of NADH upon oxidation. The structural
change results in the loss of fluorescence of the molecule. The diagram
shows the reactive site only (fortunately, the reactive site happens
to be the fluorescent site as well). Our research project: We
apply two-photon microscopy and spectroscopy to conventional redox-fluorimetry
of the brain, extending the pioneering work of Britton Chance (one-photon
redox fluorimetry) and David Piston (two-photon redox-fluorimetry).
Our goal is to develop two-photon redox fluorescence microscopy
into a functional metabolic imaging technique. The inherent high
spatial resolution of two-photon microscopy enables the visualization
of metabolic states within single neurons and subcellular mitochondrial
populations deep within living brain tissue. This spatial resolution
far exceeds that of previous techniques. |
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