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Title: Quantitative two-photon redox fluorescence microscopy of neurometabolic dynamics
Authors: Vishwasrao, Harshad
Keywords: spectroscopy
Issue Date: 13-Dec-2004
Abstract: The fluorescence of the intracellular electron donor reduced nicotinamide adenine dinucleotide (NADH) is a well established probe of cellular metabolic state. This technique, called redox fluorimetry, can be combined with two-photon microscopy to provide functional imaging deep in living neural tissue with a spatio-temporal resolution far exceeding that of conventional functional imaging techniques (e.g. fMRI, PET). This resolution offers a new opportunity to explore spatio-temporal heterogeneities in the response of neural tissue to stressors such as metabolic inhibition and activity induced metabolic load. These metabolic responses were found to differ between cell types in the brain (astrocytes and neurons) and between sub-cellular compartments (mitochondria and cytosol). The time course of compartmental responses revealed that transient hypoxia caused an NADH increase followed by a post-hypoxic mitochondrial NADH hyperoxidation and a cytosolic lactate accumulation. A similar analysis of the time course of electrical activity induced metabolic responses revealed that the metabolic cost of neural activity was first met by oxidative neuronal metabolism followed by glycolytic metabolism in astrocytes. The accuracy of redox-fluorimetry is limited however, by the effect of the local environment on the fluorescence of intracellular NADH. To characterize the effect of the intracellular environment on the photophysical properties of intracellular NADH, we measure its time resolved fluorescence and rotational anisotropy decays. These decays characterize the excited state and rotational dynamics of intracellular NADH, and from them we can infer how metabolic inhibition affects the enzyme binding states of NADH and the local viscosity for NADH rotational motion. The net effect is a reduction of the average lifetime of intracellular NADH fluorescence upon metabolic inhibition, causing the fluorescence increase during inhibition to underestimate significantly the actual concentration increase. Historically, measurements of the NADH response to metabolic perturbations have not significantly described the complexity of this response. The research presented here shows that metabolism associated changes of intracellular NADH are not only spatio-temporally heterogeneous, but also entail changes in the NADH conformation and its photophysical properties. By characterizing and accounting for these effects, we make progress towards quantitative redox-fluorimetry and the development of an accurate picture of in vivo NADH dynamics.
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