In live tissues, alterations in metabolism induce changes in the fluorescence decay of the spectrally identical redox carriers NADH and NADPH. The biochemical pathways and photophysical mechanisms that contribute to these changes are largely unknown. This work combined ultrafast laser spectroscopy and live-cell imaging to investigate these phenomena. Time-resolved spectroscopy of NADH and NADPH was performed using single-photon and two-photon excitation. In solution, the fluorescence lifetimes of the two cofactors were identical. The anisotropy decay dynamics of both molecules indicated that distinct molecular configurations caused the presence of two emitting states, perhaps involving alternate cis/trans geometries of the amide group. Using a range of water/glycerol mixtures as solvents, the viscosity dependence of the non-radiative decay of NAD(P)H was shown to be well described by Kramers and Kramers-Hubbard models of activated barrier crossing. This suggested that variations in the fluorescence lifetimes of the cofactors when bound to different enzymes result from differing levels of conformational restriction of the nicotinamide ring in the binding site. Despite identical fluorescence lifetimes in solution, studies on genetically modified cell lines in which NAD kinase was overexpressed or knocked down indicated that intracellular NADPH was associated with a significantly larger fluorescence lifetime when bound to enzymes (~4.4 ns) than enzyme-bound NADH (~1.5 ns). This suggested that variations in the NAD(P)H fluorescence decay upon metabolic perturbation by pharmacological or pathological means, reported both in this work and in the literature, result from changes in the relative concentrations of NADH and NADPH. NAD(P)H FLIM was used to observe elevated NADPH concentrations in the support cells of the mammalian cochlea, highlighting the potential of the technique as a label-free method for monitoring the metabolic state of complex tissue preparations.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:626320 |
| Date | January 2013 |
| Creators | Blacker, T. S. |
| Publisher | University College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://discovery.ucl.ac.uk/1401694/ |
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