Separating NADH and NADPH fluorescence in live cells and tissues using FLIM

Thomas S. Blacker, Zoe F. Mann, Jonathan E. Gale, Mathias Ziegler, Angus J. Bain, Gyorgy Szabadkai and Michael R. Duchen ()
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Thomas S. Blacker: Centre for Mathematics and Physics in the Life Sciences and Experimental Biology, University College London
Zoe F. Mann: University College London
Jonathan E. Gale: University College London
Mathias Ziegler: University of Bergen
Angus J. Bain: University College London
Gyorgy Szabadkai: University College London
Michael R. Duchen: University College London

Nature Communications, 2014, vol. 5, issue 1, 1-9

Abstract: Abstract NAD is a key determinant of cellular energy metabolism. In contrast, its phosphorylated form, NADP, plays a central role in biosynthetic pathways and antioxidant defence. The reduced forms of both pyridine nucleotides are fluorescent in living cells but they cannot be distinguished, as they are spectrally identical. Here, using genetic and pharmacological approaches to perturb NAD(P)H metabolism, we find that fluorescence lifetime imaging (FLIM) differentiates quantitatively between the two cofactors. Systematic manipulations to change the balance between oxidative and glycolytic metabolism suggest that these states do not directly impact NAD(P)H fluorescence decay rates. The lifetime changes observed in cancers thus likely reflect shifts in the NADPH/NADH balance. Using a mathematical model, we use these experimental data to quantify the relative levels of NADH and NADPH in different cell types of a complex tissue, the mammalian cochlea. This reveals NADPH-enriched populations of cells, raising questions about their distinct metabolic roles.

Date: 2014
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DOI: 10.1038/ncomms4936

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