Certain molecules found in cells like Nicotinamide adenine dinucleotide phosphate (NADP), Nicotinamide adenine dinucleotide Hydrogenase (NADH), and flavins exhibit natural fluorescence called autofluorescence. Autofluorescence spectroscopy can be used to identify different metabolic effects caused by these compounds. Phasor analysis is an analytical technique used to assess real time emission signals that could be used to decipher cellular-level metabolic status of tissues and blood. Saccharomyces cerevisiae (baker’s yeast), a eukaryotic cell, is usually used in suspension for spectral phasor analysis. Real-time monitoring of these NADH conformations have immense biophysical and biomedical significance. For instance, NADH can be used as a metabolic indicator and endogenous biomarker, in monitoring cytotoxicity, and in investigating NADH/reduced nicotinamide adenine dinucleotide phosphate (NADPH) balance. Spectral phasor analysis can be used to study cancer pathology and enzyme kinetics which has immense medical significance. In our lab, we use the phasor analysis to study the response map of two reducing equivalents, NADH and NADPH based on the inhibition (i.e. cyanide) or activation (i.e. peroxide) of the metabolic pathways or reactions to fluctuate the amount of these metabolites for expressing the behaviours in the UV-excited autofluorescence sensing maps. We are also using spectral phasor analysis to distinguish between the emission signals from tissues and emission signals from blood. Other labs are also using spectral phasor analysis and autofluorescence to identify NADH and NADPH concentration in the cytoplasm and in the nucleus. Existence of NADH in free and bound states makes it hard to distinguish between the emission signals, and therefore some laboratories are working on finding methods to find the absolute concentration of NADH.
Authors: Sumit Tripathi, Physics and Philosophy
Ken Nguyen, Medical Laboratory Science
Advisor: Paul Urayama, Physics
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