Distinguishing cellular respiration vs. oxidative stress in turbid media using UV-excited autofluorescence spectroscopy

The reduced pyridine-nucleotides nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH) are ubiquitous metabolic cofactors playing significant, distinct roles in cellular metabolism. NADH and NADPH are primarily involved in cellular respiration and in maintaining antioxidant defenses, respectively, however their nearly identical fluorescence properties (the abbreviation NAD(P)H denotes this uncertainty) pose a challenge when interpreting and distinguishing autofluorescence signals. For sensing in turbid media such as tissue, additional challenges include the presence of multiple scattering, intrinsic absorption, and background fluorescence. Here, we assess an approach for distinguishing cellular-respiration and oxidative-stress responses when sensed in turbid media. Spectral phasor analysis, an analytical approach originally developed for the rapid segmentation of hyperspectral images, has been used on UV-excited autofluorescence for the real time monitoring of cellular NAD(P)H conformation. We showed previously that the spectral response to chemicals affecting NADH and NADPH pathways, e.g., in response to cyanide and hydrogen peroxide, does not follow two-component behavior and so could be distinguished in cell-only preparations. Here, we demonstrate pathway-level sensing in turbid media by monitoring the metabolic response of yeast cells embedded in a source for background emission. The distinguishability of UV-excited autofluorescence spectra to chemical perturbations affecting cellular respiration and oxidative stress are compared with previously reported cell-only observations.