Reduced nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH) are metabolic cofactors playing significant, distinct roles in cellular metabolism primarily involving cellular respiration and maintaining antioxidant defenses, respectively. Signals from NADH and NADPH are a significant component of cellular autofluorescence and are useful for metabolic sensing. For in vivo sensing of tissues, challenges include the presence of non-specific background fluorescence as well as perturbations of intrinsic NADH/NADPH fluorescence due to scattering and absorption. Here, we assess an approach for cellular metabolic sensing in turbid media based on a spectral phasor analysis of UV-excited autofluorescence. Recent studies of autofluorescence emission during chemically induced metabolic response in cell-only environments showed that two-component spectral behavior, i.e., spectral change acting as a superposition of two spectra, depended on whether one or multiple metabolic pathways were affected. In this way, the spectral response to chemicals affecting NADH and NADPH pathways, e.g., in response to cyanide and hydrogen peroxide, could be distinguished. Here, we demonstrate pathway-level sensing in turbid media by monitoring the autofluorescence response of yeast cells embedded in tissue-like environments containing background emission, scattering, and absorption. Additionally, we discuss the potential for sensing cellular metabolism in tissue.
Authors: Nick Majer, Taylor Phillips
Advisors: Paul Urayama, Physics, Karthik Vishwanath, Physics
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