Spectral Phasor Analysis On Nanosecond-Gated Autofluorescence Reveals Real Time Information On Cellular Nad(P) H Conformation During Chemically-Induced Metabolic Response

Cellular reduced pyridine nucleotides NADH and NADPH are metabolic co-factors that exist in multiple conformations, significant because the proportions of these forms respond differently to metabolic conditions. Real time monitoring approaches sensitive to conformation support the use of NADH and NADPH as metabolic indicators and endogenous biomarkers. Here, we demonstrate an approach combining nanosecond-gated spectral detection with spectral phasor analysis on UV-excited autofluorescence in Saccharomyces cerevisiae suspensions. The approach provides real time, dynamical information on cellular pyridine nucleotide conformation to chemically-induced metabolic change. We demonstrate the ability to sense a broad range of responses, including concentration-dependent-differences in the acute toxicity response to cyanide, discrimination between NADH- and NADPH-linked responses, and the ability to sense metabolic response under pressurized (several hundred atm) conditions. Emphasis is placed on interpreting the non-two-component behavior in the spectral response in terms of the affected biochemical pathways.