Autofluorescence from NADH Conformations Associated with Different Metabolic Pathways Monitored Using Nanosecond-Gated Spectroscopy and Spectral Phasor Analysis

Abstract

Cellular NADH conformation is increasingly recognized as an endogenous optical biomarker and metabolic indicator. Recently, we reported a real-time approach for tracking metabolism on the basis of the quantification of UV-excited autofluorescence spectrum shape. Here, we use nanosecond-gated spectral acquisition, combined with spectrum-shape quantification, to monitor the long excited-state lifetime autofluorescence (usually associated with protein-bound NADH conformations) separately from the autofluorescence signal as a whole. We observe that the autofluorescence response induced by two NADH-oxidation inhibitors—cyanide and ethanol—are similar in Saccharomyces cerevisiae when monitored using time-integrated detection but easily distinguished using time-gated detection. Results are consistent with the observation of multiple NADH conformations as assessed using spectral phasor analysis. Further, because well-known oxidation inhibitors are used, changes in spectrum shape can be associated with NADH conformations involved in the different metabolic pathways, giving bioanalytic utility to the spectral responses.