Using Spectral Phasor Analysis Of Uv-Excited Autofluorescence To Reveal Small Differences Between Chemically-Induced Mitochondrial Responses

Spectral phasor analysis on nanosecond-gated, UV-excited autofluorescence is being developed for the real time monitoring of cellular metabolism. Previous studies have shown that the response of autofluorescence spectrum shape to additions of chemical agents (e.g., cyanide, ethanol, glucose) is due to the changing ensemble of cellular reduced nicotinamide adenine dinucleotide (NADH) forms. Here we apply the monitoring technique to investigate the autofluorescence response of Saccharomyces cerevisiae suspensions to additions of respiratory inhibitors, including cyanide (a complex IV inhibitor) and 2-thenoyltrifluoroacetone (TTFA, a complex II inhibitor). Using spectral phasor analysis to assess whether observed spectral changes are consistent with a two-state model and using nanosecond-gated spectral detection to isolate the long excited-state lifetime signal, we find evidence for the spectral detection of a concentration-dependent cyanide action and for the ability to sense complex-II inhibition despite the presence of fluorescence quenching behavior from TTFA. Results demonstrate how the quantification of spectrum shape can be useful in the real time, non-invasive detection of small differences between mitochondrial responses and contribute to the development of NADH as a metabolic indicator and biomarker.