Real-Time Quantification of Time-Gated Autofluorescence Spectrum Shape to Track Mitochondrial Metabolism

Abstract

Recent studies by various groups report the use of NADH as an intrinsic optical biomarker and metabolic indicator. Using measurements of excited-state lifetime or anisotropy decay, different forms of intracellular NADH were identified and monitored, with the response of NADH to metabolic conditions being more complex than a simple concentration change. Recently, we report an approach for the real-time tracking of UV-excited autofluorescence based on the rapid quantification of spectrum shape. Here, we show that nanosecond-gated spectral acquisition - combined with spectrum-shape quantification (such as with spectral phasor analysis) - can be used to track the physiologic response of a cellular system (Saccharomyces cereviseae) to the addition of mitochondrial functional modifiers (e.g., cyanide) and metabolic substrates (e.g., ethanol and glucose). We demonstrate that time-gated detection allows for the rejection of short excited-state-lifetime emission. We observe that the spectrum shape of long excited-state-lifetime autofluorescence exhibits a different response to chemical additions than that of the time-integrated autofluorescence, even allowing for the discrimination between nominally similar responses. Results are consistent with the existence of multiple NADH forms, as changes in autofluorescence spectrum shape cannot be accounted for by a two spectra model. Time-gated spectroscopy combined with rapid spectrum-shape analysis may lead to new and useful approaches for the real-time tracking of cellular metabolic state.