Local redox conditions in cells imaged via non-fluorescent transient states of NAD(P)H

The autofluorescent coenzyme nicotinamide adenine dinucleotide (NADH) and its phosphorylated form (NADPH) are major determinants of cellular redox balance. Both their fluorescence intensities and lifetimes are extensively used as label-free readouts in cellular metabolic imaging studies. Here, we introduce fluorescence blinking of NAD(P)H, as an additional, orthogonal readout in such studies. Blinking of fluorophores and their underlying dark state transitions are specifically sensitive to redox conditions and oxygenation, parameters of particular relevance in cellular metabolic studies. We show that such dark state transitions in NAD(P)H can be quantified via the average fluorescence intensity recorded upon modulated one-photon excitation, so-called transient state (TRAST) monitoring. Thereby, transitions in NAD(P)H, previously only accessible from elaborate spectroscopic cuvette measurements, can be imaged at subcellular resolution in live cells. We then demonstrate that these transitions can be imaged with a standard laser-scanning confocal microscope and two-photon excitation, in parallel with regular fluorescence lifetime imaging (FLIM). TRAST imaging of NAD(P)H was found to provide additional, orthogonal information to FLIM and allows altered oxidative environments in cells treated with a mitochondrial un-coupler or cyanide to be clearly distinguished. We propose TRAST imaging as a straightforward and widely applicable modality, extending the range of information obtainable from cellular metabolic imaging of NAD(P)H fluorescence.