A fraction of NADH in solution is "dark": Implications for metabolic sensing via fluorescence lifetime.

The metabolic cofactor and energy carrier NADH (nicotinamide adenine dinucleotide, reduced) has fluorescence yield and lifetime that depends strongly on conformation, a fact that has enabled metabolic monitoring of cells via FLIM (Fluorescence Lifetime Microscopy). Using femtosecond fluorescence upconversion, we show that this molecule in solution participates in ultrafast self-quenching along with both bulk solvent relaxation and spectral relaxation on 1.4 and 26 ps timescales. This, in effect, means up to a third of NADH is effectively "dark" for FLIM in the 400-500 nm observation window commonly employed. Methods to compensate for, avoid or measure dark species corrections are outlined.