The redox pair based on nicotinamide adenine dinucleotide (NADH/NAD + ) is a critical indicator of many cellular metabolic pathways. As such, fluorescence lifetime imaging (FLIM) of NADH is often used as an indicator of cellular redox levels. NADH is naturally autofluorescent, making it a powerful label-free imaging tool. Typically, in living cells, NADH exists primarily in a “free” form vs. a protein-bound form (oft presumed bound to membrane-associated dehydrogenases). Free NADH has a complex short fluorescence decay centered at ∼ 0.3–0.6 ns, while the bound forms have typical lifetimes of ∼1.6–4 ns. Modern detectors have faster response and are coupled to fast electronics; with these detectors and time taggers we can go beyond the ∼100 ps limit of traditional FLIM and seek ultrafast lifetimes. To resolve these ultrafast lifetimes we collect a true instrument response function (IRF) via observing SHG within urea crystals, which we found, has a full width half max (FWHM) of 24 ps. We employ a picosecond time-correlated single-photon counting (TCSPC) apparatus consisting of a fast time tagger (SPC-180NX, Becker-Hickl) and hybrid detectors (HPM-100-06, Becker-Hickl). This measured IRF (NOT just risetime synthetic) enables us to better deconvolve and dissect the free NADH lifetimes in PBS. We split the familiar ∼0.4 ns lifetime (a composite of 250 ps and 690 ps lifetimes reported previously), but we also recover the more recently discovered “dark” (30 ps decay time) population. This lifetime is in rough agreement with published cuvette-based upconversion studies at subpicosecond resolution. Most importantly, this measurement is now also tenable in the cellular imaging environment, and the potential information available from free dark state(s) or highly quenched bound environments of NADH in metabolism is hence available to be exploited.
Link et al. (Sun,) studied this question.