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The cofactors NADH and NADPH, hereafter NAD(P)H [NAD(P)= nicotinamide adenine dinucleotide (phosphate)], belong to the principal endogenous indicators of energetic cellular metabolism. Since the metabolic activity of cells is given by the ratio between the concentrations of free and protein-bound NAD(P)H, the development of autofluorescence techniques which accurately measure the modifications to this ratio is particularly significant. Hitherto the methods applied in the monitoring of cellular metabolism have provided either imprecise results, due to interference of the NAD(P)H signal by perturbing factors, or they have required a complicated internal calibration. We employ biexponential fluorescence lifetime imaging (FLIM) in order to discriminate between the free and protein-bound NAD(P)H without any previous calibration. Thus, we have obtained directly, and for the first time, a high-resolution map of cellular metabolism, that is, an image of the contribution of the protein-bound NAD(P)H to the cumulative NAD(P)H fluorescence signal. Moreover, we demonstrate that protein-NAD(P)H complexes characterised by different fluorescence lifetimes are not uniformly distributed all over the cell, as assumed until now, but are concentrated in certain cellular regions. The different fluorescence lifetimes indicate either different protein-NAD(P)H complexes or different bond strengths between NAD(P)H and the protein in these complexes. Since an important aspect in biological applications is to monitor the dynamics of the relevant processes (such as cellular metabolism), rapid dynamical techniques, for example, rapid biexponential fluorescence lifetime imaging, are needed. Furthermore, it is necessary to reduce the evaluation effort as much as possible. Most of the evaluation techniques in multiexponential FLIM are time-expensive iterative methods. The few exceptions are connected with a loss of information, for example, global analysis; or a loss in accuracy, for example, the rapid evaluation technique (RLD). We implement for the first time in FLIM a noniterative, nonrestrictive method originally developed by Prony for approximations of multiexponential decays. The accuracy of this method is verified in biexponential FLIM experiments in time-domain on mixtures of two chromophores both in homogenous and in heterogeneous media. The resulting fluorescence lifetimes agree (within error margins) with the lifetimes of the pure substances determined in monoexponential FLIM experiments. The rapidity of our evaluation method as compared to iterative pixel-by-pixel methods is evidenced by a reduction of the evaluation time by more than one order of magnitude. Furthermore, the applicability of this method for the biosciences is demonstrated in the investigation of cellular metabolism by means of NAD(P)H endogenous fluorescence.

An important goal in single molecule fluorescence correlation spectroscopy is the theoretical simulation of the fluorescence signal stemming from individual molecules and its autocorrelation function. The simulation approaches developed up to now are based exclusively on continuous-wave (cw) illumination and consequently on cw-excitation. However, this approximation is no longer valid in the case of two-photon excitation, for which pulsed illumination is usually employed. We present a novel theoretical model for the simulation of the fluorescence signal of single molecules and its autocorrelation function with consideration of the time dependence of the excitation flux and thus of all illumination-dependent photoprocesses: two-photon excitation, induced emission and photobleaching. Further important characteristics of our approach are the consideration of the dependence of the photobleaching rate on illumination and the low intersystem-crossing rates of the studied coumarins. Moreover, using our approach, we can predict quantitatively the effect of the laser pulse width on the fluorescence signal of a molecule, that is, the contributions of the photobleaching and saturation effects, and thus we can calculate the optimal laser pulse width. The theoretical autocorrelation functions were fitted to the experimental data, and we could ascertain a good agreement between the resulting and the expected parameters. The most important parameter is the photobleaching constant sigma, the cross section of the transition Sn<--S1, which characterises the photostability of the molecules independent of the experimental conditions. Its value is 1.7 x 10(-23) cm2 for coumarin 153 and 5 x 10(-23) cm2 for coumarin 314.