FLUORESCENCE OF REDUCED NICOTINAMIDES USING ONE-PHOTON AND 2-PHOTON EXCITATION

We examined the steady-state and time-resolved emission of NADH and NA MH resulting from one-photon and two-photon excitation. Similar emissi on spectra were observed for both modes of excitation. The fundamental anisotropy of NADH is near 0.54 for two-photon excitation from 690 to 740 nm, which is 46% higher than the value of 0.37 observed for one-p hoton excitation. This observation of a higher anisotropy with two-pho ton excitation was consistent with INDO/SDCI calculations of the one- and two-photon transitions. Minor differences in the multi-exponential decays of NADH were observed for one- and two-photon excitation, but presently available resolution does not allow us to conclude the decay s are distinct. NADH-LADH-IBA complex formation led to an order of mag nitude larger of the average lifetimes of NADH fluorescence resulting from one- and two-photon excitation. Fluorescence intensity and fluore scence anisotropy decays of NADH was double-exponential for both modes of excitation and show that the observed heterogeneity of the fluores cence decay kinetics of reduced nicotinamides arises from the inherent photoprocess of the dihydronicotinamide chromophore and not due to an y intramolecular interactions with adenine part of NADH. Such interact ions are responsible for the depolarization of NADH fluorescence obser ved for excitation wavelength below 300 nm for OPE and 600 nm for TPE, respectively. NADH displays a low cross-section for two-photon excita tion which suggests that fluorescence from NADH will be moderately dif ficult to observe with two-photon fluorescence microscopy, and may not interfere with observations of TPIF of other extrinsic probes used to label cells.