Kierdaszuk B, Malak H, Gryczynski I, Callis P, Lakowicz J R
Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore 21201, USA.
Biophys Chem. 1996 Nov 29;62(1-3):1-13. doi: 10.1016/s0301-4622(96)02182-5.
We examined the steady-state and time-resolved emission of NADH and NAMH resulting from one-photon and two-photon excitation. Similar emission 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-photon excitation. This observation of a higher anisotropy with two-photon 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 decays are distinct. NADH-LADH-IBA complex formation led to an order of magnitude larger of the average lifetimes of NADH fluorescence resulting from one- and two-photon excitation. Fluorescence intensity and fluorescence anisotropy decays of NADH was double-exponential for both modes of excitation and show that the observed heterogeneity of the fluorescence decay kinetics of reduced nicotinamides arises from the inherent photoprocess of the dihydronicotinamide chromophore and not due to any intramolecular interactions with adenine part of NADH. Such interactions are responsible for the depolarization of NADH fluorescence observed for excitation wavelength below 300 nm for OPE and 600 nm for TPE, respectively. NADH displays a low cross-section for two-photon excitation which suggests that fluorescence from NADH will be moderately difficult to observe with two-photon fluorescence microscopy, and may not interfere with observations of TPIF of other extrinsic probes used to label cells.
我们研究了单光子和双光子激发产生的NADH和NAMH的稳态和时间分辨发射。两种激发模式下观察到相似的发射光谱。对于690至740 nm的双光子激发，NADH的基本各向异性接近0.54，比单光子激发观察到的0.37的值高46%。双光子激发下更高各向异性的这一观察结果与单光子和双光子跃迁的INDO/SDCI计算一致。单光子和双光子激发下观察到NADH的多指数衰减存在微小差异，但目前可用的分辨率不允许我们得出衰减是不同的结论。NADH-LADH-IBA复合物的形成导致单光子和双光子激发产生的NADH荧光平均寿命增大一个数量级。对于两种激发模式，NADH的荧光强度和荧光各向异性衰减都是双指数的，表明观察到的还原型烟酰胺荧光衰减动力学的异质性源于二氢烟酰胺发色团固有的光过程，而不是由于与NADH腺嘌呤部分的任何分子内相互作用。这种相互作用分别导致了单光子激发在300 nm以下和双光子激发在600 nm以下激发波长时观察到的NADH荧光去极化。NADH双光子激发的截面较低，这表明用双光子荧光显微镜观察NADH的荧光会有一定难度，并且可能不会干扰用于标记细胞的其他外源性探针的双光子诱导荧光观察。
