Krasieva Tatiana B, Giedzinski Erich, Tran Katherine, Lan Mary, Limoli Charles L, Tromberg Bruce J
Laser Microbeam and Medical Program, Beckman Laser Institute, University of California, Irvine, 1002 Health Sciences Road East, Irvine, CA 92612, USA.
J Innov Opt Health Sci. 2011 Jul 1;4(3):289-300. doi: 10.1142/S1793545811001629.
Two-photon excited fluorescence (TPEF) spectroscopy and imaging were used to investigate the effects of gamma-irradiation on neural stem and precursor cells (NSPCs). While the observed signal from reduced nicotinamide adenine dinucleotide (NADH) was localized to the mitochondria, the signal typically associated with oxidized flavoproteins (Fp) was distributed diffusely throughout the cell. The measured TPEF emission and excitation spectra were similar to the established spectra of NAD(P)H and Fp. Fp fluorescence intensity was markedly increased by addition of the electron transport chain (ETC) modulator menadione to the medium, along with a concomitant decrease in the NAD(P)H signal. Three-dimensional (3D) neurospheres were imaged to obtain the cellular metabolic index (CMI), calculated as the ratio of Fp to NAD(P)H fluorescence intensity. Radiation effects were found to differ between low-dose (≤ 50 cGy) and high-dose (≥ 50 cGy) exposures. Low-dose irradiation caused a marked drop in CMI values accompanied by increased cellular proliferation. At higher doses, both NAD(P)H and Fp signals increased, leading to an overall elevation in CMI values. These findings underscore the complex relationship between radiation dose, metabolic state, and proliferation status in NSPCs and highlight the ability of TPEF spectroscopy and imaging to characterize metabolism in 3D spheroids.
采用双光子激发荧光（TPEF）光谱和成像技术研究γ射线辐照对神经干细胞和前体细胞（NSPCs）的影响。虽然观察到的来自还原型烟酰胺腺嘌呤二核苷酸（NADH）的信号定位于线粒体，但通常与氧化型黄素蛋白（Fp）相关的信号则在整个细胞中呈弥漫性分布。所测得的TPEF发射光谱和激发光谱与已确定的NAD（P）H和Fp光谱相似。向培养基中添加电子传递链（ETC）调节剂甲萘醌后，Fp荧光强度显著增加，同时NAD（P）H信号随之降低。对三维（3D）神经球进行成像以获得细胞代谢指数（CMI），其计算方法为Fp与NAD（P）H荧光强度之比。结果发现，低剂量（≤50 cGy）和高剂量（≥50 cGy）照射的辐射效应有所不同。低剂量照射导致CMI值显著下降，同时细胞增殖增加。在较高剂量下，NAD（P）H和Fp信号均增加，导致CMI值总体升高。这些发现强调了NSPCs中辐射剂量、代谢状态和增殖状态之间的复杂关系，并突出了TPEF光谱和成像技术在表征3D球体代谢方面的能力。
