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.

中文翻译：

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使用双波长本征信号双光子激发荧光研究伽马辐照对神经干细胞和前体细胞代谢状态的影响

双光子激发荧光 (TPEF) 光谱和成像用于研究伽马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，但通常与氧化黄素蛋白 (Fp) 相关的信号在整个细胞中广泛分布。测得的 TPEF 发射和激发光谱与已建立的 NAD(P)H 和 Fp 光谱相似。通过将电子传输链 (ETC) 调节剂甲萘醌添加到培养基中，Fp 荧光强度显着增加，同时 NAD(P)H 信号也随之降低。对三维 (3D) 神经球进行成像以获得细胞代谢指数 (CMI)，计算为 Fp 与 NAD(P)H 荧光强度的比率。发现低剂量（≤ 50 cGy）和高剂量（≥ 50 cGy）照射之间的辐射效应不同。低剂量照射导致 CMI 值显着下降，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 值整体升高。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 光谱和成像表征 3D 球体代谢的能力。