The auto-fluorescent coenzymes reduced nicotinamide dinucleotide (NADH) and oxidized flavin adenine dinucleotide (FAD) allow label-free detection of cellular metabolism. The optical redox ratio, which is traditionally computed as the ratio of NADH and FAD intensities, allows quantification of cell redox state. In addition to multiple formulations of the optical redox ratio from NADH and FAD intensity measurements, a fluorescence lifetime redox ratio (FLIRR) based on the fractions of protein-bound NADH and FAD was developed to overcome the limitations of experimental factors that influence fluorescence intensity measurements. In this paper, we compare fluorescence-intensity computations of the optical redox ratio with the fluorescence lifetime redox ratio for quiescent and activated T cells. Fluorescence lifetime images of NAD(P)H and FAD of T cells were acquired with a two-photon fluorescence lifetime microscope. Metabolic perturbation experiments, including inhibition of glycolysis, oxidative phosphorylation, glutaminolysis, and fatty acid synthesis revealed differences between the intensity and lifetime redox ratios. Statistical analysis reveals that the FLIRR has a lower standard deviation and skewness (two-tail T-test, P value = 0.05) than the intensity redox ratio. Correlation analysis revealed a weak relationship between FLIRR and intensity redox ratio for individual cells, with a stronger correlation identified for activated T cells (Linear regression, R-value = 0.450) than quiescent T cells (R-value = 0.172). Altogether, the results demonstrate that while both the fluorescence lifetime and intensity redox ratios resolve metabolic perturbations in T cells, the endpoints are influenced by different metabolic processes.

中文翻译：

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荧光强度和终生氧化还原比检测T细胞中的代谢扰动

自体荧光辅酶还原的烟酰胺二核苷酸（NADH）和氧化的黄素腺嘌呤二核苷酸（FAD）允许无标记的细胞代谢检测。光学氧化还原比，传统上计算为NADH与FAD强度之比，可以量化细胞的氧化还原状态。除了来自NADH和FAD强度测量的光学氧化还原比的多种配方外，还开发了基于蛋白质结合的NADH和FAD分数的荧光寿命氧化还原比（FLIRR），以克服影响荧光强度测量的实验因素的局限性。在本文中，我们比较了静态和活化T细胞的光学氧化还原比与荧光寿命氧化还原比的荧光强度计算。用两光子荧光寿命显微镜获取T细胞的NAD（P）H和FAD的荧光寿命图像。代谢扰动实验，包括抑制糖酵解，氧化磷酸化，谷氨酰胺分解和脂肪酸合成，揭示了强度和终生氧化还原比之间的差异。统计分析表明，FLIRR的标准偏差和偏斜度（二尾T检验，P值= 0.05）低于强度氧化还原比。相关分析显示单个细胞的FLIRR与强度氧化还原比之间的关系较弱，与静止的T细胞（R值= 0.172）相比，活化T细胞（线性回归，R值= 0.450）的相关性更强。共，