Apoptosis is very important for the maintenance of cellular homeostasis and is closely related to the occurrence and treatment of many diseases. Mitochondria in cells play a crucial role in programmed cell death and redox processes. Nicotinamide adenine dinucleotide (NAD(P)H) is the primary producer of energy in mitochondria, changing NAD(P)H can directly reflect the physiological state of mitochondria. Therefore, NAD(P)H can be used to evaluate metabolic response. In this paper, we propose a noninvasive detection method that uses two-photon fluorescence lifetime imaging microscopy (TP-FLIM) to characterize apoptosis by observing the binding kinetics of cellular endogenous NAD(P)H. The result shows that the average fluorescence lifetime of NAD(P)H and the fluorescence lifetime of protein-bound NAD(P)H will be affected by the changing pH, serum content, and oxygen concentration in the cell culture environment, and by the treatment with reagents such as H2O2 and paclitaxel. Taxol (PTX). This noninvasive detection method realized the dynamic detection of cellular endogenous substances and the assessment of apoptosis.

Reduced nicotinamide adenine dinucleotide (NADH) plays a crucial role in many biochemical reactions in human metabolism. In this work, a flow-mediated skin fluorescence (FMSF)-postocclusion reactive hyperaemia (PORH) system was developed for noninvasive and in vivo measurement of NADH fluorescence and its real-time dynamical changes in human skin tissue. The real-time dynamical changes of NADH fluorescence were analyzed with the changes of skin blood flow measured by laser speckle contrast imaging (LSCI) experiments simultaneously with FMSFPORH measurements, which suggests that the dynamical changes of NADH fluorescence would be mainly correlated with the intrinsic changes of NADH level in the skin tissue. In addition, Monte Carlo simulations were applied to understand the impact of optical property changes on the dynamical changes of NADH fluorescence during the PORH process, which further supports that the dynamical changes of NADH fluorescence measured in our system would be intrinsic changes of NADH level in the skin tissue.

细胞微环境的稳定是保持细胞正常增殖、 代谢和功能活动的重要条件, 微环境成分的异常变化可使细胞发生病变。 采用荧光光谱技术研究离体白细胞在多糖微环境中荧光发射特性的变化规律和发光机制, 并进一步的分析了多糖对白细胞的生物活性的影响。 实验结果表明: 当白细胞受波长为407 nm的激光照射时, 发射位于450 nm的荧光。 在加入脂多糖或葡聚糖时, 白细胞的荧光发射峰的位置不会变化, 峰值受到影响。 脂多糖的加入会减弱白细胞荧光峰强度, 且荧光强度随脂多糖浓度（0～500 μg·mL-1范围内）的增加而持续减弱。 而葡聚糖可以一定程度增加白细胞的荧光强度, 浓度越高, 荧光强度越大。 分析认为白细胞发射的450 nm荧光来自发射物质烟酰胺腺嘌呤二核苷酸(NADH)。 白细胞内NADH随着离体时间的增长, 被氧化成不发荧光的烟酰胺腺嘌呤磷酸二核苷酸（NAD+）, 导致细胞荧光峰值下降, 从而引起细胞凋亡。 加入脂多糖产生的羟自由基（·OH）会与NADH发生氧化反应, 因此脂多糖加速了NADH的消耗, 导致白细胞荧光减弱, 加快细胞凋亡。 而葡聚糖主要是由葡萄糖单体组成, 葡聚糖的加入会将NAD+还原成NADH, 因此延缓了白细胞的凋亡。 分析认为脂多糖可以加速白细胞的凋亡, 提高细胞发生炎症甚至是肿瘤的机率, 而葡聚糖对白细胞有保护作用。 该研究为研究肿瘤的发生和发展过程以及治疗提供有价值的参考。

烟酰胺, 又称为维生素PP, 是辅酶Ⅰ和辅酶Ⅱ的组成部分, 是许多脱氢酶的辅酶。 庚二酸是一种广泛使用的共晶体前体。 烟酰胺与庚二酸形成的药物共晶体具有两种不同的多晶型, 分别为晶型Ⅰ和晶型Ⅱ。 不同的药物晶型常常具有不同的物理化学性质, 这些差异可能会对药物的溶出速率、 稳定性以及药效具有较大影响, 因此在药物领域中寻找合适的技术手段鉴别药物的不同晶型非常重要。 利用太赫兹时域光谱在室温下对烟酰胺-庚二酸的两种共晶体在0.2～2.2 THz范围内进行检测, 发现两者的特征吸收峰具有明显的差异, 晶型Ⅰ在1.51, 1.73, 1.94, 2.01和2.17 THz有五个特征吸收峰, 其中在1.94, 2.01和2.17 THz处是三个吸收强度较大的峰, 而晶型Ⅱ在1.66, 1.74, 1.88, 2.02和2.16 THz有五个特征吸收峰, 与晶型Ⅰ不同的是在2.02和2.16 THz处有两个强度较大的吸收峰。 利用密度泛函理论对烟酰胺-庚二酸两种共晶体进行理论计算, 计算结果表明实验峰与理论峰基本对应, 对吸收峰指认归属表明特征吸收峰来源于分子骨架的振动与包含氢键的振动。 研究结果表明太赫兹时域光谱技术对于区分药物共晶体的多晶型现象具有重要的应用。

Photodynamic therapy (PDT) gains wide attention as a useful therapeutic method for cancer. It is mediated by the oxygen and photosensitizer under the specific light irradiation to produce the reactive oxygen species (ROS), which induce cellular toxicity and regulate the redox potential in tumor cells. Nowadays, genetic photosensitizers of low toxicity and easy production are required to be developed. KillerRed, a unique red fluorescent protein exhibiting excellent phototoxic properties, has the potential to act as a photosensitizer in the application of tumor PDT. Meantime, the course of tumor redox metabolism during this treatment was rarely investigated so far. Thus here, we investigated the effects of KillerRed-based PDT on tumor growth in vivo and examined the subsequent tumor metabolic states including the changes of nicotinamide adenine dinucleotide hydrogen (NADH) and flavoprotein (Fp), two important metabolic coenzymes of tumor cells. Results showed the tumor growth had been significantly inhibited by KillerRedbased PDT treatment compared to control groups. A home-made cryo-imaging redox scanner was used to measure intrinsic fluorescence and exogenous KillerRed fluorescence signals in tumors. The Fp signal was elevated by nearly 4.5-fold, while the NADH signal decreased by 66% after light irradiation, indicating that Fp and NADH were oxidized in the course of KillerRedbased PDT. Furthermore, we also observed correlation between the fluorescence distribution of KillerRed and NADH. It suggests that the KillerRed protein based PDT might provide a new approach for tumor therapy accompanied by altering tumor metabolism.

Hypoxia is closely related to many diseases and often leads to death. Early detection and identification of the hypoxia causes may help to promptly determine the right rescue plan and reduce the mortality. We proposed a new multiparametric monitoring method employing mitochondrial reduced nicotinamide adenine dinucleotide (NADH) fluorescence, regional reflectance, regional cerebral blood flow (CBF), electrocardiography (ECG), and respiration under six kinds of acute hypoxia in four categories to investigate a correlation between the parameter variances and the hypoxia causes. The variation patterns of the parameters were discussed, and the combination of NADH and CBF may contribute to the identification of the causes of hypoxia.

The aim of this ex vivo study was to explore the potential of using the fluorescence lifetime of intracellular reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) as a label-free indicator to characterize the differences between human leukemic myeloid cells and normal mononuclear cells (MNC). The steady-state and time-resolved autofluorescence of two human leukemic myeloid cell lines (K562, HL60) and MNC were measured by a spectrofluorimeter. According to excitation–emission matrix (EEM) analysis, the optimal emission of NAD(P)H in these cells suspensions occurred at 445 nm. Furthermore, the fluorescence lifetimes of NAD(P)H in leukemic myeloid cells and MNC were determined by fitting the time-resolved autofluorescence data. The mean fluorescence lifetimes of NAD(P)H in K562, HL60, and MNC cells were 5.57± 1.19, 4.45 ± 0.71, and 7.31 ± 0.60 ns, respectively. There was a significant difference in the mean lifetime of NAD(P)H between leukemic myeloid cells and MNC (p < 0:05). The difference was essentially caused by the change in relative concentration of free and protein-bound NAD(P)H. This study suggests that the mean fluorescence lifetime of NAD(P)H might be a potential labelfree indicator for differentiating leukemic myeloid cells from MNC.

Function-specific homeostasis (FSH) has been defined as a negative-feedback response of a biosystem to maintain its interior function-specific conditions so that the function is perfectly performed. There is no photobiomodulation of intranasal low intensity laser therapy (ILILT) on a function in its FSH, but ILILT could modulate a function far from its FSH. This rehabilitation has been found to be mediated by the ratio of intracellular nicotinamide adenine dinucleotide (NAD+) and its reduced form NADH, NAD+/NADH, and then sirtuin 1 (SIRT1). There might be FSH-specific NAD+/NADH (FSN) and SIRT1 activity (FSA). ILILT might enhance NAD+/NADH and SIRT1 activity until they arrive at FSN and FSA, respectively. The NAD+/NADH and SIRT1 activity of related cells of many athletic diseases such as upper respiratory tract infection, asthma, osteoarthritis, exercise-induced muscle damage, wound, traumatic brain injury, and osteoporosis are lower than FSN and FSA, respectively. Therefore, there may be therapeutic effects of ILILT on those athletic diseases. Furthermore, many phenomena and the ILILT mechanism have been integrated to support the prophylaxis effects of ILILT on the swine-origin influenza A (H1N1).

Mitochondrial redox states provide important information about energy-linked biological processes and signaling events in tissues for various disease phenotypes including cancer. The redox scanning method developed at the Chance laboratory about 30 years ago has allowed 3D highresolution (～ 50 × 50 × 10μm³) imaging of mitochondrial redox state in tissue on the basis of the fluorescence of NADH (reduced nicotinamide adenine dinucleotide) and Fp (oxidized flavoproteins including flavin adenine dinucleotide, i.e., FAD). In this review, we illustrate its basic principles, recent technical developments, and biomedical applications to cancer diagnostic and therapeutic studies in small animal models. Recently developed calibration procedures for the redox imaging using reference standards allow quantification of nominal NADH and Fp concentrations, and the concentration-based redox ratios, e.g., Fp/(Fp+NADH) and NADH/(Fp+NADH) in tissues. This calibration facilitates the comparison of redox imaging results acquired for different metabolic states at different times and/or with different instrumental settings. A redox imager using a CCD detector has been developed to acquire 3D images faster and with a higher in-plane resolution down to 10 μm. Ex vivo imaging and in vivo imaging of tissue mitochondrial redox status have been demonstrated with the CCD imager. Applications of tissue redox imaging in small animal cancer models include metabolic imaging of glioma and myc-induced mouse mammary tumors, predicting the metastatic potentials of human melanoma and breast cancer mouse xenografts, differentiating precancerous and normal tissues, and monitoring the tumor treatment response to photodynamic therapy. Possible future directions for the development of redox imaging are also discussed.

研究了室温条件下结构相似的烟酸和烟酰胺以及烟碱的太赫兹光谱。 测量了它们的时间分辨光谱， 通过傅里叶变换获得了它们的频谱， 从而得到了频谱响应和折射率色散关系。 实验结果表明, 烟碱在太赫兹波段无明显的特征频谱响应， 而烟酸和烟酰胺在太赫兹波段存在明显的特征频谱响应。 用密度泛函理论（DFT）计算了烟酸和烟酰胺的太赫兹频谱， 对它们的吸收峰产生的原因做了初步分析， 认为烟酸吸收峰的产生是由于分子内部的扭转和摇摆所造成的， 而烟酰胺的吸收峰（除1.93 THz以外）是由于分子间的相互作用和光声子模式而造成的。 结果表明， 数值模拟和实验结果相结合， 可以用来分析烟酸和烟酰胺的分子结构和分子振动模式。