Single Molecule Fluorescence Imaging and Its Application to the Study of DNA Condensation

Single molecule fluorescence imaging incorporated with optical tweezers and a laminar flow cell has been used to monitor the kinetic process of DNA condensation induced by spermidine. It was found that at least two steps were involved in the condensation process of the hydrodynamically-stretched linear DNA; a lag period followed by a rapid collapse of DNA. The lag time increased with the flow speed and the collapse time remained short within the range of the flow speed studied. The effect of salt concentration on the condensation process was examined, and the results suggest that the longer lag time observed in the higher salt buffer probably results from the displacement of bound cations and rearrangement of spermidine on the DNA. The flow-speed dependence of the lag time suggests that a nucleation event at the free end of the DNA, i.e. formation of a loop, may play a vital role in the kinetic process of condensation.     

Detection and Analysis of Protein Aggregation with Confocal Single Molecule Fluorescence Spectroscopy

The misfolding and aggregation of proteins is a common phenomenon both in the cell, in in vitro protein refolding, and the corresponding biotechnological applications. Most importantly, it is involved in a wide range of diseases, including some of the most prevalent neurodegenerative disorders. However, the range of methods available to analyze this highly heterogeneous process and the resulting aggregate structures has been very limited. Here we present an approach that uses confocal single molecule detection of FRET-labeled samples employing four detection channels to obtain information about diffusivity, anisotropy, fluorescence lifetimes and Förster transfer efficiencies from a single measurement. By combining these observables, this method allows the separation of subpopulations of folded and misfolded proteins in solution with high sensitivity and a differentiation of aggregates generated under different conditions. We demonstrate the versatility of the method with experiments on rhodanese, an aggregation-prone two-domain protein.     

The Influence of Surface Trapping and Dark States on the Fluorescence Emission Efficiency and Lifetime of CdSe and CdSe/ZnS Quantum Dots

To investigate the influence of surface trapping and dark states on CdSe and CdSe/ZnS quantum dots (QDs), we studied the absorption, fluorescence intensity and lifetime by using one-and two-photon excitation, respectively. Experimental results show that both one- and two-photon fluorescence emission efficiencies of the QDs enhance greatly and the lifetime increase after capping CdSe with ZnS due to the effective surface passivation. The lifetime of one-photon fluorescence of CdSe and CdSe/ZnS QDs increase with increasing emission wavelength in a supralinear way, which is attributed to the energy transfer of dark excitons. On the contrary, the lifetime of two-photon fluorescence of bare and core-shell QDs decrease with increasing emission wavelength, and this indicates that the surface trapping is the dominant decay mechanism in this case.     

基于三维荧光光谱和PSO-SVM对胭脂红含量的测定

胭脂红是一种应用广泛的食品色素，在各种食品、饮料的添加剂里都有它的身影，过量食用人工合成色素会严重危害健康。食物中色素一般都是多种联用，各种色素之间会相互产生干扰，这加大了对食品中色素检测的难度，模拟食品中多种色素共存的环境，采用荧光光谱技术，结合PSO-SVM算法，建立一种测定混合溶液中胭脂红含量的方法。从试剂公司购买胭脂红和苋菜红固体粉末，选择胭脂红为待检测色素，苋菜红为干扰色素，配成不同浓度的胭脂红单色溶液以及加入苋菜红后的混合溶液样本，其中胭脂红的浓度在0.1~30 μg·mL-1之间，干扰色素苋菜红的浓度在0.1~10 μg·mL-1之间随意添加。运用Edinburgh Instruments 公司生产的FS920稳态荧光光谱仪, 测得胭脂红单色溶液与加入苋菜红后混合溶液的荧光光谱图，分析得到胭脂红的最佳激发波长为λ_ex＝326 nm，最佳发射波长为λ_em＝430 nm。各选取6组不同浓度的单色样本以及混合色素样本，其中，胭脂红的物质浓度同为3，4，5，6，7和8 μg·mL-1，苋菜红的物质浓度都定在2 μg·mL-1。观察6组样本在激发波长λ_ex=326 nm时的发射光谱和荧光强度的关系。单色样本中，胭脂红浓度与荧光强度线性关系良好；而在混合溶液中，随着胭脂红浓度的增加，荧光强度呈现出先降后增再降的过程，光谱线型、强度与各组分浓度间存在复杂的非线性关系，得以证明混合溶液的荧光光谱并不是由各组分光谱简单的叠加，而是在吸收光谱的过程中，胭脂红溶液与苋菜红溶液存在竞争和相互影响。配取25组胭脂红、苋菜红混合溶液，从中选择7个作为预测样本，其余18组作为训练样本。7 个预测样本中胭脂红的浓度分别为 1.0，2.0，4.0，6.0，9.0，12和15 μg·mL-1，干扰物质苋菜红的物质浓度在0.1~10 μg·mL-1之间。选择各组样本在最佳激发波长λ_ex＝326 nm 下对应的荧光强度，作为检测模型的输入，以胭脂红的预测浓度作为输出。对PSO参数初始化设置后，训练输出SVM的最佳参数c和g，将所得的最佳参数输入PSO-SVM模型, 得到7组预测样本的浓度预测结果分别为：1.146 9，1.860 6，3.854 4，6.146 9，9.133 8，11.857 6和14.859 8 μg·mL-1。分析PSO-SVM的预测结果，得到胭脂红平均回收率为100.84%，预测均方根误差(RMSEP)为1.03×10-4，模型输出与真实值之间的相关系数是0.999。在同等条件下，采用误差逆向传播算法(BP)预测得到的7组样本浓度分别为：1.140 1，2.139 8，3.188 2，6.436 2，8.882 7，11.860 1和12.664 3 μg·mL-1，其平均回收率为98.56%，均方根误差为4.65×10-3，输出值与真实值之间的相关系数为0.972。与误差逆向传播算法(BP)的预测结果相比较，PSO-SVM 相关系数高出2.7%，平均回收率高出0.6%，均方根误差降低了将近一个数量级。分析结果表明，通过荧光光谱技术与PSO-SVM相结合的方法，能够有效的避开干扰色素的影响，准确的测定混合溶液中胭脂红的含量，并且效果相比较于BP更加理想。     

胭脂红分子结构的量子化学计算及荧光光谱机理研究

运用Gaussian 09W量子化学程序包,对胭脂红分子进行基态和激发态几何结构构型优化,计算得到激发态电子结构、分子前线轨道和发射光谱等信息;应用英国Edinburgh FLS920P 光谱仪,实验测定胭脂红溶液的荧光光谱。比较荧光发射光谱的计算值与实验值,吻合较好,说明优化所得构型基本合理。进一步对比胭脂红分子基态和激发态结构,分析荧光光谱产生的机理,发现胭脂红分子激发态几乎呈平面结构,为强荧光物质,其荧光由139→137轨道的跃迁产生。     

Quantification of Low Concentrations of DNA Using Single Molecule Detection and Velocity Measurement in a Microchannel

We present a novel method for quantifying low concentrations of DNA based on single molecule detection (SMD) for molecular counting and flow measurements inside a microchannel. A custom confocal fluorescence spectroscopic system is implemented to detect fluorescent bursts emitted from stained DNA molecules. Measurements are made one molecule at a time as they flow through a femtoliter-sized laser focal probe. Durations of single molecule fluorescent bursts, which are found to be strongly related to the molecular transit times through the detection region, are statistically analyzed to determine the in situ flow speed and subsequently the sample volume flowing through the focal probe. Therefore, the absolute concentration of a DNA sample can be quantified based on the single molecule fluorescent counts from the DNA molecules and the associated probe volume for a measured time course. To validate this method for quantifying low concentrations of biomolecules, we tested samples of pBR322 DNA ranging from 1 pM to 10 fM (∼3 ng/ml to 30 pg/ml). Besides molecular quantification, we also demonstrate this method to be a precise and non-invasive way for flow profiling within a microchannel.     

基于单光子探测及脉冲位置调制的激光通信关键技术研究

为了实现高灵敏度的空间激光通信,并提高传输信道的抗干扰能力,将单光子探测技术和脉冲位置调制技术相结合,采用门控电路与反馈淬灭电路相结合的方式淬灭单光子探测器雪崩,设计了插入帧头法用于脉冲位置调制解调。用现场可编程门阵列进行了脉冲位置调制解调过程的仿真,验证了插入帧头法的有效性与可行性。在此基础上搭建了1 550 nm的脉冲位置调制激光通信实验,同时测试了单光子探测器在不同参数下的性能。结果表明,当探测效率为25%,触发延时为8.00 ns,门宽为5.0 ns,死时间为0.1μs时,单光子探测器性能最佳。最后测试了不同调制速率下单光子探测器的探测灵敏度,结果表明,当通信码速率为1 Mbps时,通信灵敏度为-51.8 dBm;当通信码速率为4 Mbps时,通信灵敏度为-41.0 dBm,实现了高灵敏度的空间激光通信。     

光谱法研究3H-吲哚探针分子与Triton X-100胶束间的相互作用

用光谱法研究了荧光分子2－（对－己基胺基）苯基－3,3－二甲基－5－乙酯基－3H吲哚基－甲基二辛基磺化铵（A）与Triton X-100胶束间的相互作用,测定了A在不同浓度的Trtiton X-100溶液中的吸收光谱,荧光光谱和荧光寿命,实验得到了A与胶束的结合常数Ks,A在胶束中所处环境的极性及其在胶束中发生的pH效应,结果表明,A与胶束间存在较强的相互作用,A在胶束中所处环境的极性及其在胶束中发生的pH效应,结果表明：A与胶束间存在较强的相互作用;A在胶束中所处环境的极性接近于甲醇的极性,并推测出了A的不同基团在胶束中所处的不同位置,同时发现：在Triton X-100胶束中A可探测到两个位置,但当Triton X-100的浓度过大时,A只能探测到一个位置。     

直肠癌组织与直肠正常组织的荧光光谱研究

用荧光光谱仪对12例离体直肠癌组织和对应的直肠正常组织的荧光光谱(激发光的波长为320 nm)进行了研究。并与相应的红外光谱结果进行了对比。结果表明直肠正常组织的荧光强度明显高于直肠癌组织的荧光强度。直肠组织在波长为492和544 nm的荧光强度的差别可用于区分直肠正常和直肠癌组织。     

Concept for the Traceability of Fluorescence (Beads) in Flow Cytometry: Exploiting Saturation and Microscopic Single Molecule Bleaching

We have determined the fluorescence yield of stained micro beads, used for calibration purposes in flow cytometry, as function of the irradiance of the exciting laser beam. A rate equation model has been applied to derive the number of fluorescence molecules carried by each micro bead. To derive in situ photo-physical properties of the specific dye, required for the rate equation model, we discuss an approach based on flow cytometric sorting of micro beads, which have passed two laser beams with properly chosen different irradiances, and subsequent observation of single molecule bleaching employing high sensitivity microscopy. The feasibility of our approach is demonstrated presenting first results concerning saturation of fluorescence of beads in flow and single molecule bleaching by high sensitivity microscopy.     

水杨酸与牛血清蛋白相互作用的荧光光谱研究

应用荧光光谱研究了水杨酸与牛血清蛋白 (BSA)分子间的相互作用。研究表明 :水杨酸对BSA内源荧光的猝灭机制属于形成化合物所引起的静态猝灭 ,猝灭常数Ksv 为 1 0 97× 10 4 (mol·L- 1 ) - 1 ;水杨酸与BSA反应的结合常数为 7 377× 10 4 ,结合位点数为 1,当水杨酸浓度较低 (摩尔比小于 1∶1)时 ,它与Trp残基或其附近基团结合但并不引起Trp残基微环境的改变 ;根据F rster非辐射能量转移理论 ,计算了授体 受体间的结合距离和能量转移效率     

应用荧光猝灭法研究尼莫地平与牛血清白蛋白的相互作用

应用荧光光谱(FS)和紫外光谱(UV)研究了尼莫地平与牛血清白蛋白(BSA)之间的相互作用。尼莫地平与BSA的结合常数K_A为5.01×104(26 ℃)和4.46×104(36 ℃),尼莫地平在BSA上的结合位点数为1.08±0.01。根据Frster非辐射能量转移理论,求出了尼莫地平与BSA之间的结合距离为3.14 nm(26 ℃)和3.10 nm(36 ℃)。实验表明静态猝灭和非辐射能量转移是 导致尼莫地平对BSA荧光猝灭的两大原因。通过计算热力学参数,可知该药物与牛血清白蛋白的相互作用是一个吉布斯自由能降低的自发过程,且二者之间的作用力以静电相互作用为主。     

The Mean Single Molecule Rate (mSMR) in the Analysis of Fluorescence Fluctuations: Measurements on DNA Mixtures of Defined Composition

Journal of Fluorescence - We present a method for the evaluation of fluorescence fluctuations on the basis of Mandel’s Q parameter, using sampling time-dependent factorial cumulants. By...     

荧光探针技术研究阿特拉津与ct-DNA的相互作用

利用分子荧光探针和紫外吸收光谱技术研究了阿特拉津（Atrazine）与小牛胸腺DNA（ct-DNA）之间的相互作用。实验中选用溴化乙锭（EB）为荧光探针,考察了阿特拉津浓度、磷酸盐、离子强度以及碘化钾对系统荧光的影响。结果表明,阿特拉津对ct-DNA-EB体系的荧光存在猝灭现象,并同时存在静态和动态两种猝灭方式。KI对ct-DNA-Atrazine体系的荧光猝灭及阿特拉津使ct-DNA紫外吸收的增色和红移现象表明阿特拉津与ct-DNA之间存在嵌插作用。磷酸盐、离子强度对ct-DNA-EB-Atrazine体系的荧光强度影响表明,阿特拉津与ct-DNA的磷酸基之间存在非特征性的静电作用,并且高离子强度不利于这种作用。     

基于荧光光谱及矩阵分析的植物油鉴别技术

利用FS920荧光光谱仪测量42个油样(包括36个纯植物油样,3个调和油样和3个混合油样)的荧光光谱,并对其数据矩阵(EEMs)进行归一化处理,确定了植物油特征激发波长及矩阵分析模型。综合分析植物油在特定范围内(激发波长为250～550 nm,发射波长为260～750 nm)的等高线光谱图和特征发射谱线图,将植物油划分为三类;将矩阵分析模型应用于纯植物油鉴别,分类正确率100%;为验证矩阵分析的定量判别能力,对三种混合油样进行分析,得到接近实际配比的分析结果;对市售三种调和油样本进行分析,得出调和油以大豆和菜籽油为基底的结论。通过对植物油荧光光谱的图谱特征和其矩阵模型的分析,证实荧光光谱技术和矩阵分析法对植物油进行分析和种类鉴别的有效性。     

基于荧光光谱技术和GA-BP神经网络的对羟基苯甲酸甲酯钠含量的测定

对羟基苯甲酸甲酯钠是一种使用广泛的防腐剂,食用过量将会危害身体健康,因此各国对对羟基苯甲酸甲酯钠的用量有严格规定。采用FS920荧光光谱仪分析了对羟基苯甲酸甲酯钠在水和橙汁溶液中的荧光光谱特性,发现水溶液的荧光特征峰位于λ_ex/λ_em=380/510 nm,橙汁溶液两个荧光特征峰,分别位于λ_ex/λ_em=440/530 nm和470/530 nm,最佳激发波长为440 nm。从实验结果可以看出两者的特征峰发生了明显的变化。经分析得出,对羟基苯甲酸甲酯钠橙汁溶液与对羟基苯甲酸甲酯钠水溶液相比,荧光特征峰发生变化是由橙汁的荧光特性干扰引起的。为了准确测定鲜橙汁中对羟基苯甲酸甲酯钠含量,根据对羟基苯甲酸甲酯钠橙汁溶液在激发波长λ_ex=440 nm时的相对荧光强度和对羟基苯甲酸甲酯钠含量的关系,基于GA-BP神经网络构建了橙汁中对羟基苯甲酸甲酯钠含量检测数学模型,当网络训练过程中误差精度达到10-3时,网络输出与期望的相关系数为0.996,预测样本的平均回收率为99.52%,平均相对标准偏差为0.86%,预测结果较为理想。结果证明,当浓度范围为0.02~1.0 g·L-1时,荧光光谱技术和GA-BP神经网络相结合的方法能够准确地测定鲜橙汁中对羟基苯甲酸甲酯钠的含量,此方法具有新颖简便性,同时有望应用于一般饮品中对羟基苯甲酸酯类钠盐含量的快速测定。     

多糖对白细胞荧光发射特性及变化机制研究

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...     

Steady-State and Time-Resolved Fluorescence Spectroscopic Studies on Interaction of the N-terminal Region with the Hairpin Loop of the Phytocystain Scb

The steady-state and time-resolved fluorescece spectroscopy is one of the most powerful method to detect and analyze subtle conformation change and interaction between peptide elements in protein. Phytocystatin Scb isolated from sunflower seeds includes a single Trp residue at position 85. In an attempt to investigate the interaction of the N-terminal region of Scb with the first and second hairpin loops by fluorescence spectroscopy of Trp residue, two Scb mutants in which single Trp locates at position 52 and 58, respectively, and their N-terminal removed mutants were generated. The N-terminal truncation changed the fluorescence decay kinetics of Trp52 from the triple exponential to double. Furthermore, the time-resolved fluorescence anisotropy residue indicated that the segmental motion of Trp52 was significantly enhanced by its N-terminal truncation. In contrast, Trp58 and Trp85 had little influence. The N-terminal successive truncations of Scb and its mutants resulted in the weaken inhibitors to papain. These results suggested that the N-terminal region of Scb interacts with the peptide segment preceding the first hairpin loop, thereby stabilizing the conformation of the hairpin loop structure.     

污染水体溶解有机物诱导荧光猝灭特性的实验研究

溶解有机物的含量是衡量水质的常用指标,利用荧光方法可以实现对自然水体溶解有机物的浓度进行高灵敏在线遥测。文章利用三维荧光光谱技术对溶解有机物进行了荧光表征,实验研究了溶解有机物浓度与荧光强度的关系,以及水体酸碱度和金属离子对荧光强度的影响。研究表明,当溶解有机物浓度在小于10 mg·L-1,pH值在6.5～7.5范围内时,荧光强度与浓度具有很好的线性关系;金属离子对溶解有机物诱导荧光有较大猝灭作用,不同金属离子对诱导荧光强度的猝灭效果有显著不同。     

初始物料组成对堆肥理化、生物和光谱学性质的影响

在中国,厌氧发酵的渣滓——沼渣的主要处理方式是作为有机肥直接施用于土壤,此方式对土壤存在不利影响,本研究采用畜禽粪便与沼渣混和堆肥的方式处理沼渣,可避免传统沼渣处理方式的缺陷,改善土壤质量。初始原料组成对堆肥过程有显著影响。为揭示初始物料组成对堆肥理化、生物和光谱学性质的影响,进行了15组不同物料配比的沼渣、猪粪和鸡粪混合堆肥试验。对堆肥理化和生物指标进行了测定,并采用紫外-可见光谱（UV-Vis）、同步和三维荧光光谱（3D-EEM）结合区域体积积分（FRI）和平行因子分析（PARAFAC）识别特征光谱参数,表征堆肥光谱学特性,采用典型对应分析（CCA）分别建立堆肥理化生物学性质和光谱学性质与初始物料组成的关系。结果显示：堆肥理化、生物学性质和光谱学性质都与初始物料组成有较高相关性,前4个排序轴分别合并解释了83.9%和97.5%的样本总变异。影响堆肥理化、生物学性质和光谱学性质的环境因子排序分别为：猪粪量＞鸡粪量＞沼渣量和沼渣量＞猪粪量＞鸡粪量。富碳原料有利于堆肥腐熟,高比例富氮原料不会导致堆肥氨氮累积。在沼渣混合物料堆肥中,低比例的沼渣有利于腐殖质形成。对沼渣与畜禽粪便混合堆肥发酵效果的评价应综合考虑理化、生物学指标和光谱学参数。