Quenchers Induce Wavelength Dependence on Protein Fluorescence Lifetimes

We have analysed the picosecond resolved fluorescence emission decay of horseradish peroxidase A2 and of HEW lysozyme acquired with a streak camera. Analyses of the fluorescence decay data of both proteins revealed that the dynamics of the decay is dependent on the emission wavelength. Our data strongly indicates that resonance energy transfer occurring between aromatic residues and different protein fluorescence quencher groups, and the nature of the quencher groups, are the causes of the observed wavelength dependent mean lifetime distribution. Using the global analysis data to calculate the fluorescence mean lifetime at each wavelength revealed that for lysozyme, the mean fluorescence lifetime increased with observation wavelength, whereas the opposite was the case for peroxidase. Both proteins contain strong fluorescence quencher groups located in close spatial proximity to the protein’s aromatic residues. Lysozyme contains disulfide bridges as the main fluorescence quencher whereas peroxidase contains a heme group. Both for lysozyme and horseradish peroxidase there is a clear correlation between the observed fluorescence mean lifetime of the protein at a particular emission wavelength and the respective quencher’s extinction coefficient at the respective wavelength. Furthermore, our study also reports a comparison of the analyses of the fluorescence data done with three different methods. Analyses of the fluorescence decay at 10 different fluorescence emission wavelengths revealed significant differences in both fluorescence lifetimes and the pre-exponential factor distributions. Such values differed from the values recovered from the integrated decay curves and from global analyse.     

Asymmetrical Distribution of Intrinsic Fluorescence Lifetimes in Proteins

Dynamic fluorescence measurements of proteins in solution are often interpreted in terms of continuous distributions of lifetimes, which reflect the intrinsic structural heterogeneity of these systems. In several cases a single Gaussian or Lorentzian symmetric distribution has been used to fit the data. In this paper we describe a new nonsymmetric Lorentzian function which contains three free parameters (the center, the left, and the right widths) like the double-discrete exponential model (the two lifetimes and one preexponential factor). Simulated data in the frequency domain have been used to compare the fits obtained with these different approaches, introducing a new parameter, , which quantitatively measures the asymmetry of the distribution or the ratio of the two preexponential factors, in the continuous and discrete models, respectively. Real measurements of a mixture of independent fluorophores, as well as of protein fluorescence decays, have also been performed and analyzed in terms of the new asymmetric function. The data have also been fitted with traditional discrete methods (such as the two- and the three-exponential decay) and with another asymmetric function, namely, the skewed Gaussian distribution.     

Study of Preferential Solvation in Binary Mixtures by Means of Frequency-Domain Fluorescence Spectroscopy

The preferential solvation of 8-N,N-(dimethylamino)-11H-indeno[2,1-a]pyrene, Py(S)DMA, in its transient charge transfer (CT) state in binary solvents such as toluene/DMSO liquid mixtures was studied by means of frequency-domain fluorometry. The data obtained were considered within the following kinetic scheme: the preferential solvation was described by the system of consecutive reversible reactions of which each step is associated with the absorption of one DMSO molecule in the first solvation shell of the fluoresent Py(S)DMA dipolar CT molecule. The rate constants of the first two reversible elementary processes (i.e., the decay of solvation complexes of Py(S)DMA with one and two polar molecules, k _–1 = 1.1 10⁹ s^–1 and k _–2 = 1.4 10⁹ s^–1) were determined.     

乙醇水溶液荧光发射的时域和频域特性研究

针对乙醇水溶液荧光发射的四个特征参量进行了研究,得出了该溶液发射荧光光子的时域和频域特征参量。发射光谱和激发光谱表明乙醇水溶液中含有三个结构不同的发光物质,其发射峰分别位于290 nm,305 nm和330 nm处,与其相对应的最佳吸收峰为265 nm,280 nm和236 nm。荧光强度随溶液中乙醇与水体积混合比的变化规律也证实了三种不同发光结构的存在。在荧光光谱峰值波长处分别监测其荧光强度随时间的衰变过程,将获得的荧光衰减动力学曲线采用指数方法拟合并进行解卷积处理,测试的荧光寿命分别对应8 ns,12 ns,25 ns。结合乙醇水溶液荧光发射的四个特征参量可以看出:乙醇分子和水分子发生团簇作用形成了三个新的分子结构从而可发射具有不同能量的荧光光子。该研究结果能为乙醇水分子的团簇结构研究提供参考。     

Rapid Frequency-Domain FLIM Spinning Disk Confocal Microscope: Lifetime Resolution,Image Improvement and Wavelet Analysis

A spinning disk confocal attachment is added to a full-field real-time frequency-domain fluorescence lifetime-resolved imaging microscope (FLIM). This provides confocal 3-D imaging while retaining all the characteristics of the normal 2-D FLIM. The spinning disk arrangement allows us to retain the speed of the normal 2-D full field FLIM while gaining true 3-D resolution. We also introduce the use of wavelet image transformations into the FLIM analysis. Wavelets prove useful for selecting objects according to their morphology, denoising and background subtraction. The performance of the instrument and the analysis routines are tested with quantitative physical samples and examples are presented with complex biological samples.     

A Polar Representation and the Morse Index Theorem

In this Letter we study the behavior of the eigenvalues of an operator defined by the action associated to a generic quadratic time-dependent Hamiltonian. This is done using a polar representation of the solutions of the corresponding linear Hamiltonian system. A proof of the Morse index theorem is given.     

Ruby Crystal for Demonstrating Time- and Frequency-Domain Methods of Fluorescence Lifetime Measurements

We present experiments that are convenient and educational for measuring fluorescence lifetimes with both time- and frequency-domain methods. The sample is ruby crystal, which has a lifetime of about 3.5 milliseconds, and is easy to use as a class-room demonstration. The experiments and methods of data analysis are used in the lab section of a class on optical spectroscopy, where we go through the theory and applications of fluorescence. Because the fluorescence decay time of ruby is in the millisecond region, the instrumentation for this experiment can be constructed easily and inexpensively compared to the nanosecond-resolved instrumentation required for most fluorescent compounds, which have nanosecond fluorescence lifetimes. The methods are applicable to other luminescent compounds with decay constants from microseconds and longer, such as transition metal and lanthanide complexes and phosphorescent samples. The experiments, which clearly demonstrate the theory and methods of measuring temporally resolved fluorescence, are instructive and demonstrate what the students have learned in the lectures without the distraction of highly sophisticated instrumentation.     

Parameter Recovery in Frequency-Domain Time-Resolved Fluorescence Spectroscopy; Resolution of the Prototropic Forms of 5-Carboxyfluorescein in the Physiological pH Range

In time-resolved fluorescence spectroscopy, the resolution of fluorescence species becomes increasingly difficult as their respective lifetimes get closer. For a biexponential decay, a factor of 1.4 between the two decay times is commonly accepted as the practical resolution limit. The goal of the present contribution is to characterize the fluorescence probe 5-carboxyfluorescein using frequency-domain time-resolved fluorescence spectroscopy (FD-TRFS). To resolve the different prototropic forms of this probe, the limit above had to be overcome. For this purpose, the standard global analysis method was used, and special emphasis was put on the errors associated with the recovered parameters. In particular, a Monte Carlo simulation was performed to estimate these errors and the results of this analysis were compared with those delivered by software packages widely used in the field. The lifetimes of the trianionic and dianionic forms of 5-carboxyfluorescein were 4.01 ± 0.06 and 3.03 ± 0.09 ns, respectively, and the pK _a for this acid–base equilibrium was determined to be 6.9 ± 0.3.     

扩展目标夏克-哈特曼波前传感器的频域迭代算法研究

扩展目标夏克-哈特曼波前传感器(SH-WFS)子图像之间偏移量的计算是影响波前传感精度的关键,通常采用相关算法来实现,并通过抛物线插值达到亚像元精度。子图像间的相对偏移量计算也可采用频域相移量估算的方法进行计算,频域算法还可通过迭代进一步提高算法精度。对频域迭代算法进行了理论分析、仿真和实验研究,结果表明,频域迭代算法在信噪比高于4…1时,具有比抛物线插值法更高的计算精度;在信噪比较低时,与抛物线插值法精度相近。     

Measurements of Key Parameters for Birefringent Single-Mode Optical Fibers by Optical Frequency-Domain Interferometry

This Letter describes measurements of modal birefringence (MB) and polarization mode dispersion (PMD) of a birefringent single-mode optical fiber for use at 1.5 #x03BC;m in optical wavelength. Optical frequency-domain interferometry based on a fixed analyzer method is applied to the measurement of orthogonal polarization components of light guided in the fiber. An amplified spontaneous emission source having an optical wavelength range between 1520 and 1560 nm is used. A channeled interference spectrum is obtained to measure MB and PMD in turn. These two parameters are not frequency-dependent in the above optical wavelength range and MB = 4.0 #x00D7; 10^#x2212;4 and PMD = 1.33 ps/m are obtained.     

Fluorescence quenching of newly synthesized biologically active coumarin derivative by aniline in binary solvent mixtures

The fluorescence quenching of newly synthesized coumarin (chromen-2-one) derivative, 4-(5-methyl-3-furan-2-yl-benzofuran-2-yl)-7-methyl-chromen-2-one (MFBMC) by aniline in different solvent mixtures of benzene and acetonitrile was determined at room temperature (296 K) by steady-state fluorescence measurements. The quenching is found to be appreciable and positive deviation from linearity was observed in the Stern-Volmer (S-V) plots in all the solvent mixtures. This could be explained by static and dynamic quenching models. The positive deviation in the S-V plot is interpreted in terms of ground-state complex formation model and sphere of action static quenching model. Various rate parameters for the fluorescence quenching process have been determined by using the modified Stern-Volmer equation. The sphere of action static quenching model agrees very well with experimental results. The dependence of Stern-Volmer constant K_SV, on dielectric constant ε of the solvent mixture suggests that the fluorescence quenching is diffusion-limited. Further with the use of finite sink approximation model, it is concluded that these bimolecular quenching reactions are diffusion-limited. Using lifetime (τ_o) data, the distance parameter R′ and mutual diffusion coefficient D are estimated independently.     

氨基甲酸酯类农药荧光分析研究

分析了西维因、克百威、残杀威等几种常用的氨基甲酸酯类农药的分子结构并揭示了它们的荧光特性,利用稳态荧光光谱仪对西维因和克百威标准溶液进行了荧光光谱实验,结果表明,氨基甲酸酯类农药在一定的溶剂条件下能够受激发产生荧光,而且荧光光谱清晰、分辨率高,受干扰较小,利用荧光光谱分析法对它们进行检测是可行的。     

Histons: New Quasi-particles and Time-Resolved Fluorescence Shift of Polar Disordered Media

With statistical thermodynamics methods, it is shown that the evolution of a polar luminescence probe + polar disordered medium system after pulse excitation is described by an equation for damped vibrations. From this it follows that in the solvation shell of the excited solute probe, not only synchronous librations but also cooperative jump reorientations of solvent molecules take place, and their damping motion is caused by localized dielectric friction. This collective molecular dynamics is examined as the motion of a quasi-particle called a histon. The histon moment of inertia and mass are determined. The concept of l- and j-histons are also introduced. In terms of this model the full correlation functions for nonequilibrium solvent dynamics are derived. The behavior of steady-state and time-resolved spectra is examined and it is found that the limiting short-wavelength position of the steady-state spectrum at a low temperature and that for time-dependent fluorescence at t 0 are not identical. The histon model has been used for interpretation of the emission band collapse that is observed in long-time evolution experiments. Some other problems of spectral broadening are also discussed. As predicted, sharp expansion of the fluorescence band takes place on an ultrafast time scale due to dephasing of l-histons.     

Frequency-Domain Measurements of Diffusing Photon Propagation in Solid Phantoms

Photon propagation in a highly scattering medium such as living tissue is well described by the photon diffusion equation. In a homogeneous and isotropic case, the equation has analytically been solved for a medium with a simple geometrical shape. We report frequency-domain measurements using slab-shaped solid phantoms. Comparison of the results with computer simulations proves the validity of extrapolated boundary conditions.     

玄参汤剂的荧光光谱分析

测量了相应的激发光谱以及溶液的pH值,发现同种玄参汤剂中的荧光峰值波长随着激发波长的增加而增加;冷浸玄参汤剂的荧光峰为441 nm,而沸煮玄参汤剂的为532 nm;相应激发光谱由双峰结构向单峰结构转变,并且激发峰值也有明显的红移;同时其pH值由5.5变化到4.1。结合有机物混合体系中的荧光产生机理,解释了上述现象的产生原因：玄参汤剂中的不同荧光分子之间的相互作用以及相同荧光分子内部的能量转移;加热所导致的荧光大分子含量的增加和荧光分子间氢键二聚体的形成。文章对中药玄参的药理学研究具有一定的参考价值,为玄参汤剂质量鉴定提供了一种有效的测量方法。     

一种新型微通道分析系统——发光二极管诱导荧光检测器

报道了一种新型发光二极管诱导荧光检测器。该系统采用共线性光学结构、通过柱上检测可与微通道分析系统在线联用。以异硫氰酸荧光素(FITC)及其标记的氨基酸为标样、结合毛细管电泳和流动注射技术评价了该系统。对FITC标记的苯丙氨酸浓度检测限为10-8 mol·L-1(柱上检测,0.05 mm内径),在1×10-7～2×10-5 mol·L-1有很好的线性关系(r2 =0.999),6次连续进样所得峰高值相对标准偏差(RSD)小于3 %。     

掺染料微结构光纤荧光传感器的理论分析

针对一般光纤荧光传感器收集荧光能力不足的缺陷,设计了一种在微结构聚合物光纤的空气孔内填充掺有机染料高折射率液体的荧光传感器.使用可调节边界条件傅里叶分解法计算了这种微结构光纤的模场分布,分析了光纤结构参量和液体折射率对荧光捕获分数的影响,结果表明,使用小纤芯半径和高于纤芯折射率的液体可以增强激发光的吸收效率,增大荧光捕获分数,提高光纤荧光传感器的灵敏度.     

Lifetimes of metastable states in Sr II

The lifetimes of the 4d ²D _3/2,5/2 levels of Sr⁺ have been determined both experimentally and theoretically. The experiment was performed at an ion storage ring utilising collinear laser excitation. The calculation was performed by the Hartree-Fock method including relativistic effects and core polarisation. The obtained lifetimes (which are about 0.4 s) are discussed in detail and compared with earlier published results. In addition, calculated lifetimes of a large number of excited states in Sr⁺ are included. Received 15 February 2000 and Received in final form 20 March 2000     

含萘荧光单体和聚合物的紫外和荧光分析

分别用紫外分光光度法和荧光光谱法对含萘荧光单体2-(甲基丙烯酰氧)-N,N-二甲基-N-(1-萘甲基)乙基氯化铵和相应的聚合物进行检测。在λmax=222nm下测定不同浓度的含萘荧光单体和聚合物的吸光度,线性相关系数均为0.9995。同时在λex=222nm和λem=332nm条件下研究两者的荧光光谱,样品浓度与荧光强度成良好线性关系,相关系数可达0.9998。对比两种分析方法表明,两者的荧光测定检出限相对较低,操作简单快捷,准确性和可行性高,可很好地监测水处理剂中聚合物的含量,从而实现水处理系统在线检测和自动控制。     

一种用于化学分析的新方法——荧光毛细分析法

提出了一种新方法——荧光毛细分析法(fluorescence capillary analysis,FCA)。FCA法用毛细管做为荧光反应和测定的载体,实现了微量样品的微量分析。本方法特点是操作简便,试样、试剂用量少(<10 μL);毛细管及毛细生物反应器可循环使用,测定成本极低,能大大减少环境污染。该研究基于FCA法测定了核黄素、还原型辅酶Ⅰ,基于固定化酶FCA法测定了酒中乙醇,基于固定化核苷酸探针基因FCA法测定了靶核苷酸,都初步得到了满意的结果。FCA法将可以应用到临床医学、药学、生化、染料工业、发酵食品、环境检测等各个领域中。