Spectral Properties of Thioflavin T and Its Complexes with Amyloid Fibrils

Comparative analysis of the absorption and fluorescence spectra and fluorescence excitation spectra of thioflavin T (ThT) in various solvents and in the composition of amyloid fibrils has shown that ThT, when excited in the region of the long-wavelength absorption band, fluoresces in the spectral region with a maximum at 478–484 nm. The appearance in aqueous and alcohol solutions of a fluorescence band with a maximum near 440 nm has been attributed to the presence in the composition of the ThT preparations of an impurity with an absorption band in the 340–350-nm range. The literature data showing that in glycerol ThT has a wide fluorescence spectrum with two maxima are due to the artifact connected with the use of a high concentration of the dye. It has been suggested that the cause of the low quantum yield of ThT aqueous and alcohol solutions is the breakage of the system of conjugated bonds due to the reorientation of the benzothiozole and benzaminic rings of ThT in the excited state with respect to one another. The main factor determining the high quantum yield of fluorescence of ThT incorporated in fibrils is the steric restriction of the rotation of the rings about one another under these conditions. The suggestions made have been verified by the quantum-chemical calculation of the ThT molecule geometry in the ground and excited states.     

Monitoring Membrane Protein Conformational Heterogeneity by Fluorescence Lifetime Distribution Analysis Using the Maximum Entropy Method

Due to the inherent difficulty in crystallizing membrane proteins, approaches based on fluorescence spectroscopy have proved useful in elucidating their conformational characteristics. The ion channel peptide gramicidin serves as an excellent prototype for monitoring membrane protein conformation and dynamics due to a number of reasons. We have analyzed conformational heterogeneity in membrane-bound gramicidin using fluorescence lifetime distribution analysis of tryptophan residues by the maximum entropy method (MEM). MEM represents a model-free and robust approach for analyzing fluorescence lifetime distribution. In this paper, we show for the first time, that fluorescence lifetime distribution analysis using MEM could be a convenient approach to monitor conformational heterogeneity in membrane-bound gramicidin in particular and membrane proteins in general. Lifetime distribution analysis by MEM therefore provides a novel window to monitor conformational transitions in membrane proteins.     

Analysis of Simulated Fluorescence Intensities Decays by a New Maximum Entropy Method Algorithm

A new algorithm for the Maximum Entropy Method (MEM) is proposed for recovering the lifetime distribution in time-resolved fluorescence decays. The procedure is based on seeking the distribution that maximizes the Skilling entropy function subjected to the chi-squared constraint χ ²?~?1 through iterative linear approximations, LU decomposition of the Hessian matrix of the lagrangian problem and the Golden Section Search for backtracking. The accuracy of this algorithm has been investigated through comparisons with simulated fluorescence decays both of narrow and broad lifetime distributions. The proposed approach is capable to analyse datasets of up to 4,096 points with a discretization ranging from 100 to 1,000 lifetimes. A good agreement with non linear fitting estimates has been observed when the method has been applied to multi-exponential decays. Remarkable results have been also obtained for the broad lifetime distributions where the position is recovered with high accuracy and the distribution width is estimated within 3 %. These results indicate that the procedure proposed generates MEM lifetime distributions that can be used to quantify the real heterogeneity of lifetimes in a sample.     

Thioflavin T Displays Enhanced Fluorescence Selectively Inside Anionic Micelles and Mammalian Cells

Thioflavin T (ThT) has been widely employed to detect amyloid fibrils in tissues and recently in presence of SDS micelles. However, the contribution of membranes or micelles to ThT fluorescence has never been investigated. In this paper, we show for the first time that the anionic micellar microenvironment of SDS has a profound impact on the absorption and fluorescence spectra of ThT in sharp contrast to cationic (CTAB) and neutral micelles (Triton X-100 & Tween 20). Unlike CTAB or Triton X-100 or Tween 20 micelles, formation of SDS micelles shifts the λ_max for ThT absorption from 412 nm in buffer to 428 nm inside the micelle, with a 28% increase in the peak molar absorptivity and a ∼13 fold increase in ThT fluorescence (λ_max = 489 nm). Extending these observations to cell plasma membranes, we show that ThT can quickly enter and appear selectively fluorescent inside mammalian cells like BHK21 and HT29, against a dark background owing to negligible fluorescence from free ThT in aqueous medium. The above results suggest that ThT can be a useful probe for live cell imaging and for selectively labeling micelles on the basis of the charge in the polar headgroup. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Photo-physical and lasing characterisation of a polyparaphenylenevinylene (PPV) neat film

The wave-guided travelling-wave laser action (amplified spontaneous emission) of a neat film of poly(p-phenylenevinylene) (PPV) on a quartz glass substrate prepared by a sulfinyl precursor technique is studied. The samples are transversally pumped with picosecond excitation pulses (wavelength 347.15 nm, duration 35 ps). Lasing occurs at 550 nm. The optical constants of the neat films are determined by transmittance measurements exploiting the multiple beam interference in the transparency region. A fluorescence spectroscopic characterisation is carried out determining the fluorescence quantum distribution, fluorescence quantum yield, degree of fluorescence polarisation, and fluorescence lifetime. The emitting chromophore size (emitting singlet exciton extension) is determined by the ratio of exciton radiative lifetime to repeat-unit based radiative lifetime. The obtained size of about two repeat units is discussed in a disordered solid-state polymer model.     

Characterization of human immunodeficiency virus-1 (HIV-1) rev by (time-resolved) fluorescence spectroscopy

Fluorescence spectroscopy has been applied to the single tryptophan-containing regulatory protein Rev of human immunodeficiency virus (HIV-1). The fluorescence emission was found to have a maximum at 336 nm which refers to a surrounding of the chromophore of intermediate polarity. Fluorescence transients recorded at the maximum of fluorescence were found to decay nonexponentially. A bimodal lifetime distribution is obtained from exponential series analysis (ESM) with centers at 1.7 and 4.5 ns. Two microenvironments for tryptophan are suggested to be responsible for the two lifetime distributions. No innerfilter effect occurred in a Rev solution up to a concentration of 40 μM. A data quality study of ESM analysis as function of collected counts in the peak channel maximum (CIM) showed that, for reliable reconvolution, at least 15,000 CIM are necessary. The widths of the two distributions are shown to be temperature dependent. The broadening of the lifetime distributions when the temperature is raised to 50°C is interpreted as extension of the number of conformational substates which do not interconvert on the fluorescence time scale. The thermal deactivation (temperature quenching) is reflected in a constant decrease in the center of the short-lived lifetime distribution.     

Photochemical Processes in Aqueous Solutions of Immunoactive 8-Azasteroid

Based on an analysis of the absorption, fluorescence excitation, and fluorescence spectra, and the duration of the excited states of an aqueous solution (pH 7.4) of 2,3-dimethoxy-8-azagona-1,3,5(10),13-tetraen-12,17-dione molecules, the existence of two centers emitting longwave (L centers) and shortwave (S centers) fluorescence has been established. Irradiation of the solution accelerates the accumulation of L centers and considerably reduces the quantity of S centers that represent the initial molecule. Analysis of the induced-absorption spectra and their kinetics with picosecond time resolution shows that ionization of the molecule studied is the initial photochemical act and formation of a dehydro derivative of the initial molecule or protonation of carbonyl groups is the next act.     

Diphenylhexatriene-phosphatidylcholine fluorescence in POPC vesicles: Comparison of the exponential-series and the maximum-entropy methods

We have investigated the time-resolved fluorescence of diphenylhexatriene (DPH) covalently linked to phosphatidylcholine (PC) in palmitoyloleoylglycerophosphocholine (POPC) vesicles with special consideration of the comparison of two methods for distributional lifetime analysis: the exponential-series method (ESM) and the maximum-entropy method (MEM). Generally, both methods were found to reveal equivalent results on high-quality data. Different are the shapes of the recovered distributions (symmetry and width) as well as the time effort for the numerical analysis.     

Fluorescence of organic dyes in lipid membranes: Site of solubilization and effects of viscosity and refractive index on lifetimes

The fluorescence decay of several organic dye molecules intercalated in egg phosphatidylcholine lipid membrane vesicles is consistent with the existence of two or three prominent lifetime components rather than a single continuous distribution of lifetimes. The major lifetime components are identified with different sites of solubilization in the membrane. The variation of the lifetime of the membrane-bound dye was studied as a function of the sucrose concentration, which varied the viscosity and refractive index of the aqueous solution. The combined effect of viscosity and refractive index on the lifetime of the dye was used to identify the site of solubilization of the dye in the membrane. The study was useful to identify dye molecules on the surface which are exposed to the aqueous phase, for which the fluorescence lifetime increased systematically with sucrose (viscosity effect). More importantly, it was possible in a few cases to identify the dye molecules which are oriented in the membrane phase, and the fluorescence lifetime decreased systematically with sucrose (refractive index effect). Anomalous values of order parameters determined from the refractive index effect are explained in terms of an orientational distribution of the linear dye molecule weighted in favor of mutually orthogonal orientations.     

Maximum entropy: a complement to Tikhonov regularization for determination of pair distance distributions by pulsed ESR

Tikhonov regularization (TIKR) has been demonstrated as a powerful and valuable method for the determination of distance distributions of spin-pairs in bi-labeled biomolecules directly from pulsed ESR signals. TIKR is a direct method, which requires no iteration, and, therefore, provides a rapid and unique solution. However, the distribution obtained tends to exhibit oscillatory excursions with negative portions in the presence of finite noise, especially in the peripheral regions of the distribution. The Shannon-Jaynes entropy of a probability distribution provides an intrinsic non-negativity constraint on the probability distribution and an unbiased way of obtaining information from incomplete data. We describe how the maximum entropy regularization method (MEM) may be applied to solve the ill-posed nature of the dipolar signal in pulsed ESR. We make use of it to suppress the negative excursions of the distance distribution and to increase the tolerance to noise in the dipolar signal. Model studies and experimental data are investigated, and they show that, with the initial or "seed" probability distribution that is required for MEM taken as the TIKR result, then MEM is able to provide a regularized solution, subject to the non-negativity constraint, and it is effective in dealing with noise that is problematic for TIKR. In addition we have incorporated into our MEM method the ability to extract the intermolecular dipolar component, which is embedded in the raw experimental data. We find that MEM minimization, which is implemented iteratively, is greatly accelerated using the TIKR result as the seed, and it converges more successfully. Thus we regard the MEM method as a complement to TIKR by securing a positive pair distance distribution and enhancing the accuracy of TIKR.     

用于光通信的高速响应有机电致发光器件

采用直接光强调制的方法,建立了一种新型有机电致发光器件(OLED)的光电信号传输体系,研究了发光层掺杂、发光面积和预置电压对OLED响应速度的影响。结果发现:与发光层为单独的Alq3的器件相比较,掺杂rubrene的发光层的荧光寿命较短,响应较快;减小OLED的发光面积能提高OLED的响应速度,并在0.02 mm2的发光面积上实现了100 Mbit/s的信号传输速度;同时,预置直流电压也能改善OLED的响应速度。最后,提出将柔性OLED与聚合物波导及有机光电二极管结合,实现了一种全有机的柔性光电子体系。     

33 Hz脉冲电场辐照对胰岛素淀粉样变性的影响

蛋白质结构失稳后会发生错误折叠、集聚或纤维化,不仅丧失正常功能,有时甚至会产生生物毒性,导致相关疾病。纤维化了的蛋白质是神经退行性疾病以及二型糖尿病等的主要诱因,它在溶液中具有和淀粉类似的特性,因此蛋白质的纤维化又被称为淀粉样变性。电磁辐射是引起蛋白质结构失稳的一个常见因素。经电场辐照后,蛋白质会发生去折叠和集聚,改变自发折叠机制。同时,经受电场辐照的扰动而失稳的蛋白质,纤维化过程也会受到一定的影响。以胰岛素为研究对象,利用硫磺素T(ThT)染色的荧光光谱法、透射电子显微镜(TEM)以及圆二色谱(CD)法,分别从纤维化的宏观动力学过程、成熟纤维丝微观形貌以及原纤维二级结构组成等不同角度,探究经33 Hz脉冲电场(PEF)不同电场强度及不同持续时间辐照后蛋白质在体外淀粉样变性机制的变化情况。结果表明,胰岛素溶液经过33 Hz PEF辐照后,淀粉样变性初期产生前体蛋白的成核期延长,原纤维丝聚集延长过程(即中间产物的寿命)缩短,形成的成熟纤维丝长度变短且整齐成簇无分支。这些效应随着辐照电场强度和辐照时间的增加而有不同程度的加强。研究结果一致说明PEF辐照对胰岛素的淀粉样变性有一定的抑制作用。     

Electron density distribution in Si and Ge using multipole, maximum entropy method and pair distribution function analysis

The local, average and electronic structure of the semiconducting materials Si and Ge has been studied using multipole, maximum entropy method (MEM) and pair distribution function (PDF) analyses, using X-ray powder data. The covalent nature of bonding and the interaction between the atoms are clearly revealed by the two-dimensional MEM maps plotted on (100) and (110) planes and one-dimensional density along [100], [110] and [111] directions. The mid-bond electron densities between the atoms are 0.554 e/?³ and 0.187 e/?³ for Si and Ge respectively. In this work, the local structural information has also been obtained by analyzing the atomic pair distribution function. An attempt has been made in the present work to utilize the X-ray powder data sets to refine the structure and electron density distribution using the currently available versatile methods, MEM, multipole analysis and determination of pair distribution function for these two systems.      

基于时间相关单光子计数的荧光寿命成像技术

采用时域法中的时间相关单光子计数方法记录荧光寿命,时间相关单光子计数采用多波长通道同时记录荧光光子数,可以提高计数效率和信息量,还可以在稳态图像中分离不同荧光团,形成4维图像。并采用多光子激发技术,利用长波长光源发出的两个或多个光子可以激发出一个短波长的光子。多个光子必须几乎同时到达激发点,才能提供被激发分子足够的能量以产生荧光。多光子激发波长较长,生物组织对其散射减小,因而可以穿透到更深层的组织,从而提高荧光成像深度和空间分辨力,并减少对活体样品的损伤。     

Different Conformation of Thiol Protease Inhibitor During Amyloid Formation: Inhibition by Curcumin and Quercetin

Cystatins are thiol proteinase inhibitors ubiquitously present in mammalian body and serve various important physiological functions. In the present study, we examined the effects of acid denaturation on newly identified thiol protease inhibitors from the lungs of Capra hircus (Goat) with a focus on protein conformational changes and amyloid fibril formation. Acid denaturation as studied by CD (Circular Dichroism) and fluorescence spectroscopy showed that purified inhibitor named GLC (Goat Lung Cystatin) populates three partly unfolded species, a native like state at pH?3.0, a partly unfolded intermediate at pH2.0, and unstructured unfolded state at pH?1.0, from each of which amyloid like fibrils grow as assessed by thioflavin T (ThT) spectroscopy. The result showed, native like structure formed at pH?3.0 is more responsive towards amyloid formation when compare to other conformation of proteins. Morphology of the protein species incubated for amyloid process was observed using transmission electron microscopy (TEM). Moreover, anti-fibrillogenic effects of curcumin and quercetin were analysed using ThT binding assay. Curcumin and quercetin produced a concentration dependent decline inThT fluorescence suggesting deaggregation of the fibrils. When added prior to amyloid fibril initiation 50 μM curcumin inhibited amyloid aggregation. However, more quercetin is needed to prevent the same extent of fibrillation. Implications for therapeutics in view of polyphenols as essential nutrients are suggested in lung diseases.     

Photoluminescence in ZnO-CdO-SiO2 glasses

Photoluminescence (PL) experiments have been carried out in glasses from the ZnO-CdO-SiO₂ system. The glasses exhibit broad bands in the PL spectra centered between 540 and 560 nm for emissions and at about 250 nm for excitations. Decay time curves for the different glass samples appear, within experimental errors, quite similar. The curves are not a simple exponential and have been discomposed as a sum of three exponentials with lifetime components of about 0.025, 0.1, and 0.52 ms. The results seem to indicate that luminescence is dominated by short ordering, nuclei and/or crystalline centers.     

A Spectroscopic Study of 2-[4′-(Dimethylamino)phenyl]-benzothiazole Binding to Insulin Amyloid Fibrils

The spectroscopic properties of 2-[4′-(dimethylamino)phenyl]-benzothiazole (BTA-2) in solution and in the presence of amyloid fibrils were investigated using absorption and fluorescence spectroscopy. Solution studies show that BTA-2 forms micelles in aqueous solutions, but that the dye can be solvated upon the addition of acetonitrile (CH₃CN). BTA-2 binds to amyloid fibrils in solution leading to a characteristic blue-shift in the emission spectrum and an increase in fluorescence intensity. However, in solutions with increasing CH₃CN concentration, there was a marked decrease in binding of the BTA-2 to fibrils. Studies demonstrating the effect of BTA-2 concentration on binding were performed. A comparison with the standard amyloid fluorescent marker, thioflavin T (ThT), showed that BTA-2 is more fluorescent, making it an excellent dye to label amyloid samples.     

激光诱导荧光寿命及其测量

激光诱导荧光特性的研究可用于包括心血管病在内的多种疾病的诊断。荧光发射包括光谱（频域）和时间（时域）两方面的信息，后者表现为荧光寿命。在很多情况下，测量荧光寿命是比测量光谱更为有效的诊断方法。本文从理论上讨论了荧光寿命问题，并介绍两种测量方法，可用于测量人体正常组织和病变组织的激光诱导荧光寿命     

基于激光诱导叶绿素荧光寿命成像技术的植物荧光特性研究

针对植物荧光遥感探测中信号易受干扰的问题, 提出了一种用于评估植物生长状况及环境监测的荧光寿命成像技术. 采用凹透镜对355 nm波长的激光扩束, 再照射植物激发叶绿素荧光, 由增强型电荷耦合器件接收荧光信号. 采用时间分辨测量法, 连续用相同激光脉冲照射植物以激发相同的荧光信号, 同时不断改变激光脉冲触发探测器启动的延时时间, 从而能够得到完整的离散荧光信号分布图像. 对植物特定位置点产生的离散荧光信号进行拟合, 再运用一种改进型的迭代解卷积法可反演高精度的荧光寿命; 进而反演图像各点的荧光寿命以生成植物的荧光寿命分布图. 该方法所绘制的荧光寿命图比荧光强度图能更准确地反映植物内部的叶绿素含量, 并对活体植物叶绿素荧光寿命的物理特性进行了初步研究, 证明叶绿素荧光寿命与植物生理状态存在一定关联; 并且叶绿素荧光寿命与活体植物所处环境存在着复杂的关系. 未来将与生物物理学家们合作, 继续探寻叶绿素荧光寿命与植物生存环境的关系.     

Fluorescence properties of Bi4Ge3O12(BGO) single crystals under laser excitation: Excited state dynamics and saturation effects

Laser-excited techniques were used to investigate the optical properties of bismuth germanate crystals. Absorption, reflectivity, excitation, emission, lifetime, time-resolved fluorescence, photoconductivity, thermally stimulated conductivity measurements were performed at various temperatures on single crystals of different origins.The absorption is shown to occur in bismuth and germanate centers while both intrinsic and perturbed Bi³⁺ ions together with impurities contribute to the total fluorescence.The emission mechanism at room temperature involves a thermally activated energy migration, and at low temperature localized emitting centers. Formation of deep holes in the wide emission band at room temperature reveals saturation effects on various luminescent centers, promoted by energy migration. Trapped exciton models are proposed to explain the excited state dynamics occurring at low and room temperature.