Tyrosyl Fluorescence Decays and Rotational Dynamics in Tyrosine Monomers and in Dipeptides

Picosecond time-correlated single-photon counting was used to measure fluorescence lifetimes and fluorescence anisotropy decays of tyrosine and the tyrosine–alanine and tyrosine–leucine dipeptides. After excitation of tyrosine at 287 nm two emitting species were observed, one at 303 nm with a lifetime of 3.3 ns and another at 340 nm with a lifetime of 360 ps. The rotational correlation time of tyrosine at 303 nm is 38 ps in water at pH 7 and depends linearly on viscosity with a slope of 44 ps/cP, consistent with Stokes–Einstein–Debye theory. We calculated a value of 45 ns for the radiative lifetime of tyrosine, yielding a fluorescence quantum yield of 0.07. The dipeptides Tyr–Ala and Tyr–Leu exhibit two- or three-exponential decays. The amplitudes of the decay components for three-exponential fits correlate closely with the populations of rotamers in these peptides as determined by NMR. The quenching of dipeptide fluorescence is shown to depend on the solvent polarity, strongly supporting the hypothesis that tyrosyl fluorescence in peptides is quenched by charge transfer. The rotational correlation times of tyrosine, Tyr–Ala, and Tyr–Leu increase linearly with the van der Waals volumes. However, rotational relaxation is somewhat faster than expected from Stokes–Einstein–Debye theory with stick boundary conditions.     

Hyper Rayleigh Scattering Yields Improved Response Function in Analysing 2-Photon Excited Fluorescence

An accurate instrumental response function is needed to conclusively deconvolute fluorescence data based on time-correlated single-photon counting (TCSPC) and multiphoton excitation. Routinely the response function is measured as Rayleigh scattering (RS) from a colloidal solution, even if the excitation is a multiphoton event. Present work demonstrates that a response function obtained as hyper Rayleigh scattering (HRS) provides a better choice for deconvolution of 2-photon excited fluorescence decays. The 1- and 2-photon response functions were monitored as RS and HRS from colloidal gold particles at 800 and 400 nm, respectively.     

基于超短脉冲的癌细胞荧光光谱

研究用于癌症诊断与治疗的光敏剂血卟啉(hematoporphyrin derivative,HPD)的超快光动力学过程。采用超短脉冲激光光谱技术和皮秒时间相关单光子计数系统,测量经血卟啉培养的活体癌细胞与正常细胞的荧光光谱、荧光寿命特性及荧光峰值强度随时间的变化,观测到:癌细胞样品在645nm处具有特征发射光谱峰;癌细胞与正常细胞样品荧光寿命的快成分分别为150,300ps;癌细胞与正常细胞的荧光峰值强度经12h分别衰减10%和55%。对测量所得的荧光光谱曲线及时间分辨荧光衰减曲线分析,计算出:在癌细胞内部血卟啉浓度增大了约2个数量级;癌细胞与正常细胞的荧光寿命分别为824,1798ps;血卟啉在癌细胞与正常细胞样品中滞留时间分别为17,6d。测量结果确认了荧光光谱技术诊断与治疗癌症的可行性,并对发展超短脉冲激光光谱技术早期诊断与治疗癌症具有重要的指导意义和临床应用价值。     

Comparison Between Fluorescence Correlation Spectroscopy and Time-Resolved Fluorescence Anisotropy as Illustrated with a Fluorescent Dextran Conjugate

The motional properties of rhodamine green alone and conjugated to 10-kDa dextran have been studied by fluorescence correlation spectroscopy (FCS) and time-resolved fluorescence anisotropy (TRFA). With FCS the translational diffusion times of the fluorescent particles can be determined, which are directly proportional to the shear viscosity as shown in aqueous solutions of different sucrose concentrations. With TRFA the rotational correlation times of the fluorescent particles can be determined. TRFA experiments in the case of fluorescent dextran reveal a distinct restricted internal motion of the fluorescent probe independent of the slower overall rotation of the polysaccharide. The fast depolarization is most likely due to internal motion and not to energy transfer between different rhodamine green molecules in the same dextran, since a higher viscosity of the solvent increases the correlation time for internal motion proportionally. FCS and TRFA yield complementary information in the sense that the correlation time for overall dextran rotation can be accurately determined from the translational diffusion coefficient.     

Study of Macromolecular Chain Dynamics in Polymer Complexes by Time-Resolved Fluorescence Spectroscopy

Poly(methyl methacrylate)s labeled with the anthracene fluorophore were prepared by free radical, anionic, and coordination polymerization yielding atactic and syndiotactic polymers. Unlabeled isotactic poly(methyl methacrylate) was prepared by anionic polymerization. Time-resolved fluorescence spectroscopy was used to study polymer association in solution. The time-dependent decays of fluorescence anisotropy show that stereocomplexation causes an increase in rotational correlation times of anthracene fluorophores both embedded in the polymer backbone and attached at the end of the side chain of polymer molecules. The rotational correlation time of anthracene fluorophore in dimethylformamide as a part of stereocomplex is 11.9 and 30 ns in the side chain and embedded in the polymer backbone, respectively, and shorter than 3 ns in noncomplexing solvent.     

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。     

Time-Resolved Fluorescence Anisotropy Measurements Made Simple

Time-resolved anisotropy measurements were performed using simple instrumentation with the aim to demonstrate the speed and ease of the experiment. Subsequent data analysis examples involved common, easily adaptable procedures.     

On the Fluorescence Depolarization and the Calculation of Rotational Relaxation Times of Rigid Rod Molecules

The rotational relaxation times of rod like molecules like poly[arylene-ethynylene]s and their low molecular weight model compounds calculated from a simple model agree well with the experimental ones as long as the axial ratio of the corresponding rotational ellipsoid is less than 8. For the polymer (axial ratio > 10) the fluorescence depolarization cannot be described by rotational motion perpendicularly to the long molecular axis. One has to take into consideration bending motions in connection with energy transfer along the bent backbone.     

Fluorescence lifetime measurement from a designated single-bunch in the BEPCⅡ colliding mode

Fluorescence lifetime measurement in the time domain requires excitation from a well separated single bunch using synchrotron light sources. In the colliding mode of the Beijing Electron Positron Collider Ⅱ (BEPCⅡ), a hybrid filling pattern was realized such that a single bunch was placed in the middle of a large gap between two multi-bunch groups. Detection of fluorescence lifetime, based on the excitation of the light pulse from this designated single-bunch, was established at Beamline 4B8 of the Beijing Synchrotron Radiation Facility (BSRF). The timing signal of the BEPCII was utilized as a trigger to gate this fluorescence event. L-Tryptophan amino acid, a known lifetime standard, was selected to assess the lifetime measurement performance. The measured lifetime was consistent in both colliding and single-bunch mode with the time resolution down to 450 ps. Moreover, both the bunch purity and the fine structure of the hybrid filling pattern were characterized.     

Conformational differences of oxytocin and vasopressin as observed by fluorescence anisotropy decays and transient effects in collisional quenching of tyrosine fluorescence

We used gigahertz frequency-domain fluorometry to examine the tyrosyl fluorescence intensity and anisotropy decays of the single-tyrosine cyclic peptide hormones oxytocin and vasopressin. Acrylamide quenching and a distance-dependent quenching model for collisional quenching were used to evaluate the extent of tyrosyl exposure to the quencher and to provide increased resolution of the picosecond anisotropy decays. Analysis of the intensity decays using a lifetime distribution model shows different distributions for oxytocin and vasopressin. We found that the tyrosyl fluorescence of lysine-vasopressin, as revealed both by the lifetime Stern-Volmer plots and from the quenching analysis, is quenched more effectively than oxytocin. ForN-acetyltyrosinamide (NATyrA), oxytocin, and lysine-vasopressin, we recovered apparent diffusion coefficients for quenching of 4.7×10^–6, 0.44×10^–6, and 4.3×10^–6 cm²/s, respectively, the lower value for oxytocin suggesting a shielded environment for its tyrosyl residue. Tyrosyl anisotropy decays were recovered by global analysis of progressively quenched samples. Compared with oxytocin, vasopressin displayed a longer correlation time for overall rotational diffusion and a higher amplitude for picosecond segmented motions of its tyrosyl residue. All the data are consistent with a more extended and flexible solution structure for vasopressin than for oxytocin.Dedicated to Professor Alfons Kawski on the occasion of his 65th birthday.     

Fluorescence anisotropy decay study of self-association of bacterial luciferase intermediates

The fluorescence dynamics parameters of the fluorescent transient flavin-luciferase species from the typesVibrio fischeri andPhotobacterium leiognathi are presented. The fluorescence anisotropy decay is a single exponential function for both types. The correlation time is 70 ns for theP. leiognathi fluorescent transient intermediate (2°C, aqueous buffer, pH 7.0), consistent with the rotational correlation time of the luciferase macromolecule (77 kD) to which the flavin fluorophore is rigidly attached. In contrast, for theV. fischeri species the observed correlation time for the anisotropy decay function is 133 ns. This suggests that protein self-association occurs in theV. fischeri case and this is confirmed by filtration, where the fluorescent transient fromV. fischeri does not pass through a 100,000 molecular weight cutoff membrane, whereas theP. leiognathi species does. The filtration method also demonstrates self-association in the luciferase peroxyflavin and photoflavin fromV. fischeri. A monomer-dimer equilibrium also explains the previously reported high correlation times for theV. harveyi luciferase-flavin species. It is proposed that the self-association competes with the lumazine protein interaction in the bioluminescence reaction.     

New Perspectives of Fluorescence Correlation Spectroscopy

The principle of fluorescence correlation spectroscopy is outlined. The technique has been applied to a mutant of the well-known green fluorescent protein. A comparative study has been made with time-resolved fluorescence anisotropy. The latter experiment shows that the fluorophore is rigidly bound inside the protein matrix follows the rotation of the whole protein and does not show any fast restricted motion. It is evident from fluorescence correlation spectroscopy that some excited-state reaction plays a role, since the autocorrelation traces show a significant effect on the incident laser power. Other potential applications of fluorescence correlation spectroscopy are presented as taken from very recent publications.     

多元散射校正在荧光谱分析中的应用研究

针对废水中氨基酸荧光分析过程中光谱数据去噪声处理过程,提出了基于多元散射校正思想建立相对荧光强度与浓度关系模型的方法,建立了荧光测量中多波段波谱系列线性模型。研究结果表明,实验数据进行线性拟合,色氨酸呈现出良好线性关系,通过多元散射校正方法进行光谱数据处理,有效降低了散射及测量环境噪声的影响,提高了光谱数据的信噪比,校正后数据更加准确的表征了色氨酸相对荧光强度与浓度数据之间的线性相关性。建立的多波段波谱系列线性模型为现场检测仪器研制过程中滤波片的选择及混合溶液的在线解析提供了理论依据。     

Pressure Effects on Submicrosecond Phospholipid Dynamics Using a Long-Lived Fluorescence Probe

The effects of applied external hydrostatic pressure on submicrosecond lipid motions in DPPC⁴ bilayers have been examined using coronene (a long-lived planar fluorescent molecule) and DPH. Steady-state fluorescence emission anisotropy (EA) values () obtained for probe-labeled DPPC SUVs measured at different fixed temperatures above T _c as a function of increasing hydrostatic pressure reveal pressure-induced lipid phase transition profiles. For coronene-labeled samples, the observed lipid melt profiles are broad and shifted to higher midpoint EA pressure values (P _1/2) compared with corresponding DPH-labeled SUVs at the same temperature. The data suggest lipid motions occurring on the submicrosecond time scale, detected only by using a long-lived fluorescence probe, which occur well above the normally reported fluid–gel lipid phase transition. Slopes of the pressure-to-temperature equivalence plots (dP _1/2/dT = 39 bar/K) obtained for DPH-or coronene-labeled DPPC SUVs are identical within experimental error and reflect probe independence. For DPH, the slope of the P _1/2(T) plot provides the expected phase transition phospholipid volume change. However, intercept values (at P _1/2 = 1 bar) or apparent phase transition temperatures obtained from the equivalence plots for the two probes are not equal. Differences appear to arise due to the very disparate fluorescence lifetime values of the two probes, which result in rotational sensitivity of coronene to gel lipid volume fluctuations occurring during the extended time window provided by coronene fluorescence.     

Steady-State and Time-Resolved Fluorescence Polarization Behavior of Acenaphthene

Steady-state and time-resolved fluorescence polarization studies have been carried out on acenaphthene (ACE) in low-temperature glass solutions and at room temperature. In the low-temperature glass the fluorescence polarization values vary considerably with both emission and excitation wavelength. There is a time dependence (on the nanosecond time scale) of the fluorescence anisotropy, r(t), at 77 K, which has a strong dependence upon the excitation and emission wavelengths. Under these conditions, the time-dependent decay of the anisotropy is not attributable to chromophoric motion. The observations are consistent with emission from two closely lying and interconverting excited states. Rate constants for the photophysical processes involved have been determined by fitting the data using a model proposed by Fleming et. al. The results are discussed with particular reference to the care required in using dynamic fluorescence polarization measurements to determine energy transfer rates in systems containing this chromophore.     

Two-Photon Excited Fluorescence and Molecular Reorientations in Liquid Solutions

Theoretical expressions are derived that relate the two-photon excited fluorescence depolarisation experiments to the molecular symmetry and the rotational motions of fluorescent molecules. Diffusive rotational motions in liquid solvents are considered, as well as the influence of fast unresolved motions (e.g. librations). The results obtained are compared with one-photon excited fluorescence depolarisation experiments. The derived theoretical expressions can be applied for detailed analyses of the molecular rotation in solvent. Several of the results are useful for determining and assigning the components of two-photon absorption tensors.     

Fluorescence Dynamics of DNA Condensed by the Molecular Crowding Agent Poly(Ethylene Glycol)

Condensation of extended DNA into compact structures is encountered in a variety of situations, both natural and artificial. While condensation of DNA has been routinely carried out by the use of multivalent cations, cationic lipids, detergents, and polyvalent cationic polymers, the use of molecular crowding agents in condensing DNA is rather striking. In this work, we have studied the dynamics of plasmid DNA condensed in the presence of a molecular crowding agent, polyethylene glycol (PEG). Steady-state and time-resolved fluorescence of the recently established condensation-indicating DNA binder, YOYO-1 [G. Krishnamoorthy, G. Duportail, and Y. Mely (2002), Biochemistry 41, 15277–15287] was used in inferring the dynamic aspects of DNA condensates. It is shown that DNA condensed by PEG is more flexible and less compact when compared to DNA condensed by binding agents such as polyethyleneimine. The relevance of such differences in dynamics toward functional aspects of condensed DNA is discussed.     

Molecular Dynamics of Biological Probes by Fluorescence Correlation Microscopy with Two-Photon Excitation

We report on the application of fluorescence correlation microscopy under two-photon excitation of fluorophores of biological interest: FITC–dextran (MW, from 20 to 150 kDa), green fluorescent protein (MW, 27 kDa), and fluorescein (MW, 330 Da). Under these experimental conditions, the translational diffusion coefficients of these molecules in aqueous solutions derived from the fluorescence intensity autocorrelation function were determined for the first time and were found to be 24 × 10^–7, 8.2 × 10^–7, and 3 × 10^–7 cm² s^–1 for 150-kDa FITC–dextran, green fluorescent protein, and fluorescein, respectively. These results are discussed in connection with previously reported results obtained by different methods. The great sensibility of the system has been applied to single-molecule detection of the smaller fluorophore, fluorescein.     

基于皮秒时间分辨的癌细胞荧光寿命研究

研究了用于癌症诊断与治疗的光敏剂血卟啉(hematoporphyrin derivative,HPD)的超快光动力学过程,采用超短脉冲激光光谱技术和皮秒时间相关单光子计数系统,测量了经血卟啉培养的活体癌细胞与正常细胞的皮秒时间分辨荧光光谱及荧光峰值强度随时间衰变曲线,观测到：癌细胞与正常细胞样品荧光寿命的快成分分别为150和300 ps;癌细胞与正常细胞的荧光峰值强度经12 h分别衰减10%和55%。经对测量所得的荧光衰减曲线进行分析,计算出癌细胞与正常细胞的荧光寿命分别为824和1798 ps;血卟啉在癌细胞与正常细胞样品中滞留时间分别为17天和6天。结果表明癌细胞与正常细胞对血卟啉亲和性及对血卟啉滞留的稳定性有显著差异,测量结果确认了荧光光谱技术诊断与治疗癌症的可行性,并对实时监测生物样品微弱超快荧光具有重要的指导意义和临床应用价值。     

Rotational Motion of a Solute Molecule in a Highly Viscous Liquid Studied byC NMR: 1,3-Dibromoadamantane in Polymeric Chlorotrifluoroethene

The viscosity-dependent retarding effect of a polymeric solvent on the rotation of small solute molecules is investigated by¹³C NMR relaxation measurements. It is found that the relaxation data of 1,3-dibromoadamantane in highly viscous polymeric chlorotrifluoroethene can be explained neither by isotropic nor by realistic anisotropic tumbling in a single environment. The experimental data are rationalized in terms of fast exchange between at least two environments with correlation times differing by up to two orders of magnitude. The study shows that a uniform retardation of molecular tumbling by a polymeric solvent, desirable for shifting the NMR observation window in studies of intramolecular mobility, is not always feasible.