Two photon-induced fluorescence intensity and anisotropy decays of diphenylhexatriene in solvents and lipid bilayers

We measured the fluorescence intensity and anisotropy decays of 1,6-diphenyl-1,3,5-hexatriene (DPH)-labeled membranes resulting from simultaneous two-photon excitation of fluorescence. Comparison of these two-photon data with the more usual one-photon measurements revealed that DPH displayed identical intensity decays, anisotropy decays, and order parameters for one- and two-photon excitation. While the anisotropy data are numerically distinct, they can be compared by use of the factor 10/7, which accounts for the two-photon versus one-photon photoselection. The increased time 0 anisotropy of DPH can result in increased resolution of complex anisotropy decays. Global analysis of the one- and two-photon data reveals consistency with a single apparent angle between the absorption and the emission oscillators. The global anisotropy analysis also suggests that, except for the photoselection factor, the anisotropy decays are the same for one-and two-photon excitation. This ideal behavior of DPH as a two-photon absorber, and its high two-photon cross section, makes DPH a potential probe for confocal two-photon microscopy and other systems where it is advantageous to use long-wavelength (680- to 760-nm) excitation.     

新型多光子成像技术研究进展

相比于传统的光学成像技术,近年来获得快速发展的新型多光子成像技术具有穿透深度大,组织光损伤小,信噪比高,且可方便进行光学层析成像的特点,故而被广泛应用于包括脑、肿瘤、胚胎在内的多种活体组织成像中。本综述回顾了新型多光子成像技术的诞生与发展历程,包括微型化双光子成像技术、双光子内窥技术和三光子成像技术,概括分析了其基本原理与成像特点,讨论了这一领域具有代表性的最新研究成果,重点总结了其在生物学基础研究领域和临床医学诊断中的主要应用,并展望了其未来的应用与发展前景。可以预见,随着激光器和光探测技术的不断进步,多光子成像技术将会得到更大的发展与更加广泛的应用。     

Review of fluorescence anisotropy decay analysis by frequency-domain fluorescence spectroscopy

This didactic paper summarizes the mathematical expressions needed for analysis of fluorescence anisotropy decays from polarized frequency-domain fluorescence data. The observed values are the phase angle difference between the polarized components of the emission and the modulated anisotropy, which is the ratio of the polarized and amplitude-modulated components of the emission. This procedure requires a separate measurement of the intensity decay of the total emission. The expressions are suitable for any number of exponential components in both the intensity decay and the anisotropy decay. The formalism is generalized for global analysis of anisotropy decays measured at different excitation wavelengths and for different intensity decay times as the result of quenching. Additionally, we describe the expressions required for associated anisotropy decays, that is, anisotropy decays where each correlation time is associated with a decay time present in the anisotropy decay. And finally, we present expressions appropriate for distributions of correlation times. This article should serve as a reference for researchers using frequency-domain fluorometry.     

Fluorescence of horse liver alcohol dehydrogenase using one- and two-photon excitation

We examined the steady-state and time-resolved emission of liver alcohol dehydrogenase resulting from one-photon and two-photon excitation. Previous studies with one-photon excitation revealed that the two nonidentical tryptophan residues display different emission spectra and decay times. The use of two-photon excitation resulted in similar emission spectra, multiexponential intensity decays, time-resolved emission spectra, and anisotropy decays as was observed for one-photon excitation. These results suggest that both nonidentical tryptophan residues are excited to a similar extent for one- and two-photon excitation. However, the limiting anisotropy (r ₀) with two-photon excitation from 585 to 610 nm is below 0.1 and appears distinct from that observed previously forN-acetyl-l-tryptophanamide.Abbreviations LADH liver alcohol dehydrogenase - -NAD⁺ -nicotinamide adenine dinucleotide - OPE one-photon excitation - OPIF one-photon induced fluorescence - TPE two-photon excitation - TCSPC time-correlated single photon counting - TPIF two-photon induced fluorescence     

Determination of DPH order parameters in unoriented vesicles

This article reviews the determination of orientational order parameters in non-macroscopically oriented membranes from the data obtained with the fluorescent probe all-trans-1,6-diphenyl-1,3,5-hexatriene (DPH). Special attention is paid to the effect of microheterogeneity in the probe environment on the recovered values of the order parameters. An effort is made to accommodate new findings in the existing picture of orientational order in membranes.     

Three-photon excitation ofp-quaterphenyl with a mode-locked femtosecond Ti:sapphire laser

We observed emission fromp-quaterphenyl (p-QT) at 360 nm when exposed to the focused light from a femtosecond (fs) Ti:sapphire laser at 850 nm. This wavelength is too long to allow two-photon excitation of p-QT. The emission intensity of p-QT was found to depend on the cube of the laser power at 850 nm, suggesting that excitation occurs due to a three-photon process. The same emission spectrum and single exponential decay times were observed for three-photon excitation at 850 nm as for two-photon excitation at 586 nm and for one-photon excitation at 283 nm. The same rotational correlation times were observed for one-, two-, and three-photon excitation, but higher time-zero anisotropies were observed for two- and three-photon excitation. The steady-state anisotropies for one-, two-, and three-photon excitation are precisely consistent with cos², cos⁴, and cos⁶ excitation photoselection, where is the angle between the electric field of the incident light and the absorption dipole. These experiments were performed with 3×10^–5 M solutions of p-QT. Use of such low concentrations was possible because p-QT displays one of the highest apparent cross sections we have observed to date for three-photon excitation. The spatial distribution of the excited fluorescence was less for three-photon excitation than for two-photon excitation of Coumarin 102 at the same 850-nm excitation wavelength. The high cross section, photostability, and clear cos⁶ photoselection of p-QT make it an ideal three-photon standard for spectroscopy and microscopy.     

Fluorescence Studies on Potential Antitumoral Heteroaryl and Heteroannulated Indoles in Solution and in Lipid Membranes

Fluorescence properties of three potential antitumoral compounds, a 3-(dibenzothien-4-yl)indole 1, a phenylbenzothienoindole 2 and a 3-(dibenzofur-4-yl)indole 3, were studied in solution and in lipid aggregates of dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylethanolamine (DOPE) and egg yolk phosphatidylcholine (Egg-PC). The 3-(dibenzofur-4-yl)indole 3 exhibits the higher fluorescence quantum yields in all solvents studied (0.32 ≤ Φ_F ≤ 0.51). All the compounds present a solvent sensitive emission, with significant red shifts in alcohols. The results point to an ICT character of the excited state, more pronounced for compound 1. Fluorescence (steady-state) anisotropy measurements of the compounds incorporated in lipid aggregates of DPPC, DOPE and Egg-PC indicate that the three compounds are deeply located in the lipid bilayer, feeling the difference between the rigid gel phase and fluid phases.     

钠蒸气中的双光子共振三光子电离和双光子混合共振三光子电离

在钠原子－分子混合样品中，分别运用双光子共振三光子电离以及双光子混合共振三光子电离，测得了钠原子４Ｄ态的电离流时间衰减曲线。由电离流与多光子动力学参数的关系，得到了４Ｄ态的光电离截面。     

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.     

Investigation of the Dynamic Properties of the Lipid Layer of Intraplastid Membranes by Lipophilic Fluorescent Probes

The dynamic properties of the lipid layer of intraplastid membranes have been studied by analyzing the stationary and kinetic spectral polarization characteristics of the fluorescence of lipophilic probes of pyrene and diphenylhexatriene (DPH). Based on the data of the decay of the fluorescence anisotropy of pyrene, the value of the microviscosity of lipids in the membranes of prolamellar bodies (PLB) and protilakoids of etioplasts has been calculated. The pyrene molecules built into the membranes of etioplasts have a relatively high rotational mobility (stationary anisotropy r _s < 0.1). The DPH molecules rotate with difficulty in etioplast membranes (r _s > 0.3). After photoreduction of protochlorophyllide (Pd) in vivo, the rotation of the pyrene and DPH molecules in the membranes of prolamellar bodies becomes easier and this leads to a decrease in r _s. Illumination raised the degree of excimerization of the pyrene immersed into lipids (_exc = 337 nm), and the microsurrounding of the molecules of the probe in lipids became more hydropholic (the relationship between the vibronic maxima at 373 and 387 nm decrease). The set of data obtained points to a decrease in the microviscosity of the lipid layer of the membranes of prolamellar bodies as a result of illumination of sproutings.     

钾蒸汽中双光子激发产生2~3∏_g—α~3∑_u~+扩散带受激辐射

本文报道了在K_2-K中通过双光子共振激发原子或双光子激发分子产生2~3∏_(?)—a~3∑_u~+扩散带辐射的实验结果,并对激发机制以及扩散带辐射随激发能量和温度的变化进行了讨论.     

线型三原子分子在强激光场中多光子激发的李代数方法

使用二次非谐振子模型和Magnus近似方法构造了在强激光场下线型三原子分子的振动哈密顿并推导出时间演化算符的代数形式,在此基础上研究了线型DCN分子的跃迁几率随外场的频率和强度的变化关系,表明了多光子跃迁谱的特点.     

飞秒激光双光子微细加工技术及研究现状

飞秒激光双光子微细加工技术以其特有的高精度三维微加工优势,成为微型机械加工领域新的发展方向之一。介绍了飞秒激光双光子微细加工技术的原理和应用的现状。结合目前已有的微细加工技术,对双光子微细加工技术的特点加以评述。简要报道了利用飞秒激光双光子微细加工技术的一些研究进展。探讨了飞秒激光双光子微细加工技术今后的发展方向及其存在的基本问题。     

On the interpretation of fluorescence anisotropy decays from probe molecules in lipid vesicle systems

Measurements of fluorescence depolarization decays are widely used to obtain information about the molecular order and rotational dynamics of fluorescent probe molecules in membrane systems. This information is obtained by least-squares fits of the experimental data to the predictions of physical models for motion. Here we present a critical review of the ways and means of the data analysis and address the question how and why totally different models such as Brownian rotational diffusion and wobble-in-cone provide such convincing fits to the fluorescence anistropy decay curves. We show that while these models are useful for investigating the general trends in the behavior of the probe molecules, they fail to describe the underlying motional processes. We propose to remedy this situation with a model in which the probe molecules undergo fast, though restricted local motions within a slowly rotating cage in the lipid bilayer structure. The cage may be envisaged as a free volume cavity between the lipid molecules, so that its position and orientation change with the internal conformational motions of the lipid chains. This approach may be considered to be a synthesis of the wobble-in-cone and Brownian rotational diffusion models. Importantly, this compound motion model appears to provide a consistent picture of fluorescent probe behavior in both oriented lipid bilayers and lipid vesicle systems.     

Er~(3+)/Tm~(3+)/Yb~(3+)共掺杂BaGa2ZnO5粉末的上转换发光

对Er~(3+)/Tm~(3+)/Yb~(3+)共掺杂BaGa_2ZnO5粉末的上转换发光特性进行了研究。首先,通过不同的制备工艺研究了BaGa2ZnO5粉末的生成条件。XRD分析表明,由于ZnO的高温分解,必须密封烧结并增加ZnO用量才能制备出BaGa2ZnO5。其次,测量了Er~(3+)/Tm~(3+)/Yb~(3+)共掺杂BaGa2ZnO5粉末在980nmLD激发下的上转换发射光谱,发现存在8个较强上转换发射峰,其中480nm和798nm对应于Tm~(3+)离子能级跃迁,其他上转换峰对应于Er~(3+)离子能级跃迁。另外,测量了980nmLD不同激发功率对应的上转换发射峰强度,并根据激发功率-上转换强度曲线拟合得到了各发射峰对应的多光子吸收过程。最后,根据上转换发射光谱和CIE1931标准数据计算了样品粉末的色坐标为(x=0.4151,y=0.4794) ,比较接近白光色坐标,说明所制备的粉末样品具有产生上转换白光的前景。     

基于双光子激发荧光的单分子探测技术研究

介绍采用双光子激发荧光方法进行单分子探测的原理和自行研制的实验装置,激发光聚焦和荧光收集采用共焦方式。选择香豆素C445水溶液作为研究对象,从样品流速、浓度、激光功率、信噪比和检测限等方面探讨了双光子激发荧光的特性。该谱仪目前己达到探测灵敏区内C445的平均分子数为1.5个的检测限     

Characterization of nanometric carbon materials by time-resolved fluorescence polarization anisotropy

In this work, time-resolved fluorescence polarization anisotropy (TRFPA) technique has been applied to the determination of the average size of asphaltenes and combustion-generated carbon nanoparticles. The characteristic depolarization time of fluorescence light following photon absorption is related to the typical particle size through the Stoke–Einstein rotational diffusion equation. The TRFPA technique employed in our experiment achieves sub-nanosecond time resolution, roughly corresponding to sub-nanometer accuracy in determining the particle size. The technique has been applied to both asphaltene and carbon nanoparticles, the former being a component of petroleum, whereas the latter result from combustion processes. Therefore, a complete and reliable characterization of such particles is of great importance in oil industry and atmospheric physics, respectively. Although the TRFPA technique has been developed and widely used on molecular and biological samples, it proves to be a very powerful tool for measuring the size of asphaltene and soot particles as small as few nanometers with a resolution of the order of 0.1 nm.