Direct Observation of Lipid Domains in Free-Standing Bilayers Using Two-Photon Excitation Fluorescence Microscopy

The direct observation of temperature-dependent lipid phase equilibria, using two-photon excitation fluorescence microscopy on giant unilamellar vesicles (GUVs) composed of different lipid mixtures, provides novel information about the physical characteristics of lipid domain coexistence. Physical characteristics such as the shape, size, and time evolution of different lipid domains are not directly accessible from the traditional experimental approaches that employ either small and large unilamellar vesicles or multilamellar vesicles. In this review article, we address the most relevant findings reported from our laboratory regarding the direct observation of lipid domain coexistence at the level of single vesicles in artificial and natural lipid mixtures. In addition, key points concerning our experimental approach will be discussed. The unique advantages of the fluorescent probe 6-dodecanoyl-2-dimethylaminonaphthalene (LAURDAN) under two-photon excitation fluorescence microscopy is particularly addressed, especially, the possibility of obtaining information on the phase state of different lipid domains directly from the fluorescent images.     

激光波长对荧光激发谱分布影响研究

激光器是DNA测序仪中的重要部件,寻求合适波长的激光器可有效提高DNA测序仪性能。分析了DNA检测所用的四种荧光染料FAM、JOE、TAMRA和ROX的激发谱和发射谱,设计实验技术方案利用488nm、505nm和515nm三种不同波长的激光器分别对其进行激发。在相同的积分时间和相同的功率下,488nm激光器对于荧光染料ROX的激发强度太低,515nm激光器对于荧光染料FAM的激发强度太低,而505nm激光器对于四种荧光染料的激发强度均比较强。实验结果表明在三种不同波长中,505nm激光器对四种荧光染料的激发效果最佳,可以替代目前大多数DNA测序仪中的氩离子激光器。     

Effect of Polystyrene Microsphere Surface to Fluorescence Lifetime Under Two-Photon Excitation

Molecular assays such as immunoassays are often performed using solid carriers and fluorescent labels. In such an assay format a question can be raised on how much the fluorescence of the label is influenced by the bio-affinity binding events and the solid carrier surface. Since changes in fluorescence intensity as labels bind to surfaces are notoriously difficult to quantify other approaches are preferred. A good indicator, independent of the fluorescence intensity of the label, is the fluorescence lifetime of the marker fluorophore. Changes in fluorescence lifetime reliably indicate the presence of dynamic quenching, energy transfer or other de-excitation processes. A microsphere based assay system is studied under two-photon excitation. Changes in fluorescence lifetime are studied as labeled protein conjugates bind on microsphere surfaces – both direct on the surface and with a few nanometer distance from the surface. Fluorescence signal is measured from individual polystyrene microspheres and the fluorescence lifetime histogram is simultaneously recorded. The results indicate that self-quenching and quenching by the polystyrene surface are both present in such a system. However, the effect of the surface can be avoided by increasing the distance between the surface and the label. Typical distances achieved by a standard sandwich type of assay, are already sufficient to overcome the surface induced quenching in fluorescence detection.     

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.     

飞秒激光诱导的Mn2+掺杂锗酸盐玻璃上转换发光

研究了室温下的飞秒激光诱导过渡金属Mn2+掺杂锗酸盐玻璃的上转换发光现象。样品的激发光谱与发射光谱表明,激发过程是能级6A(6S)→4E(4G)/4A1(4G)的跃迁,该过程源于Mn2+离子的4T1(4G)→6 A1(6 S)跃迁。由409 nm单色光激发样品得到的发射光谱与800 nm飞秒激光激发的光谱相一致。通过分析飞秒激光泵浦功率密度与荧光强度的依赖关系以及飞秒激光辐照样品前后的吸收谱,认为该上转换过程为双光子同时吸收过程。随着Mn2+浓度的提高,上转换发光的中心波长发生红移。     

拉曼激发激光诱导电子荧光流场测量系统中标记过程的研究

研制了一套拉曼激光发激光诱导电子荧光空气流场测速系统,利用Nd:YAG激光器的二次谐波及其抽运的氧气受激拉曼散射作为标记光源,以ArF准分子激光作为荧光再现光源,并用像增强CCD摄像机（ICCD）记录荧光图像,成功地获得了纯氧及空气中的标记线的荧光图像,并进行了氧气喷流速度测量的初步研究。     

DHL细胞中5-ALA代谢PpIX的双光子荧光光谱

双光子激发生物组织荧光,激发光仅作用于焦点区域,对生物样品的光漂白性和光毒性都很小,因而双光子荧光显微技术已成为细胞生物学研究的一种新技术。文章采用波长为820 nm飞秒激光激发孵育有5-ALA的DHL细胞,在激光扫描显微镜的Lambda模式中获得单个DHL细胞的双光子荧光光谱,并测量DHL细胞内积聚的卟啉九(PpIX)特征荧光值。获得了浓度分别为2, 4和10 mmol·L-1的5-ALA溶液中,细胞代谢的PpIX含量随孵育时间的变化情况。DHL细胞内积聚的PpIX处于动态变化过程,并呈现出两阶段性的特点：细胞内积聚的PpIX含量随着孵育时间增长而增加,在3 h附近达到最大值,随后随着孵育时间增长反而下降。结果表明,基于激光扫描显微的双光子荧光光谱可成为DHL细胞等白血病细胞摄取5-ALA并生成PpIX的动力学研究的有效方法。     

Femtosecond pulse duration measurement by two-photon fluorescence detection

The two-photon absorption induced fluorescence in a methanolic rhodamine 6G solution is used to measure the pulse duration of a cw femtosecond Kerr–lens mode-locked Ti:sapphire laser by a Michelson-interferometer-type autocorrelator arrangement. Interference-free autocorrelation traces are obtained by correlating perpendicular polarized pulses. The two-photon polarization parameter of rhodamine 6G is determined by contrast ratio measurement. The time resolution limits are discussed.     

Fluorescence Enhancement of CdSe Q-Dots with Intense Femtosecond Laser Irradiation

Effects of intense femtosecond (fs) laser irradiation on the optical properties of cadmium selenide (CdSe) nanocrystals are studied. We present the changes in emission and absorption of laser (800 nm, 110 fs, Ti–Sapphire) irradiated CdSe nanocrystals dispersed in dimethylformamide (DMF). It is observed that the absorbance of CdSe nanocrystals capped with trioctylphosphine (TOP) increases with the number of laser pulses. The trap state luminescence intensity of these crystals degrades, whereas the band edge luminescence intensity shows an increase as a function of the fs laser irradiation. We also report strong two photon absorption and reduction in the trap state luminescence intensity after irradiation with the laser pulses.     

双光子激发荧光成像技术对小鼠植入前胚胎的实时观测

双光子激发的蓝移效应可使利用同一束超快激光同时激发多种不同荧光特性的生物荧光染料的设想得以实现。选取锁模飞秒掺钛蓝宝石(Ti-sapphire)激光器输出的730nm激光,分别激发Hoechst33342,Fluo-4,PI和Indo-1四种常用生物荧光染料,分别利用(455±15)nm,(540±15)nm,(580±16)nm和(500±15)nm四种滤光片获得特异性荧光图像。结合双光子激发荧光成像技术穿透深,光损伤小,信噪比好等优势,选取合适的荧光染料组,应用单束激光激发、双染双通道成像方法,对小鼠植入前胚胎内细胞中的钙信号和染色体进行三维、四维实时成像,为探究小鼠植入前胚胎发育规律提供一种全新的多参数观测手段。     

Two-Photon Laser-Induced Fluorescence Spectra of Argon in a Grimm-Style Glow Discharge Tube

Two-photon excited laser-induced fluorescence (LIF) spectra of argon atom were successfully observed in a Grimm-style glow discharge tube, which has widely been applied to depth profiling of the elemental composition on various film-like samples by emission spectrometry. The LIF signal of an argon atomic line at 641.63 nm was observed when the glow discharge argon plasma was illuminated by a pulsed Ti:sapphire laser radiation of 7–10 mJ/pulse at 753.39 or 795.66 nm without focusing of the laser beam.     

Nuclear Excitation by X-Ray Emission of Femtosecond Laser Plasma

The proposed model of laser plasma emission spectrum formation enables us to determine the absolute value of the laser pulse to plasma emitted radiation conversion factor, profile of the emission spectrum, and frequency distribution of the intensity and energy in the emitted spectra. This is of interest for laser plasma diagnostics and provides a means for direct calculation of the number of excited nuclei in dependent on the parameters of laser pulse. This revised version was published online in August 2006 with corrections to the Cover Date.     

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。     

Fluorescence of Styryl Dyes-DNA Complexes Induced by Single- and Two-Photon Excitation

The series of novel monomer and homodimer styryl dyes based on (p-dimethylaminostyryl) benzothiazolium residues were synthesized and studied as possible fluorescent probes for nucleic acids detection. Spectral-luminescent and spectral-photometric properties of obtained dyes in the unbound state and in DNA presence were studied. Fluorescence emission induced by two-photon excitation of dye-DNA complexes in aqueous buffer solution was registered. Two-photon absorption cross section values of the studied dyes in DNA presence were evaluated.     

NO分子双光子诱导荧光的自吸收现象

以皮秒Nd:YAG激光器抽运的光学参量发生/放大器为激发光源,获得了在452.4 nm波长激光激发下,NO分子的双光子激光诱导色散荧光光谱(LIDFS)。并由此得到了NO分子基电子态的振动基频和振动非谐性系数分别为ω″_e=(1 904.7±7.3) cm-1, ω″_eχ″_e=(14.2±1.2) cm-1, ω″ey″_e=-(0.021 8±0.009 1) cm-1。在NO分子双光子LIDFS实验研究中,首次观测到了NO分子强烈的自吸收现象,在较高样品气压条件下,色散荧光光谱图中对应A 2Σ(v′＝0)→X 2Π(v″＝0)跃迁的谱线消失。实验结果表明,自吸收现象导致的该跃迁谱线强度的变化,随样品气压、激光场和样品作用区到荧光接收窗口距离的减小而减小。     

Non-Linear Excitation and Decay of Low-Energy Nuclear Isomers Produced under Femtosecond Laser–Plasma Interaction

The properties of internal electronic conversion of nucleus excited in hot dense laser plasma are considered. The important role of dynamics of plasma charge state in residual gas is shown. We are also considering the role of multi-quanta photoexcitation of low-energy nuclear levels in this plasma. This revised version was published online in August 2006 with corrections to the Cover Date.     

飞秒脉冲激光对YAG晶体的辐照作用的研究

采用波长为800nm，脉冲宽度为120fs的飞秒激光对提拉法和温梯法生长的YAG晶体进行辐照。并对辐照后的样品进行吸收光谱及电子顺磁共振谱(EPR)的检测。结果表明，提拉法的晶体在经过辐照后，255nm吸收峰减弱，同时在370nm处产生新的宽峰吸收，其产生原因是由于样品内部分Fe^3 转化成Fe^2 ，并有少量F^ 心生成。对比两种不同方法生长的晶体，温梯法生长的YAG晶体的抗激光辐照能力略优于提拉法生长的晶体。     

Fluorescence and Electronic Structure of the Laser Dye DCM in Solutions and in Polymethylmethacrylate

The photophysical properties and polarization of the fluorescence of the laser dye 4-dicyanomethylene-2-methyl-6-(p(dimethylamino)styryl)-4H-pyran (DCM) in solutions of different polarities and in polymethylmethacrylate (PMMA) at 293 and 77 K were investigated by stationary and pulsed fluorescent spectroscopy. Based on the data of polarized fluorescence, an oscillator model has been suggested, according to which the electronic absorption spectrum of DCM in the 240–500-nm range is formed by at least three electron transitions. The quantum yield of fluorescence _f increases linearly with increase in the polarity of a solvent in a toluene dimethylsulfoxide (DMSO) mixture from 0.08 (toluene) to 0.80 (DMSO). An increase of _f to 0.90 was also observed on increase in the rigidity of the medium by freezing a solution of DCM in n-propanol at 77 K or introducing of a dye to the polymeric matrix of PMMA at room temperature ( _f = 0.76). The fluorescence-decay kinetics of DCM in toluene, n-propanol, and PMMA at 293 K follows a biexponential law, whereas in n-hexane and vaseline oil at 293 K and in n-propanol at 77 K it follows a monoexponential law. The mechanisms underlying radiationless deactivation of the electron-excitation energy in solutions and in polymeric media are discussed.     

飞秒激光与细胞的相互作用

近年来,高速发展的激光技术已经广泛应用于医疗领域,比如眼科手术和光学相干层析成像。尤为特殊的是,飞秒激光更可专用于对活体细胞的基础研究。飞秒激光具有别种激光无法获得的特殊性质。它对于细胞的伤害很小,因为它的线性吸收和热效应远远低于连续光。此外,由于飞秒光的超高峰值功率,它可以对细胞形成多光子电离而在细胞膜上开孔,因此可以转基因和融合细胞,同时不会太过于伤害细胞。在这个报告里,将阐述我们的以下研究：飞秒光致转基因、细胞融合以及细胞在凋亡中的动态过程。在单细胞水平上提出了一些凋亡过程的可能机制,如活性氧化合物、核管和细胞内自由钙离子的产生。