Simultaneous observation of collagen and elastin based on the combined nonlinear optical imaging technique coupled with two-channel synchronized detection method

Collagen and elastin are the most important proteins of the connective tissues in higher vertebrates. In this paper, we present a combined nonlinear optical imaging technique of second-harmonic generation and two-photon excited fluorescence to simultaneously observe the collagen and elastic fiber of dermis in a freshly excised human skin and rabbit aorta using a two-channel synchronized detection method. The obtained two-channel overlay image in the backward direction can clearly distinguish the morphological structure and distribution of collagen and elastic fibers. Tissue spectrum further confirms the obtained structural information. These results suggest that the combined nonlinear optical imaging technique coupled with two-channel synchronized detection method can be an effective tool for detecting collage and elastic fibers without any invasive tissue procedure of slicing, embedding, fixation and staining when two structural proteins are simultaneously present in the biological tissue.     

有机双光子吸收材料的研究进展

本文综述了具有优异双光子吸收/双光子荧光性质的有机功能材料的研究工作和成果。近年来,有机材料凭借其出众的性质、丰富的种类、多重功能性以及快速的非线性光学响应引起国际科学和技术界的极大兴趣。文章介绍了测量材料的非线性光学性质和超快响应的实验方法,它们已被广泛应用于有机材料的研究工作,并且取得了重大的进展。本文描述了对偶极、四极、多枝结构、大环结构以及聚合物等有机材料的非线性光学性质和超快光学响应的研究结果。基于有机材料的迷人性质,具有优异非线性光学性质和超快响应的有机材料将会在很多领域显示出巨大的应用潜力。     

Hydrophilic Labeling Reagents of Dipyrrylmethene-BF2 Dyes for Two-Photon Excited Fluorometry: Syntheses and Photophysical Characterization

Recently introduced bioaffinity assay technology, ArcDia TPX, is based on two-photon excited fluorescence (TPE) and it enables separation-free ultra-sensitive immunoassays from microvolumes. Here we present syntheses of novel two-photon excitable fluorescent labeling reagents which have been specially designed to be used as label molecules in the ArcDia TPX assay technique. The labeling reagents are based on dipyrrylmetheneboron difluoride (dipyrrylmethene-BF2) chromophore, which have been substituted with aryl, heteroaryl or arylalkenyl chemical groups to extend the pi-electron conjugation. These substitutions results in a series of dipyrrylmethene-BF2 fluorophores with different photophysical properties. Dipyrrylmethene-BF2 fluorophores have been further substituted with a dipeptide linker unit and finally activated as succinimidyl esters to enable specific coupling with primary amino groups. The dipeptide linker serves as a spacer arm between the label and a target, and enhances the solubility of the label in aqueous solutions. Study of the chemical and photophysical performance of the new labeling reagents is described. The new labeling reagents exhibit high fluorescence quantum yields, and molar absorption coefficients. The results show that the new labels with the hydrophilic dipeptide linker unit provide large two-photon excitation cross-sections, high fluorescence quantum efficiency and good solubility in aqueous solutions. The results suggest that the novel dipyrrylmethene-BF2 labels are highly applicable to bioaffinity assays based on two-photon excitation of fluorescence.     

Syntheses of Novel Dipyrrylmethene-BF2 Dyes and Their Performance as Labels in Two-Photon Excited Fluoroimmunoassay

Two-photon excitation of fluorescence (TPE) has been found a powerful tool in the field of microscopy imaging and recently also in the field of bioanalytics. The recently introduced bioaffinity assay technology, ArcDia TPX, enables separation-free ultra-sensitive immunoassays from microvolumes. This assay technique is based on the use of microspheres as a solid reaction carriers and two-photon excited fluorescence detection. In the ArcDia TPX-technology, the individual microparticles are observed and the number of bound biomolecules on the microparticle surface is quantified by two-photon excited fluorescence. Here we present synthesis and use of a novel dipyrrylmethene-BF2 fluorophore that has been designed to be used as label in ArcDia TPX assay technique. The absorption and emission wavelengths of the label are tuned to allow excitation with a 1064 nm microchip laser. The label contains two-carboxylic residues, one of which is activated as N-hydroxysuccinimide ester to enable labeling of amino residues of biomolecules. The other carboxylic group is in free form to increase solubility in aqueous solutions. This new fluorescent label is tested in a separation-free immunoassay using ArcDia TPX assay technique. The performance of the new label is compared to that of one of the brightest fluorophores available, R-phycoerythrin (RPE). According to the results, the dipyrrylmethene-BF2 label provides significantly better signal-to-background ratio, leading to higher assay sensitivity and broader dynamic range compared to that of RPE. Good solubility to aqueous solutions and high fluorescence quantum efficiency, suggests the dipyrrylmethene-BF2 label is applicable also in other fluorescence-based applications.     

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.     

不同荧光波长的双光子共焦成像分析

研究了双光子共焦显微镜中不同荧光波长对成像特性的影响,导出了不同荧光波长的三维脉冲响应函数和三维光学传递函数并进行数值计算.研究结果表明：不同荧光波长对双光子共焦显微镜的三维光学传递函数、三维脉冲响应函数和空间截止频率产生明显的影响,随着荧光波长的增大,分辨率明显下降,但不会出现单光子共焦显微镜中的失锥现象,选取适当的荧光波长进行成像,有利于进一步改善图像分辨率和成像质量.     

Fluorescence methods for studying molecular motions

The characteristics of photoluminescence are affected by the molecular motions in the local environment of the excited molecule. Hence, by knowing the lifetime of the excited state, various relaxation times of the molecular motions can be estimated from the emission characteristics. In the present paper the effect of the molecular motions manifested in the fluorescence emission, such as the rotational depolarization of fluorescence, the internal quenching of fluorescence due to internal rotation, and the inter-and intramolecular excimer formation, are reviewed and the possible applications of such phenomena to the study of molecular motions in polymer systems are discussed. The application of fluorescence methods provide unique information about the molecular motions in the nanosecond range in polymer solutions, melts, and solids.     

双光子吸收材料二苯乙烯衍生物的合成及光谱性质

设计、合成并用红外光谱、1H NMR、元素分析表征了三种用于双光子吸收材料的二苯乙烯衍生物,4,4′-双(二苯氨基-反式-苯乙烯基)联苯(BPSBP),4,4′-双(二乙氨基-反式-苯乙烯基)联苯(BESBP)和4,4′-双(9-咔唑基-反式-苯乙烯基)联苯(BCSBP)。实验结果表明三者最强的单光子吸收出现在350～400 nm之间,且单光子吸收和荧光光谱中表现出明显的溶剂化显色效应,揭示了分子内对称电荷转移的本质,双光子荧光光谱则揭示了单光子和双光子吸收具有相同的发射机理。利用双光子上转换荧光法测试发现,三种双光子吸收材料在800 nm飞秒激光的激发下具有较大的吸收截面,分别为892,617和483 GM,这表明在双光子领域有潜在的应用价值。     

反-4,4''-双(N,N-二丁胺基)二苯乙烯分子双光子吸收性质研究

对于实验室合成的反-4,4'-双(N,N-二丁胺基)二苯乙烯分子,实验测量了该分子的单光子和双光子荧光谱,然后从理论上研究了其单光子和双光子吸收特性.研究结果表明,在低能量范围内,分子的单光子吸收主要发生在分子的第一激发态,而分子的双光子吸收主要发生在分子的第二和第四激发态上.该分子在相应系列衍生物中具有最大的双光子吸收截面.分子的相关能对分子的激发态能量影响较大.我们给出了分子基态与电荷转移态的电荷转移过程,并从理论上定性解释了双光子聚合反应的聚合机理.     

Two-photon Absorption Properties of 9,10-Disubstituted 2,6-Bis(p-dihexylaminostyryl)Anthracene Derivatives. Effect of 9,10-Substituents

A series of 2,6-bis(p-dihexylaminostyryl)anthracence derivatives having phenyl, styryl, and phenylethynyl groups at 9,10-positions (1−4) have been synthesized and their two-photon cross-sections were determined. Overall, the wavelengths of the longest wavelength absorption band and emission spectra increase with increase in the conjugation length and the electron withdrawing ability of the 9,10-substituents. All compounds show two-photon cross sections in the range of 740−3940 GM at 780−960 nm, which increase significantly by the donor and acceptor groups at 9,10-positions. In addition, Ph and phenylethynyl groups are better when compared to the styryl group at the 9,10-positions in terms of the two-photon action cross section. From a practical perspective, 1a, 2a–c, and 4b showed significant two-photon action cross-section and are most useful for applications that use two-photon excited fluorescence.     

Two-Photon Excited Fluorescence Energy Transfer: A Study Based on Oligonucleotide Rulers

The use of two-photon excitation of fluorescence for detection of fluorescence resonance energy transfer (FRET) was studied for a selected fluorescent donor–acceptor pair. A method based on labeled DNA was developed for controlling the distance between the donor and the acceptor molecules. The method consists of hybridization of fluorescent oligonucleotides to a complementary single-stranded target DNA. As the efficiency of FRET is strongly distance dependent, energy transfer does not occur unless the fluorescent oligonucleotides and the target DNA are hybridized. A high degree of DNA hybridization and an excellent FRET efficiency were verified with one-photon excited fluorescence studies. Excitation spectra of fluorophores are usually wider in case of two-photon excitation than in the case of one-photon excitation [1]. This makes the selective excitation of donor difficult and might cause errors in detection of FRET with two-photon excited fluorescence. Different techniques to analyze the FRET efficiency from two-photon excited fluorescence data are discussed. The quenching of the donor fluorescence intensity turned to be the most consistent way to detect the FRET efficiency. The two-photon excited FRET is shown to give a good response to the distance between the donor and the acceptor molecules.     

取代效应对脂滴检测NAPBr染料分子的双光子吸收和激发态性质影响的理论探究

双光子荧光染料分子在生物医学成像中具有广阔的应用前景,但取代效应对分子结构以及光物理性质影响的探求相对匮乏. 本文设计并研究了一系列脂滴检测染料分子,分析了分子的光学性质以及无辐射跃迁等. 通过分子内弱相互作用和电子- 空穴布居分析,阐述了其内在机理. 结果表明,所研究的分子均具有优良的光物理性能、高效荧光量子产量、大的斯托克斯位移以及显著的双光子吸收截面等. 本工作合理地解释了实验现象并阐述了取代效应对脂滴检测NAPBr染料分子的双光子吸收和激发态性质的影响,这为设计新型的高效有机分子提供了理论指导.     

以萘为π-中心的双芪类衍生物双光子上转换荧光性能研究

研究了2个新的双光子上转换荧光分子—1,4-双-(9-乙基咔唑基)萘(简称为BECVN)和1,4-双-(4’-N,N-二甲氨基苯乙烯基)萘(简称为BMABN)的单光子、双光子光谱性质。在～375 nm Xe灯光源激发下,两样品的DMF溶液发出很强的蓝、绿色荧光(峰位492～541 nm),视感效果非常明显。BMABN分子的线性吸收/发射光谱的峰位与BECVN分子相比,均发生红移;相对荧光量子产率(Φ_f)比BECVN降低了7.4倍。在飞秒钛宝石激光器泵浦下(760 nm),两样品的DMF溶液发出强的双光子上转换荧光发射,峰位与单光子荧光峰位相比发生红移(500～556 nm)。BMABN的双光子荧光强度和双光子吸收截面分别是BECVN的3倍和30.4倍。     

Acetylene-Substituted Two-Photon Absorbing Molecules With Rigid Elongated Pi-Conjugation: Synthesis,Spectroscopic Properties and Two-Photon Fluorescence Cell Imaging Applications

Two asymmetrical molecules with substituted acetylene as central rigid elongated conjugation are reported as potential chromophores for two-photon microscopic imaging. These molecules consist of a typical D–π–A structure, have different donors (D), the same π-conjugated center (π) and the same acceptor (A). Structural characterization and spectroscopic properties, including single-photon (linear) absorption, quantum yields, single-photon fluorescence, and two-photon absorption spectra, were studied in solvents with different polarity. These acetylene-substituted molecules were found to have high two-photon absorption cross-sections (for example, 690 GM for molecule 1 in toluene), which were determined by a two-photon induced fluorescence method using a femtosecond Ti: sapphire laser as excitation source. Single- and two-photon cellular imaging experiments demonstrate that the substituted acetylene derivatives could be one kind of promising two-photon fluorescence probes for cellular imaging.     

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。     

Photobleaching with a subnanosecond laser flash

In standard fluorescence recovery after photobleaching (FRAP) applications for measuring lateral diffusion rates and adsorption/desorption kinetics of fluorescent molecules at biological or model membranes, irreversible bleaching is induced by a bright excitation flash of at least millisecond time scale. It has been presumed that the bleaching event is of a low probability and the significant bleached population that develops during the flash results from each molecule undergoing thousands of excitation/deexcitation cycles before a bleaching event occurs. In some FRAP experiments, notably polarized FRAP (PFRAP) for measuring molecular rotational diffusion rates, it is desirable to use much shorter (subnanosecond) bleaching pulses. However, subnanosecond pulses are shorter than the fluorescence lifetime, so that any fluorophore will experience at most only one visit to the excited state during the bleaching pulse. If bleaching occurs only by the same processes as in slower FRAP experiments, one would thereby expect only minimal bleaching regardless of the bleach intensity. Moreover, the ability of fast polarized pulses to imprint an anisotropic orientational pattern in the postbleach unbleached fluorophore, an ability essential for PFRAP, is not at all guaranteed, particularly if two-photon processes are involved in high-intensity short bleach pulses. In this study, bleaching depths are measured as a function of subnanosecond pulse intensity on a small labeled protein covalently immobilized on fused silica. We show that bright subnanosecond laser flashes do indeed produce significant bleaching, that both two photon effects and reversible bleaching are involved, and that polarized bleaching does produce an anisotropic orientational pattern of unbleached fluorophore. We also postulate a theoretical molecular state model which semiquantitatively accounts for the experimentally observed dependence of reversible bleaching on bleaching pulse intensity.     

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。     

Fluorescence upconversion properties of a class of improved pyridinium dyes induced by two-photon absorption

We report the fluorescence upconversion properties of a class of improved pyridinium toluene-p-sulfonates having donor–π–acceptor (D–π–A) structure under two-photon excitation at 1064 nm. The experimental results show that both the two-photon excited (TPE) fluorescence lifetime and the two-photon pumped (TPP) energy upconversion efficiency were increased with the enhancement of electron-donating capability of the donor in the molecule. It is also indicated that an overlong alkyl group tends to result in a weakened molecular conjugation, leading to a decreased two-photon absorption (TPA) cross section. By choosing the donor, we can obtain a longest fluorescence lifetime of 837 ps, a highest energy upconversion efficiency of 6.1%, and a maximum TPA cross-section of 8.74×10⁻⁴⁸ cm⁴ s/photon in these dyes.     

A new class of quantum states generated by m-fold application of Bogoliubov＇s transformation

By applying higher powers of the Bogoliubov transformation operator b^{\dagger }=ν^*a+μ^*a^{\dagger } to the two-photon coherent states (or minimum uncertainty squeezed states) we construct a new type of quantum state which we call the generalized excited two-photon coherent states. Analytic expressions for the quantum statistical properties are derived, and through numerical computation the phase space quasi-probability distributions are found. These states can exhibit highly nonclassical behaviour depending on the degree of excitation m and other parameters. For particular values of two parameters λ and ρ, these generalized states reduce to other classes of coherent states formerly reported. Our theory thus presents a much broader approach to these types of quantum states.     

9,10-Anthracene-centered oligo(p-phenyleneethynylene)s with end-capping didecylamines exhibiting enhanced two-photon fluorescence action cross-section

This study reports the synthesis and the two-photon absorption (TPA) properties of a series of 9,10-anthracene-centred oligo(p-phenyleneethynylene)s with end-capping didecylamine. The results show that both the maximal TPA cross-sections (δ_max) and the fluorescence quantum yields (Φ) of the oligomers increase with the extension of phenylethynyl bridges. Interestingly, the Φδ_max per repeating unit to is obviously enhanced by extending linearly conjugation size of the oligomers. Moreover, these oligomers all show the maximal two-photon absorption cross-sections at 800 nm and emit strong green to yellowish-green two-photon excited fluorescence. These materials may find applications in which two-photon excited fluorescence and photo-induced electron transfer are utilized.