时间分辨荧光(TRF)测量及其应用

荧光寿命和时间分辨荧光光谱是荧光光谱学的两个重要参量,随着探测技术的发展而得到更多的应用.本文简述TRF测量技术及其在化学、物理、生化和医学方面的应用,以期促进TRF在国内的广泛利用.     

飞秒时间分辨光学多道谱仪研究

超快时间分辨光谱学为研究非线性光学，固体物理，化学反应动力学，能量传递和驰豫、生物过程动力学和相干光谱技术提供了一种有效的工具。本文报导了一种飞秒时间分辨光学多道光谱设备，该设备是由一台用氩离子激光器原浦且产生１００ｆｓ光脉冲并调谐在７２０－９８０ｎｍ的飞秒掺钛兰宝石激光器，改进了光学多道器（ＯＭＡ－ＩＩ），３８６计算机及光学系统组成的。激光脉冲用ＬＢＯ晶体倍频，谱仪装置的时间分辨率约１２０ｆｓ，     

光波导耦合的表面等离子体共振光谱传感器实时监测表面生化反应

利用厚度为100 μm且局部表面覆盖有45 nm金膜的玻璃片作为消逝波表面敏感元件,自主设计并组装了光波导耦合的光谱表面等离子体共振(SPR)生化传感器.本仪器使用一对玻璃棱镜作为输入-输出耦合器,卤钨灯作为白光光源,利用时间分辨的CCD光谱分析仪对光波导输出光谱进行快速测定.这种光波导耦合的光谱SPR传感器灵敏度高,稳定性好,时间分辨率高,光谱窗口和动态探测范围宽,非常适合于表面生化反应的动力学测试.利用这种光波导耦合的光谱SPR传感器对金纳米粒子在半胱胺修饰的金膜表面的自组装过程进行了原位实时监测.     

卤代苯溶剂对镓咔咯配合物光物理性质的影响:外重原子效应

利用紫外-可见光谱、稳态和时间分辨荧光光谱以及飞秒瞬态吸收光谱探测了不同卤代苯溶剂对三种五氟苯基取代的镓咔咯(1-Ga、2-Ga、3-Ga)光物理性质的影响,结果表明卤代苯溶剂的色散力对于镓咔咯电子光谱吸收峰位置的影响起着主要作用;溶剂外重原子效应能显著降低镓咔咯的荧光量子产率。飞秒瞬态吸收光谱表明,光激发下,镓咔咯与卤代苯溶剂之间可发生电子转移反应,溶剂的重原子效应可以减缓电荷分离态复合物电荷重组速率。     

超快速反应荧光亏蚀谱微观动力学理论

用超短脉冲激发动力学光谱理论在两电子体系下模拟了飞秒时间分辨的荧光亏蚀谱, 再现了实验中所得荧光亏蚀谱的双指数衰减和凹陷两种现象, 这两种现象分别是由于探测脉冲使电子激发态上振动基态和较高振动态的分子受激发射而产生的.     

异硫氰基孔雀石绿受激拉曼光谱研究

本文报道了具有时间分辨能力的全频宽带受激拉曼（BBSRS）系统和关于异硫氰基孔雀石绿（MGITC）受激拉曼光谱（sRs）的研究．BBSRS系统的探测光为450-800nm宽带连续白光,泵浦光为280～900nm范围内连续可调谐的ps窄带可见光（带宽≈7．5cm-1,脉宽≈2．5ps）．在合适的泵浦波长下,该系统可同时获取拉曼损失和拉曼增益光谱．MGITC的SRS研究结果表明,当拉曼损失谱峰出现在最大吸收波长（≈627nm）时,共振SRS谱峰强度最大;当泵浦或增益谱峰在最大吸收波长附近时,未观察到明显的共振拉曼信号;共振峰强度随浓度增大而增大,随泵浦功率增大而迅速增大,后趋于饱和;共振和非共振峰强在延时零点附近达到最大值,并随延时绝对值的增大而减小．     

SO2的简并四波混频光谱

简并四波混频（DegenerateFour－WaveMixingDFWM）是一项非线性光谱技术·近年来，它被成功地用于气相痕量原子、分子和自由基的探测［1，2］，而受到人们的关注．DFWM具有亚多普勒的光谱分辨、良好的时间和空间分群等特点，可以实现远距离探测，能有效地排除背景光干扰．此外，由于DFWM技术是直接用于探测体系的吸收，可以应用于无荧光或荧光量子产率低的体系和激发态具有电离和预解离等竞争通道的体系，而这两种体系用传统的激光诱导荧光（LIF）和共振多光子电离（REMPI）方法很难探测．Dreier和Rakestraw曾用DFWM技术探测火…     

三维同步偏振荧光光谱研究蛋白质溶液变性时氨基酸残基的发光行为

将偏振技术、同步技术与三维技术结合起来的三维同步偏振荧光光谱(TDSPS)能分辨蛋白质溶液中的色氨酸(Trp)和酪氨酸(Tyr)残基,具有同步光谱分辨率高、三维技术信息丰富的优点．本文用TDSPS表征牛血清白蛋白(BSA)、人血清白蛋白(HSA)受各种因素影响(酸效应、碱效应、盐效应、猝灭剂等)时Trp,Tyr残基荧光光谱的变化,用于区分HSA和BSA．     

飞秒受激拉曼光谱技术

飞秒受激拉曼光谱（femtosecond stimulated raman spectroscopy,FSRS）是一种新型的非线性振动光谱技术.它使用两束重叠的窄带拉曼泵浦和宽带探测脉冲激光束,利用外差式检波方法在探测光方向上进行信号探测.FSRS既可以用来探测分子电子基态的振动动力学,也可以用来探测分子电子激发态的振动动力学,比如同质异构类反应.即使荧光背景很强的分子,也可以用FSRS来研究.FSRS可以用三阶微扰的波包图像来描绘.单从相位匹配条件出发,共有48项对应于FSRS过程,但是其中只有8项满足共振条件.可以用3种方法来描述这8项：双时间线拉曼图、封闭时间路径环路图和四波混频的能级图.进一步分析表明,这8项可以分成4类,即SRS（I）,SRS（II）,IRS（I）和IRS（II）,其中SRS代表受激拉曼散射,IRS代表反转拉曼散射.SRS（I）可以用来解释自发拉曼散射,但是其余的SRS（II）、IRS（I）和IRS（II）三项只在受激拉曼光谱中存在.FSRS光谱中SRS（I）过程产生的是斯托克斯拉曼谱线,而IRS（I）过程则产生的是反斯托克斯谱线.其余的两项SRS（II）和IRS（II）只是产生很宽的背景基线,基本和我们感兴趣的观测量不相关.使用简谐振动模型,我们可以得到三阶微扰极化率的四时间相关函数的解析表达式.我们讨论了FSRS光谱之所以能够得到高时间分辨率和高频率分辨率的物理原理.在文章中,我们还就以下研究做了计算与实验结果的比较：（a）荧光性罗丹明6G的共振FSRS谱和（b）CDCl3分子非共振脉冲泵浦光制备的相干振动态的二维FSRS谱.计算得到的结果与实验十分吻合,同时在理论上证明了CDCl3二维FSRS光谱中级联效应是占主导作用的.     

一种新型吡唑啉类染料荧光性质的研究

本文利用紫外吸收、荧光光谱、偏振荧光和时间分辨瞬态光技术,研究了1-对氨硫酰苯基-3-对氯苯基吡唑(SCPL)在不同溶剂     

Spectrum Correction in Study of Solvation Dynamics by Fluorescence Non-collinear Optical Parametric Ampli cation Spectroscopy

Femtosecond time-resolved fluorescence non-collinear optical parametric amplification spectroscopy can extract the curve of spectral gain from its parametric superfluorescence. This unique spectrum correction method enables fluorescence non-collinear optical parametric amplification spectroscopy acquiring the genuine transient fluorescence spectrum of the studied system. In this work we employ fluorescence non-collinear optical parametric amplification spectroscopy technique to study the solvation dynamics of DCM dye in ethanol solution, and confirm that genuine solvation correlation function and shift of peak frequency can be derived from transient fluorescence spectra after the spectral gain correction. It demonstrates that fluorescence non-collinear optical parametric amplification spectroscopy can benefit the research fields, which focuses on both fluorescence intensity dynamics and fluorescence spectral shape evolution.     

TIME-RESOLVED PHOTOACOUSTIC CALORIMETRY OF TRYPTOPHAN IN WATER and ORGANIC SOLVENTS

Abstract –Very little has been done in the direct study of nonradiative decays of Trp, indole and derivatives, in spite of their relatively low fluorescence quantum yields. Time-resolved photoacoustic calorimetry is an ideal technique for this kind of study: it is very sensitive in the detection of small amounts of heat released and, in principle, allows a broad band of temporal resolution. The photoacoustic apparatus used for our measurements offers a temporal window between 10 ns and 10 μs. The analysis of the waveforms, based on a particular deconvolution method, simultaneously gives the fraction of energy released and the associated lifetime. In a broad sense, time-resolved photoacoustic spectroscopy can be seen as a complementary method to traditional radiative techniques such as static, time-resolved fluorescence and flash photolysis. The present work presents studies of Trp in different solvents in order to acquire new information about the interaction of this chromophore with solvents. From our measurements the high sensitivity of Trp to solvents is confirmed. The formation of complexes is evident at the excited state (exciplexes), between the chromophore and one or more molecules of solvent. These exciplexes are characterized by having an energy different from that of the singlet and triplet of Trp. Moreover, photoacoustic measurements detect, in water, the presence of another electronic state, which seems to have characteristics similar to a triplet-like state reported in other work.     

TIME-RESOLVED PHOTOACOUSTIC CALORIMETRY OF TRYPTOPHAN IN WATER AND ORGANIC SOLVENTS

Abstract: Very little has been done in the direct study of nonradiative decays of Trp, indole and derivatives, in spite of their relatively low fluorescence quantum yields. Time-resolved photoacoustic calorimetry is an ideal technique for this kind of study: it is very sensitive in the detection of small amounts of heat released and, in principle, allows a broad band of temporal resolution. The photoacoustic apparatus used for our measurements offers a temporal window between 10 ns and 10 μs. The analysis of the waveforms, based on a particular deconvolution method, simultaneously gives the fraction of energy released and the associated lifetime. In a broad sense, time-resolved photoacoustic spectroscopy can be seen as a complementary method to traditional radiative techniques such as static, time-resolved fluorescence and flash photolysis. The present work presents studies of Trp in different solvents in order to acquire new information about the interaction of this chromophore with solvents. From our measurements the high sensitivity of Trp to solvents is confirmed. The formation of complexes is evident at the excited state (exciplexes), between the chromophore and one or more molecules of solvent. These exciplexes are characterized by having an energy different from that of the singlet and triplet of Trp. Moreover, photoacoustic measurements detect, in water, the presence of another electronic state, which seems to have characteristics similar to a triplet-like state reported in other work.     

Charge resonance character in the charge transfer state of bianthryls: effect of symmetry breaking on time-resolved near-IR absorption spectra

We study the effects of symmetry breaking on the photogenerated intramolecular charge transfer (CT) state of 9,9'-bianthryl (BA) with femtosecond time-resolved near-IR spectroscopy. The time-resolved near-IR spectra are measured in acetonitrile for a symmetric substituted derivative of 10,10'-dicyano-9,9'-bianthryl (DCBA) and asymmetric substituted derivatives of 10-cyano-9,9'-bianthryl (CBA) and 9-(N-carbazolyl)anthracene (C9A), as well as nonsubstituted BA. The transient near-IR absorption spectrum of each compound at 0 ps has a locally excited (LE) absorption band, which agrees with the transient absorption band of the corresponding monomer unit. At 3 ps after the photoexcitation, the symmetric compounds show a broad charge transfer (CT) absorption band, whereas no absorption peak appears in the spectra of the asymmetric compounds. The broad CT absorption at 1250 nm only observed for the symmetric compounds can be attributed to the charge resonance transition associated with two equivalent charge separated states.     

Intramolecular directional förster resonance energy transfer at the single-molecule level in a dendritic system

We report on the directional F?rster resonance energy transfer (FRET) process taking place in single molecules of a first (T1P4) and a second (T2P8) generation of a perylenemonoimide (P)-terrylenediimide (T)-based dendrimer in which the chromophores are separated by rigid polyphenylene arms. At low excitation powers, single-molecule detection and spectroscopy of T1P4 and T2P8 dendrimers point to a highly efficient directional FRET from P donors to the central T acceptor, optical excitation at 488 nm resulting in exclusively acceptor emission in the beginning of the detected fluorescence intensity. Donor emission is seen only upon the bleaching of the acceptor. High-resolution time-resolved single-molecule fluorescence data measured with a microchannel plate photomultiplier reveal, for T2P8, a broad range of FRET rates as a result of a broad range of distances and orientations experienced by the donor-acceptor dendrimers when immobilized in a polymer matrix. Single-molecule data from T2P8 on 488 nm excitation are indicative for the presence, after terrylenediimide bleaching, of a P-P excited dimer characterized by a broad emission spectrum peaking around 600 nm and by fluctuating fluorescence decay times. At high excitation powers, single T1P4 and T2P8 molecules display simultaneous emission from both donor and acceptor chromophores. The effect, called "exciton blockade", occurs due to the presence of multiple excitations in a single molecule.     

Ultrafast fluorescence depolarisation in the yellow fluorescent protein due to its dimerisation.

Transient absorption spectroscopy with sub-100 fs time resolution was performed to investigate the oligomerisation behaviour of eYFP in solution. A single time constant tau(AD)=2.2+/-0.15 ps is sufficient to describe the time-resolved anisotropy decay up to at least 200 ps. The close contact of two protein barrels is deduced as the exclusive aggregation state in solution. From the final anisotropy r(infinity)=0.28+/-0.02, the underlying quaternary structure can be traced back to the somewhat distorted structure of the dimers of wt-GFP. The use of autofluorescent proteins as rulers in F?rster resonance energy transfer (FRET) measurements may demand polarisation-sensitive detection of the fluorescence with high time resolution.     

Photoinduced properties of methyl ester of 2,6-dimethyl-1-amino-4-benzoic acid: Evidence of charge transfer emission from a weak primary amino donor

Photophysical properties of methyl ester of 2,6-dimethyl-1-amino-4-benzoic acid (M26DMB) have been investigated using steady state absorption and emission spectroscopy and time-resolved emission spectroscopy. Interestingly, not only in polar solvents, the molecule M26DMB having a weak primary amino donor group shows broad red-shifted emission band even in non-polar environment which in all probability arises from closely spaced local and charge transfer (CT) states. Clear dual fluorescence in polar protic solvents comprises of less intense local emission band and strong red-shifted CT band. The position of the red-shifted emission band is dependent on both the polarity and the hydrogen-bonding ability of the solvent.     

Simultaneous time and frequency detection in femtosecond coherent Raman spectroscopy. II. Application to acetonitrile

The preceding paper showed that, in principle, a high-resolution coherent Raman spectrum can be recovered using femtosecond probe pulses by combined detection in both time and frequency. This measurement is possible even when the pulses are too broad in frequency for conventional frequency-domain spectroscopy and too broad in time for conventional time-domain spectroscopy. In this paper, the method is tested on experimental coherent anti-stokes Raman spectroscopy data from acetonitrile. Compared to theoretical models, experimental data are complicated by noise and incomplete knowledge of the pulse structure. Despite these complications, most of the information in the Raman spectrum is recovered from the data: weak transitions are detected and natural-linewidth resolution is achieved across an 800 cm(-1) spectral range. However, circumstances in which experimental limitations result in missed features or ambiguities in the recovered spectrum are also identified. These results suggest where improvements in measurement and data analysis can be made.     

Fluorescence characteristics of protonated form of 6-hydroxyquinoline in Nafion film

Fluorescence characteristics of 6-hydroxyquinoline (6-HQ) have been studied at room temperature in Nafion(R) film by steady state and nano-second time-resolved fluorescence spectroscopy. The fluorescence spectrum exhibits single emission band corresponding to the protonated form of 6-HQ in this matrix. However, the decay fits with two or three exponential functions depending on the emission wavelength monitored. At blue edge of the emission, the decay fits to three-exponential function, whereas at longer wavelengths, the decay fits to bi-exponential function. Two tentative mechanisms have been proposed to explain the experimental data, viz. a closely lying charge transfer state (CT) or an excited state proton transfer (ESPT) process. The photophysical parameters appear to be sensitive to the change in microstructure due to swelling of the membrane by the solvents.     

Laser picosecond microspectrofluorometry of haematoporphyrin in cells and liposomes.

The results of a laser picosecond microspectrofluorometric study of the spectral and kinetic characteristics of haematoporphyrin (Hp) fluorescence at various sites in cultured SPEV cells and phosphatidylcholine liposomes are presented. The computer-controlled detection system is based on the single-photon counting method with picosecond time resolution. In aqueous medium, the Hp fluorescence spectrum is characterized by two bands at 615 and 675 nm. In living cells and liposomes, Hp fluorescence is red shifted to 630 and 690 nm. In addition a new band at 665 nm is detected. The dependence of this band on the incubation time and Hp concentration was investigated. The fluorescence decay kinetics of Hp in a culture medium, liposome and a cell nuclear membrane were measured. Possible Hp aggregate formation in the lipid bilayer and its implications are discussed.