双光子激发时间分辨荧光光谱测量技术

建立了一台基于高重复频率扫描相机的双光子激发时间分辨荧光光谱测量系统，能够同时测量样品的荧光光谱和寿命. 该系统的时间分辨率为6.5—200ps，光谱分辨率为1—3nm，能够实现快速数据采集以及可靠和可重复的寿命和光谱测量. 利用标准荧光染料(若丹明6G、香豆素314)及其混合溶液对该系统进行了测试，所得到的荧光光谱分布和寿命值与文献报道一致. 实验结果表明，该系统能有效区分多组分荧光团. 这为鉴别多荧光团或多组分生物组织提供了一种独特的对比方法，可用于多光谱分辨荧光寿命成像和荧光共振能量转移成像等方面. 关键词： 荧光寿命 荧光光谱 双光子激发 高重复频率扫描相机     

用于双光子激发荧光寿命显微成像的高重复频率皮秒扫描相机

建立了一台基于新研制的高重复频率皮秒扫描相机的双光子激发荧光寿命显微成像系统,重点介绍所研制的高重复频率皮秒扫描相机。为了在高时间分辨力的同时扩大时间测量范围,实现大面积两维空间高时间分辨取样测量,从而提高采样速率和更有效地发挥扫描相机的作用,设计和研制了一种大面积、高时间分辨力扫描变像管和一种重复频率高达1MHz的斜坡电压扫描电路。基于上述关键部件所研制的扫描相机具有重复频率高、扫描速度可调、时间分辨力高、工作面积大、非线性低、触发晃动小等优点。用钛宝石飞秒激光器作为激光脉冲源,通过脉冲提取器将76MHz的高重复频率降低为1MHz,采用可调延时器和标准具对扫描相机的时间分辨力、扫描速度和非线性进行标定。该系统的时间分辨力达到6.5ps,非线性为2.60%,可测量的时间范围从十几皮秒到几十纳秒。测量了若丹明6G和香豆素314两种标准荧光染料的荧光寿命,取得了与参考文献一致的实验结果。     

Simultaneous one-dimensional fluorescence lifetime measurements of OH and CO in premixed flames

A method for simultaneous measurements of fluorescence lifetimes of two species along a line is described. The experimental setup is based on picosecond laser pulses from two tunable optical parametric generator/optical parametric amplifier systems together with a streak camera. With an appropriate optical time delay between the two laser pulses, whose wavelengths are tuned to excite two different species, laser-induced fluorescence can be both detected temporally and spatially resolved by the streak camera. Hence, our method enables one-dimensional imaging of fluorescence lifetimes of two species in the same streak camera recording. The concept is demonstrated for fluorescence lifetime measurements of CO and OH in a laminar methane/air flame on a Bunsen-type burner. Measurements were taken in flames with four different equivalence ratios, namely ? = 0.9, 1.0, 1.15, and 1.25. The measured one-dimensional lifetime profiles generally agree well with lifetimes calculated from quenching cross sections found in the literature and quencher concentrations predicted by the GRI 3.0 mechanism. For OH, there is a systematic deviation of approximately 30 % between calculated and measured lifetimes. It is found that this is mainly due to the adiabatic assumption regarding the flame and uncertainty in H₂O quenching cross section. This emphasizes the strength of measuring the quenching rates rather than relying on models. The measurement concept might be useful for single-shot measurements of fluorescence lifetimes of several species pairs of vital importance in combustion processes, hence allowing fluorescence signals to be corrected for quenching and ultimately yield quantitative concentration profiles.     

Temporally and spectrally resolved sampling imaging with a specially designed streak camera

We present a novel sampling imaging technique capable of performing simultaneous two-dimensional measurements of the temporal and spectral characteristics of light-emission processes by use of a specially designed streak camera. A proof-of-principle experiment was performed with a homemade multifocal multiphoton fluorescence microscope. The system was calibrated with a Fabry-Perot etalon and a standard fluorophore solution (rhodamine 6G in ethanol) and was shown to have temporal and spectral resolution of 6.5 ps and 3 nm, respectively, as well as high accuracy and reproducibility in lifetime and spectrum measurement. Temporally and spectrally resolved images of 4 x 4 foci on the sample can be obtained with a snapshot.     

激光诱导叶绿素荧光寿命的测量及其特性分析

提出了一种用于评估植物生长状况及环境监测的激光诱导叶绿素荧光寿命测量方法. 采用波长355 nm的激光作为光源激发叶绿素荧光, 由光电倍增管接收其荧光信号, 由于被测叶绿素荧光衰减函数与激光脉冲、仪器响应函数卷积在一起, 根据它们的特性, 运用时间分辨测量法分别测得叶绿素荧光及其背景信号, 并结合一种新型解卷积算法可分离出真实的叶绿素荧光衰减函数, 从而获取叶绿素的荧光寿命. 测试结果表明: 该方法能够实现叶绿素荧光寿命的高精度实时监测, 对不同叶绿素含量的溶液荧光寿命进行了测试, 证明叶绿素含量与其荧光寿命具有相关性, 并且拟合了叶绿素含量与荧光寿命的标定曲线. 关键词： 荧光寿命 激光诱导荧光 时间分辨测量法 叶绿素含量     

Dual component fluorescence lifetime of some polymethine saturable absorbing dyes

Using the synchronously-operated streak camera, dual components of the fluorescence decay of 9-alkyl substituted polymethine saturable absorbing dyes were detected with picosecond resolution. The effect on the fluorescence lifetime of viscosity, dielectric constant and temperature in particular were examined.     

激光诱导植物荧光寿命校正技术及其特性分析

激光诱导植物荧光寿命测量法是在植物荧光光谱分析法基础上开发的一种评估植物生长状况及环境监测的新技术。根据植物叶绿素荧光信号的物理特性,利用信息仿真技术开发了一种叶绿素荧光寿命校正方法,可提高植物叶绿素荧光寿命的测量精度。利用激光诱导叶绿素荧光寿命测量系统分别测得叶绿素荧光及其背景信号,先用解卷积法叶绿素荧光信号中分离出荧光衰减函数,可获取荧光寿命估计值。再结合叶绿素荧光寿命校正技术就能反演得到高精度的植物荧光寿命。仿真与实验结果表明：该方法可实现高精度的植物荧光寿命实时监测;并对不同含量的叶绿素提取液进行了测试,构建了植物荧光寿命与叶绿素含量的对应关系模型。未来该技术可用于遥感监测海洋、湖泊、河流中藻类植物的生物含量。     

Further performance tests of a picosecond X-ray and laser induced streak camera system with fast scintillation materials

A further performance test of a fluorescence lifetime spectroscopy system with 2D-photon counting based on a streak camera was carried out. This system offers three different excitation light sources. It includes a pulsable X-ray tube with 40 kV and a laser. A pulsed UV-LED has been installed for sample excitation. The table top measurement device has a temporal resolution down to 20 ps after deconvolution and fluorescence/scintillation decay times can be evaluated in a wavelength selectable range between 200 and 850 nm. Cesium fluoride, cesium chloride and cesium bromide as fast core-valence luminescence emitters with a relatively low light yield were used in the presented performance test. The easily manageable system is able to detect low light levels and to determine the decay times in agreement with literature in most instances. Beside mentioned scintillators PbCO₃, SrI₂ and Lu₂SiO₅:Ce as a high light yield material were also measured.     

Quenchers Induce Wavelength Dependence on Protein Fluorescence Lifetimes

We have analysed the picosecond resolved fluorescence emission decay of horseradish peroxidase A2 and of HEW lysozyme acquired with a streak camera. Analyses of the fluorescence decay data of both proteins revealed that the dynamics of the decay is dependent on the emission wavelength. Our data strongly indicates that resonance energy transfer occurring between aromatic residues and different protein fluorescence quencher groups, and the nature of the quencher groups, are the causes of the observed wavelength dependent mean lifetime distribution. Using the global analysis data to calculate the fluorescence mean lifetime at each wavelength revealed that for lysozyme, the mean fluorescence lifetime increased with observation wavelength, whereas the opposite was the case for peroxidase. Both proteins contain strong fluorescence quencher groups located in close spatial proximity to the protein’s aromatic residues. Lysozyme contains disulfide bridges as the main fluorescence quencher whereas peroxidase contains a heme group. Both for lysozyme and horseradish peroxidase there is a clear correlation between the observed fluorescence mean lifetime of the protein at a particular emission wavelength and the respective quencher’s extinction coefficient at the respective wavelength. Furthermore, our study also reports a comparison of the analyses of the fluorescence data done with three different methods. Analyses of the fluorescence decay at 10 different fluorescence emission wavelengths revealed significant differences in both fluorescence lifetimes and the pre-exponential factor distributions. Such values differed from the values recovered from the integrated decay curves and from global analyse.     

基于不同泵浦波形的荧光寿命测量

该文从理论上分析了不同泵浦波形下的荧光衰减规律,提出了一种在不同泵浦波形下测量荧光寿命的新方法——双脉冲探测法,即利用探测泵浦脉冲与荧光衰减脉冲的方法测量荧光寿命。通过对样品钕玻璃及Cr:ZnSe晶体的荧光寿命测量表明:利用该方法在不同泵浦波形下能够实现可见及近红外到中红外激光介质的荧光寿命测量。因此,利用该测量方法能够方便、有效的避免通过解卷积求样品荧光寿命的繁琐过程,对测量激光介质在不同泵浦波形下的荧光寿命具有参考价值。     

一种用于荧光皮秒时间分辨光谱的测试系统

本文详细描述了一种由氩离子锁模激光器、样品池、分光系统及微微秒条纹相机构成的用于测试低强度荧光微微秒时间分辨光谱的系统。给出了典型样品的测试结果:时间分辨率为10ps,光谱范围为500Å—800Å。最后对测量结果进行了分析讨论。     

单光子调制频谱用于量子点荧光寿命动力学的研究

本文开展了基于单光子调制频谱测量量子点荧光寿命动力学特性的研究.在脉冲激光激发下,对探测到的量子点单光子荧光信号进行频谱分析以获得荧光调制频谱,研究发现特征频谱信号幅值与荧光寿命之间存在确定的非线性对应关系.这种单光子调制频谱方法能有效消除背景噪声和单光子探测器暗计数的影响,用于分析量子点荧光寿命动力学特性时在准确度以及时间分辨率方面都较目前普遍采用的荧光衰减曲线寿命拟合方法呈现出明显优势:当涨落误差为5%时,寿命测量准确度提高了一个数量级;当涨落误差和偏离误差均为5%时,对动力学测量效率以及时间分辨率提高了四倍以上.因此单光子调制频谱可以作为获取量子点在短时间尺度内激发态动力学信息的一种有效技术手段.     

一种应用PLD-PMTR技术的荧光光纤温度计

通过对光纤荧光温度传感器中影响系统准确度的荧光寿命检测技术的研究，利用双参考信号、脉冲调制激励光源的锁相检测技术对荧光光纤温度传感器的荧光寿命进行检测，推导出测量荧光寿命的数学模型.给出了应用该技术的温度测量方案及实验结果，该方法具有高的信噪比且对激励光泄露有很强的抑制作用.系统的输出信号可以准确地进行长距离传输且很容易与计算机接口.实验表明，该方法是有效和实用的，达到了系统要求.     

激光诱导荧光寿命及其测量

激光诱导荧光特性的研究可用于包括心血管病在内的多种疾病的诊断。荧光发射包括光谱（频域）和时间（时域）两方面的信息，后者表现为荧光寿命。在很多情况下，测量荧光寿命是比测量光谱更为有效的诊断方法。本文从理论上讨论了荧光寿命问题，并介绍两种测量方法，可用于测量人体正常组织和病变组织的激光诱导荧光寿命     

Linear picosecond electron-optical chronoscopy at a repetition rate of 140 MHz

A picosecond streak camera operating in synchronism with C.W. mode-locked lasers provides real-time integrated measurement of repetitive luminous phenomena with a temporal resolution of ～ 10 ps. The durations of pulses from an actively mode-locked argon ion laser (514.5 nm) have been measured to be ～ 90 ps and the system is particularly suitable for the measurement of dye fluorescence lifetimes.     

基于激光诱导叶绿素荧光寿命成像技术的植物荧光特性研究

针对植物荧光遥感探测中信号易受干扰的问题, 提出了一种用于评估植物生长状况及环境监测的荧光寿命成像技术. 采用凹透镜对355 nm波长的激光扩束, 再照射植物激发叶绿素荧光, 由增强型电荷耦合器件接收荧光信号. 采用时间分辨测量法, 连续用相同激光脉冲照射植物以激发相同的荧光信号, 同时不断改变激光脉冲触发探测器启动的延时时间, 从而能够得到完整的离散荧光信号分布图像. 对植物特定位置点产生的离散荧光信号进行拟合, 再运用一种改进型的迭代解卷积法可反演高精度的荧光寿命; 进而反演图像各点的荧光寿命以生成植物的荧光寿命分布图. 该方法所绘制的荧光寿命图比荧光强度图能更准确地反映植物内部的叶绿素含量, 并对活体植物叶绿素荧光寿命的物理特性进行了初步研究, 证明叶绿素荧光寿命与植物生理状态存在一定关联; 并且叶绿素荧光寿命与活体植物所处环境存在着复杂的关系. 未来将与生物物理学家们合作, 继续探寻叶绿素荧光寿命与植物生存环境的关系.     

Measurements of collisional quenching of hydrogen atoms in an atmospheric-pressure hydrogen oxygen flame by picosecond laser-induced fluorescence

Atomic hydrogen has been probed in a hydrogen/oxygen flame at atmospheric pressure by use of two-photon, picosecond Laser-Induced Fluorescence (LIF). The fluorescence was detected and temporally resolved by a streak camera with a temporal resolution of a few ps which allowed determination of the collisionally quenched lifetime of then = 3 level of atomic hydrogen. The measurements were performed for three flame stoichiometries, = 1.4, 2.0 and 3.5, and were spatially resolved with respect to the reaction zone. The evaluated lifetime was found to vary rapidly in the reaction zone from 60 to 105 ps just above and then levelled out at 90 ps throughout the post-flame region. Power dependence measurements of the fluorescence signal indicated the presence of other phenomena, such as saturation, ionization and photodissociation. Since an inverted population is created between then = 3 and then = 2 level, Stimulated Emission (SE) of considerable magnitude occurred and was studied temporally resolved using the streak camera. The LIF and SE signal strengths for individual laser pulses were recorded in order to analyse a possible anti-correlation between the LIF and the SE.     

Investigation of viscosity dependent fluorescence lifetime using a synchronously operated picosecond streak camera

Exciting with picosecond pulses from a mode locked cw dye laser and using the Synchroscan continuously operating streak camera, the variation in the fluorescence lifetime with viscosity over the range 0.5 cp–250 cp of some mode locking and potential mode locking polymethine dyes of the cyanine family were investigated. The measured lifetimes were found to follow a ∼2/3 or 1/3 power dependence of the viscosity η.     

一种实现显微荧光寿命图的测量方法

将脉宽120fs、重复率76MHz激光引入激光扫描显微镜的激发光路,利用其扫描系统对荧光标记样品激发扫描,将激发出的荧光从荧光探测光路引入备用的外部探测口;在探测口接一快速光电倍增管,将光电倍增管的信号送给时间相关单光子计数器,获得时间相关的荧光强度图;最后通过计算机处理获得荧光寿命图。应用此系统对青色荧光蛋白(CFP)、黄色荧光蛋白(YFP)荧光寿命进行了测量,并应用CFP、YFP实现荧光共振能量转移的测量。通过实验看出利用已有的激光扫描显微镜,配合较先进的寿命测量方法,可以很好地实现显微荧光寿命图的测量。