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。     

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.     

农药荧光寿命测试系统的原理与设计

介绍荧光的产生、荧光寿命的产生机理以及荧光寿命测量的基本原理。设计了一种利用直接记录法(光子计数法)测量农药荧光寿命的测试系统。该系统针对待测样品的特性，选用了相应的脉冲光源、光学元件和半导体探测器等器件，优化了各器件的工作参数，进行了简易而又科学的模块化设计，并对西维因农药的荧光寿命在无激励光干扰情况下进行了实际测试，测得了西维因溶液在500μg／L浓度时的荧光衰减曲线和荧光寿命(0.30～0.40ns)。结果表明，该系统具有结构简易、操作方便的优点，能测量100ps级的荧光寿命，适合于对能发荧光的农药进行荧光寿命的定量测量。     

Appropriate Gate Time for Single Molecular Photon Detection Based on Optimal Signal-to-Noise Ratio Analyses

Signal-to-noise ratio of fluorescence detection from a single molecule ＆ analysed by using time-gated techniques. It is found that the optimal signal-to-noise ratio can be obtained by choosing an appropriate gate time with a certain optical background. The dependences of molecular fluorescence lifetime and the optimal signal-to-noise ratio on the appropriate gate time are respectively discussed with two kinds of background sources~ chaotic state with uniform distribution and coherent state with exponential distribution in time domain. For chaotic state background we find that a certain range for appropriate gate time can be obtained with a definite fluorescence lifetime, larger fluorescence lifetime would lower the value of optimal signal-to-noise ratios. For coherent state background we find that there is also a narrow range of appropriate gate time when lifetime of single molecule is less than that of background photons.     

Polar Plot Representation for Frequency-Domain Analysis of Fluorescence Lifetimes

We present applications of polar plots for analyzing fluorescence lifetime data acquired in the frequency domain. This graphical, analytical method is especially useful for rapid FLIM measurements. The usual method for sorting out and determining the underlying lifetime components from a complex fluorescence signal is to carry out the measurement at multiple frequencies. When it is not possible to measure at more than one frequency, such as rapid lifetime imaging, specific features of the polar plot analysis yield valuable information, and provide a diagnostic visualization of the participating fluorescent species underlying a complex lifetime distributions. Data are presented where this polar plot presentation is useful to derive valuable, unique information about the underlying component distributions. We also discuss artifacts of photolysis and how this method can also be applied to samples where each fluorescence species shows a continuous distribution of lifetimes. Polar plots of frequency-domain data are commonly used for analysis of dielectric relaxation experiments (Cole–Cole plots), which have proved to be exceptionally useful in that field for decades. We compare this analytical tool that is well developed and extensively used in dielectric relaxation and chemical kinetics to fluorescence measurements.     

基于时间分辨荧光光谱法同时测定己酸乙酯与乙酸乙酯

提出了一种基于时间分辨荧光光谱分析法同时测定己酸乙酯和乙酸乙酯混合液中各组分浓度的方法。实验测得两种单体物质的荧光发射光谱重叠度达到78%。对于荧光发射光谱重叠度很高的物质使用普通荧光方法结合算法也很难进行定量分析。而通过量子化学计算结果,对两种单体的分子结构分析,得到己酸乙酯的荧光寿命长于乙酸乙酯。通过时间分辨荧光光谱测量得到己酸乙酯和乙酸乙酯的荧光寿命分别为1.0和5.3 ns。两种物质的荧光发射光谱重叠严重,但是其荧光衰减曲线有明显的差别,所以提出使用时间分辨荧光光谱法对两种物质进行定量分析。通过分析两种物质不同体积比下的荧光衰减曲线的变化趋势,建立两种物质体积比的预测模型,并对其进行检验。实验结果表明：该方法能够实现混合溶液中各组分体积比的测量,其测量的平均残差控制在1%以内,具有一定的实用价值。     

High-efficiency photon-counting fluorometer with a channel width of 5.0 ps

We construct a photon-counting, fluorescence lifetime measurement system with a channel width of 5.0 ps. This is a modified version of our previous fluorometer based on a simultaneous detection method of photoelectron pulse trains after pulsed excitation (Iwata in Meas Sci Technol 28:075501, 2017), which can accept multiple photons during a one period of the excitation for building a histogram. Although the resolution time is 2.0 ns as before, the channel width of 5.0 ps is of the same order as that of time-correlated single-photon counting, which is achieved by delaying the trigger timing for the fluorometer by a step to that of the excitation. This results in an increase in the precision for the lifetime estimation. Although the measurement time increases with increasing the number of delay steps, it is still rather shortened owing to the high signal-gathering and histogram-building efficiencies.     

通过光致还原调制氧化石墨烯寿命并用于微纳图形制备

氧化石墨烯因其宽带可调谐的荧光发射特性已被广泛应用于荧光成像、金属离子高灵敏检测和光电器件的制备.相比于荧光强度,氧化石墨烯荧光寿命不受材料厚度和激发功率的影响,具有更为稳定和均一的特性.本文研究了在激光还原过程中氧化石墨烯荧光寿命逐渐减小的变化行为,发现了长寿命sp~3杂化结构向短寿命sp~2杂化结构的转变.通过精确控制还原时间,结合激光直写技术,在单层氧化石墨烯薄膜上实现了二维码、条形码、图形和数字等微纳图形的制备,还在多层氧化石墨烯薄膜结构上获得了多寿命多层微纳图形.这种微纳图形的制备具有灵活无掩膜、高对比和多模式的特点,可用于高密度光学存储、信息显示和光电器件制备等诸多领域.     

Fluorescence spectroscopy of semiconductor CdTe nanocrystals: preparation effect on photostability

We report on continuous-wave and time-resolved fluorescence spectroscopy studies of CdTe water-soluble nanocrystals at room temperature. For nanocrystals spread directly on the substrate we observe large variation both in fluorescence maximum energy and fluorescence lifetime. We attribute this to the influence of the surface of the nanocrystals on the stability of excitations in the nanocrystals. As the fluorescence lifetime of the nanocrystals is monitored, we find it increases with time from 6 to 18 ns and then saturates. Placing the nanocrystals in a polymer matrix remarkably improves the photostability and all the above-mentioned effects are diminished. Upon mixing the nanocrystals with gold spherical nanoparticles we observe a decrease of the fluorescence intensity due to efficient energy transfer to the nanoparticles.     

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     

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.     

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.     

荧光寿命的快速傅里叶变换拟合方法

介绍了一种利用快速傅里叶变换算法对稀土掺杂物质的荧光寿命进行数据拟合的方法。稀土掺杂物质可用来制备多种光学传感器,用于温度、pH值等多种参量测量领域。本方法利用快速傅里叶变换(FFT)结果作为基础,从非零项的相位角的正切值得出被测的荧光寿命,具有速度快、误差小、不受本底干扰等一系列优点。以掺铒光纤为例,通过数字仿真将本方法与其它几种传统的拟合方法进行了比较。快速傅里叶变换方法的测量偏差不到Prony方法的50％,为对数似合(log-fit)方法测量偏差的1／6。另外,快速傅里叶变换方法由于不受本底噪声影响,可以不必在信号处理时去掉本底噪声,因而可以明显缩短测量时间。     

Comparison of pulsed-excitation and phase-modulation methods for estimating fluorescence lifetime values using a convolved-autoregressive model and a high-gain photomultiplier tube

A comparative study between pulsed excitation (PE) and phase modulation (PM) methods has been carried out numerically and experimentally to estimate fluorescence lifetime values in the combined use of the following two measurement techniques: (i) To enhance the sensitivity in light detection, we have connected a load resistor with a large value at the output of a photomultiplier tube (PMT), which also brings about improvement in the signal-to-noise ratio (SNR) in measurements. (ii) The application of a convolved-autoregressive (C-AR) model to time-series data obtained from such a high-gain PMT enabled us to precisely estimate the fluorescence lifetime values in a nanosecond region. Although dependent on various measurement parameters, we have found that the PM method has potential in the field of biology or biochemistry for screening fluorescent samples.     

荧光寿命显微成像技术及应用的最新研究进展

由于荧光寿命不受探针浓度、激发光强度和光漂白效应等因素影响,荧光寿命显微成像技术(fluorescence lifetime imaging microscopy, FLIM)在监测微环境变化、反映分子间相互作用方面具有高特异性、高灵敏度、可定量测量等优点,近年来已被广泛应用于生物医学等领域.然而,尽管FLIM的发明和发展已历经数十年时间,其在实际应用中仍然面临着许多挑战.例如,其成像分辨率受衍射极限限制,而其成像速度与成像质量和寿命测量精度则存在相互制约的关系.近几年来,相关硬件和软件的快速发展及其与其他光学技术的结合,极大地推动了FLIM技术及其应用的新发展.本文简要介绍了基于时域和频域的不同寿命探测方法的FLIM技术的基本原理及特点,在此基础上概述了该技术的最新研究进展,包括其成像性能的提升和在生物医学应用中的研究现状,详细阐述了近几年来研究者们通过硬件和软件算法的改进以及与自适应光学、超分辨成像技术等新型光学技术的结合来提升FLIM的成像速度、寿命测量精度、成像质量和空间分辨率等方面所做的努力,以及FLIM在生物医学基础研究、疾病诊断与治疗、纳米材料的生物医学研究等方面的应用,最后对其未来发展趋势进行了展望.     

Enhancement of rapid lifetime determination for time-resolved fluorescence imaging in forensic examination

An enhancement method of rapid lifetime determination is proposed for time-resolved fluorescence imaging to discriminate substances with approximate fluorescence lifetime in forensic examination. In the method, an image-exclusive-OR treatment with filter threshold adaptively chosen is presented to extract the region of interest from dual-gated fluorescence intensity images, and then the fluorescence lifetime image is reconstructed based on the rapid lifetime determination algorithm. Furthermore, a maximum and minimum threshold filtering is developed to automatically realize visualization enhancement of the lifetime image. In proof experiments, compared with traditional fluorescence intensity imaging and rapid lifetime determination method, the proposed method automatically distinguishes altered and obliterated documents written by two brands of highlighters with the same color and close fluorescence lifetime.     

Analysis of fluorescence decay kinetics of thioflavin t by a maximum entropy method

The application of a maximum entropy method (MEM) for analysis of time-resolved fluorescence data is discussed. A developed version of MEM has been tested using simulated kinetic data. Based on computed results, practical criteria have been established to determine whether the lifetime distribution of emitting centers is described by a discrete spectrum (a set of two or three exponentials) or by a continuous one (mono- or bimodal distribution of exponentials). The proposed method has been used to analyze the fluorescence decay kinetics of thioflavin T (ThT) intercalated into amyloid fibrils. The presence of two peaks in the lifetime distribution of emitting centers has been explained by the existence in fibrils of two types of binding centers substantially differing in microenvironment rigidity. This suggestion is supported by the results of fluorescence quenching of intercalated ThT with the quencher KI.     

Iterative Joint Estimation Procedure of Channel and PDP for OFDM Systems

The power-delay profile (PDP) estimation of wireless channels is an important step to generate a channel correlation matrix for channel linear minimum mean square error (LMMSE) estimation. Estimated channel frequency response can be used to obtain time dispersion characteristics that can be exploited by adaptive orthogonal frequency division multiplexing (OFDM) systems. In this paper, a joint estimator for PDP and LMMSE channel estimation is proposed. For LMMSE channel estimation, we apply a candidate set of frequency-domain channel correlation functions (CCF) and select the one that best matches the current channel to construct the channel correlation matrix. The initial candidate set is generated based on the traditional CCF calculation method for different scenarios. Then, the result of channel estimation is used as an input for the PDP estimation whereas the estimated PDP is further used to update the candidate channel correlation matrix. The enhancement of LMMSE channel estimation and PDP estimation can be achieved by the iterative joint estimation procedure. Analysis and simulation results show that in different communication scenarios, the PDP estimation error of the proposed method can approach the Cramér–Rao lower bound (CRLB) after a finite number of iterations. Moreover, the mean square error of channel estimation is close to the performance of accurate PDP-assisted LMMSE.     

Effects of Refractive Index and Viscosity on Fluorescence and Anisotropy Decays of Enhanced Cyan and Yellow Fluorescent Proteins

The fluorescence lifetime strongly depends on the immediate environment of the fluorophore. Time-resolved fluorescence measurements of the enhanced forms of ECFP and EYFP in water–glycerol mixtures were performed to quantify the effects of the refractive index and viscosity on the fluorescence lifetimes of these proteins. The experimental data show for ECFP and EYFP two fluorescence lifetime components: one short lifetime of about 1 ns and a longer lifetime of about 3.7 ns of ECFP and for EYFP 3.4. The fluorescence of ECFP is very heterogeneous, which can be explained by the presence of two populations: a conformation (67% present) where the fluorophore is less quenched than in the other conformation (33% present). The fluorescence decay of EYFP is much more homogeneous and the amplitude of the short fluorescence lifetime is about 5%. The fluorescence anisotropy decays show that the rotational correlation time of both proteins scales with increasing viscosity of the solvent similarly as shown earlier for GFP. The rotational correlation times are identical for ECFP and EYFP, which can be expected since both proteins have the same shape and size. The only difference observed is the slightly lower initial anisotropy for ECFP as compared to the one of EYFP.     

Determination of the fluorescence lifetime of glyoxal(C_2H_2O_2)water solution by beconvolution

This paper reports that the actual fluorescence lifetime 7.64±0.06ns of a10％-glyoxal water solution is determined for the first time from the laser-induced fluores-cence decay curve detected by the system with a response time in the order of magnitude ofnanosecond and processed by deconvolution on combination with phase plane method and iter-ative convolution method.The experimental method presented in this paper is regarded as aneffective method for measuring the fluorescence lifetime in nanoseconds or even in sub-nanoseconds by means of a detecting system with an order of magnitude of nanosecond.