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.     

Experimental study of multi-photon contamination on the measurement of fluorescent decay time

In the measurement of fluorescent lifetime based on time correlation-single photon counting technique by means of TAC, due to the contamination of multi-photons a deviation of fluorescent lifetime measured from the expected value is experimentally studied. A correction function instead of a simple exponential function is used to fit the experiment data. The validation of the correction function is checked using the experimental data of several test samples: YAP, NaI(T1) and LSO. The results show that the correction function well fits the data and the reasonable fluorescent lifetimes are obtained.     

Near-field time-resolved fluorescence studies of poly(phenylmethyl silane) with subwavelength resolution

We present an example of the first time-correlated single-photon counting (TCSPC) near-field optical measurement. The aperture size of our prepared aluminun-coated fiber-optic probe was approximately 50 nm, which represents a spatial resolution of _ex/7 for our UV measurements. Near-field fluorescence decays of poly(phenylmethyl silane) in solid thin films excited in the range 325–360 nm were obtained and the steady-state excitation spectra compared with the excitation spectral information obtained in the far field. Fluorescence decays showed single exponential lifetimes ranging from 45 to 277 ps, which was dependent on the excitation wavelength and the selected near-field tip. The proximity of the metal-coated tip to the sample may be the reason for the modulation in fluorescence lifetime.     

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。     

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.     

Optimum pulse repetition rates for single photon counting experiments

In the first part of this paper, the optimum pulse repetition rate (PRR) for fluorescence lifetime measurements based on time correlated single photon counting is obtained by minimizing the variance in the lifetime estimates, assuming a single exponential decay. This allow us, in the second part, to calculate the optimum pulse repetition rate for systems possessing a set of discrete repetition rates; such as synchronously pumped mode locked cavity dumped laser systems.     

Mean fluorescence lifetime and its error

Mean excited-state lifetime is one of the fundamental fluorescence characteristics and enters as an important parameter into numerous calculations characterizing molecular interactions, such as e.g. FRET or fluorescence quenching. Our experiments demonstrated that the intensity-weighted mean fluorescence lifetime is very robust characteristic, in contrast to the amplitude-weighted one, which value is dependent on the data quality and particularly on the used fitting model. For the first time, we also report the procedure for the error estimation for both the intensity- and amplitude-weighted mean fluorescence lifetimes. Furthermore, we present a method for estimation of the mean fluorescence lifetime directly from the fluorescence-decay curve recorded by TCSPC (Time-Correlated Single-Photon Counting) method. For its simplicity and low computational demands, it could be a useful tool in the high-throughput applications, such as FACS, FLIM-FRET or HPLC detectors.     

Fluorescence lifetime study of ECFP/EYFP labeled MBP in Förster resonance energy transfer

This paper studies the conformational change of the binding protein by a fluorescence lifetime method. As a model protein, maltose binding protein (MBP) where enhanced cyan protein (ECFP) and enhanced yellow fluorescent protein (EYFP) were genetically fused to act as a donor and an acceptor in Förster resonance energy transfer (FRET) was used. The ECFP and the EYFP were linked to the C-terminal and N-terminal regions of the MBP, respectively. In order to investigate the conformational change of the MBP, the lifetime of the ECFP, which acts as a donor in the ECFP:MBP:EYFP fusion protein, was analyzed during the FRET process. We observed that two lifetime components exist when the ECFP is linked to the MBP and that the lifetime of the ECFP is shortened when ECFP:MBP:EYFP protein undergoes a conformational change as a result of the maltose binding. In addition, we observed that the lifetime of the donor is gradually shorter in the ECFP:MBP:EYFP fusion protein as the maltose concentration increases. By a lifetime analysis and simulation study, we found that the participant rate of the ECFP:MBP:EYFP protein in FRET is the main cause of the donor lifetime shortening in relation to the increase of the maltose concentration.     

基于猝灭荧光素的一种TCSPC仪器响应函数测量方法

根据荧光物质的动态猝灭作用原理,将碘化钠饱和水溶液与碱性荧光素水溶液按照特定的比例混合,利用时间分辨设备测量猝灭后的荧光素荧光寿命。实验观察到随着碘化钠饱和溶液浓度的增加,测量得到的荧光寿命逐步从4 ns减小至24 ps左右。如将猝灭荧光素作为仪器响应函数的标准样品,与通常作为标准样品的二氧化硅纳米颗粒悬浮液进行对比,实验结果显示两者非常吻合,表明猝灭荧光素可以作为荧光衰减测试中的标准样品,进一步研究发现这种新的标准样品一方面避免了传统测量手段中需要在不同探测波长反复测量仪器时间响应函数的问题,更有效地减小了实验中颜色效应造成的实验误差。有望在时间分辨荧光光谱和荧光寿命成像等研究中得到应用。     

Experimental study of multi-photon contamination on the measurement of fluorescent decay time

In the measurement of fluorescent lifetime based on time correlation-single photon counting technique by means of TAC, due to the contamination of multi-photons a deviation of fluorescent lifetime measured from the expected value is experimentally studied. A correction function instead of a simple exponential function is used to fit the experiment data. The validation of the correction function is checked using the experimental data of several test samples: YAP, NaI(Tl) and LSO. The results show that the correction function well fits the data and the reasonable fluorescent lifetimes are obtained.     

皮秒时间相关单光子计数光谱仪的核心技术

研制的皮秒时间相关单光子计数光谱仪利用时间相关单光子计数(TCSPC)技术,采用了具有分光时间弥散动态和静态补偿特性的光栅分光系统,解决了传统分光系统的光信号时间弥散问题;使用多道谱分析仪开设时间窗口,测量荧光衰减曲线和时间分辨光谱;用荧光衰减曲线的多指数拟合方法处理数据。给出了光谱仪的原理和总体方案,介绍了系统的集成和工作流程。通过各种标准样品的试验数据分析和对比,得出系统所测荧光寿命可达到ps量级,而且具有最高的灵敏度——单光子计数,时间分辨率达到8．8ps。     

时间相关单光子计数光谱仪的优化

分析了原有时间相关单光子计数光谱仪存在的不足,提出了改进方案。研制了高速数据采集系统,采用PCI总线技术、FPGA技术,开发了高速光谱数据采集卡,取代了原有的多道分析仪,数据采集速度达到20 MB/s,比原有仪器提高了约200倍;改善了仪器的性能、减小了体积。介绍了光谱仪系统的集成和工作流程,并对仪器的性能进行分析,通过多种标准样品的试验数据分析和对比,光谱仪系统具有最高的灵敏度-单光子计数,测得荧光寿命可达到ps量级,而且可以测得时间分辨光谱。     

A Feasible Add-On Upgrade on a Commercial Two-Photon FLIM Microscope for Optimal FLIM-FRET Imaging of CFP-YFP Pairs

Fluorescence lifetime imaging microscopy (FLIM) based on time-correlated single photon counting (TCSPC) is a widely used method for fluorescence resonance energy transfer (FRET). Here we report a feasible add-on approach to upgrade a commercial two-photon FLIM microscope into a single-photon FLIM microscope which provides optimal FLIM-FRET imaging of FRET pairs consisting of cyan fluorescent proteins (CFPs) as the donor and yellow fluorescent proteins (YFPs) as the acceptor. The capability of the upgraded system is evaluated and discussed, and the imaging performance of the system is demonstrated using FLIM-FRET experiments with a representative CFP-YFP FRET pair (mCerulean-mCitrine).     

Nonclassical photon statistics in single-molecule fluorescence at room temperature

The fluorescence of single terrylene molecules in a crystalline host is investigated at room temperature by scanning confocal optical microscopy. Photon arrival times are analyzed in terms of interphoton time distributions, second order correlation functions, and the variance of the photon number probability distribution. Antibunching at short times and bunching behavior for longer times is observed, associated with sub- and super-Poissonian statistics, respectively. A rate-equation analysis of the molecular level populations indicates an accelerated reverse intersystem crossing.     

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     

Frequency Domain Fluorescence Lifetime Study of Crude Petroleum Oils

Frequency domain (FD) fluorescence lifetime data was collected for a series of 20 crude petroleum oils using a 405 nm excitation source and over a spectral range of ~426 to ~650 nm. Average fluorescence lifetimes were calculated using three different models: discrete multi-exponential, Gaussian distribution, and Lorentzian distribution. Fitting the data to extract accurate average lifetimes using the various models proved easier and less time consuming for the FD data than with Time Correlated Single Photon Counting (TCSPC) methods however the analysis of confidence intervals to the computed average lifetimes proved cumbersome for both methods. The uncertainty in the average lifetime was generally larger for the discrete lifetime multi-exponential model when compared to the distribution-based models. For the lifetime distributions, the data from the light crude oils with long lifetimes generally fit to a single decay term. Heavier oils with shorter lifetimes required multiple decay terms. The actual value for the average lifetime is more dependant on the specific fitting model employed than the data acquisition method used. Correlations between average fluorescence lifetimes and physical and chemical parameters of the crude oils were made with a view to developing a quantitative model for predicting the gross chemical composition of crude oils. It was found that there was no significant benefit gained by using FD over TCSPC other than more rapid data analysis in the FD case. For the FD data the Gaussian distribution model for fluorescence lifetime gave the best correlations with chemical composition allowing a qualitative correlation to some bulk oil parameters.

延时分幅扫描单光子计数荧光光谱技术

设计建立了一套延时分幅扫描单光子计数荧光光谱装置,以雪崩光电二极管SPCM-AQR-15为荧光探测器,T914P单光子计数模件为信号接收仪器,通过对T914P的触发信号加延时,在每个延时下对荧光信号进行分幅扫描,再将所有延时的分幅扫描信号叠加在一起,可以将快速的微弱信号很好地再现出来,时间分辨率提高了16.7倍,采用此技术对光合作用CP43色素蛋白复合物进行了研究,荧光光谱解叠可分辨出三个窄谱带:681nm、684nm、729nm,并分别在670nm,680nm和685nm荧光发射处,采用全局优化拟合法处理,获取了3个寿命组分:422_ps,582_ps,3.75_ns,其中422_ps反应了Chla671到Chla679和Chla682能量传递过程;582_ps代表Chla679到Chla682的能量传递;3.75_ns代表Chla682包括荧光在内的所有去激发速率之和.     

基于SiPM和TCMPC的时间分辨拉曼散射测量技术研究

报道了一种基于硅光电信增管(SiPM)的时间相关多光子计数(TCMPC)技术并将其应用于时间分辨拉曼散射测量。相比于常规基于光电倍增管(PMT)或单光子雪崩二极管(SPAD)的时间相关单光子(TCSPC)技术,由于SiPM可以分辨信号脉冲的具体光子数,基于SiPM的TCMPC技术消除了信号脉冲包含的光子数必须小于等于1的限制,光子计数效率提高了10倍以上,大大节省了测量时间。此外,多光子测量比单光子测量能够得到更好的时间分辨率,时间分辨拉曼散射系统的仪器响应函数(IRF)从单光子81.4 ps缩短至双光子59.7 ps,因而可以用更窄的时间门限抑制荧光本底等噪声对拉曼散射测量的影响。使用TCMPC技术测量CCl₄在0.5和1.5 p.e.两个不同光子数阈值的拉曼峰的峰本比,后者较高的光子数阈值能进一步降低SiPM暗计数噪声的影响,增加了拉曼信号测量的信噪比,测量得到的CCl₄ 459 cm-1拉曼峰的峰本比是前者的6.4倍。将所述新的拉曼散射测量技术与基于PMT和锁相放大器(LIA)的传统拉曼散射测量技术进行了比较研究,前者由于可以使用仅有数十皮秒的测量门限,可以有效抑制荧光、环境杂散光和SiPM暗计数等噪声的影响,所得光谱具有更好的峰本比,测得CCl₄的459 cm-1拉曼峰和Si的一阶拉曼峰的峰本比分别是后者的3.9倍和5.5倍。     

Stochastic quantum trajectories in a QND measurement of photon number

An analysis is presented of the time evolution of an optical field during a quantum nondemolition measurement of photon number using the cross-Kerr interaction between the signal and probe fields. It is shown that the signal field state collapses into a Fock state only asymptotically (in the infinite time limit), remaining in a superposition of two Fock states (Fock-state qubit) throughout most of the measurement period. Estimates are obtained both for the time required to measure photon number to the desired accuracy and for the Fock-state qubit lifetime.