瞬时与延迟叶绿素荧光及820nm光反射动力学同步测量技术在光合作用研究中的应用

在光合作用研究中,通过分析瞬时叶绿素荧光诱导动力学曲线,可以获得围绕光系统Ⅱ(photosystemⅡ,PSⅡ)发生的原初光化学反应的信息。通过分析延迟叶绿素荧光诱导动力学和衰减动力学曲线,可以探寻发生电荷重组而产生延迟荧光的不同基团,从而更加直接地了解PSⅡ的状态。而820 nm光反射可以用来检测发生在光系统Ⅰ(photosystemⅠ,PSⅠ)的原初光化学反应。文章简要介绍了这三种动力学的发生原理及其在光合作用研究中的优缺点,并举例说明了瞬时荧光、延迟荧光及820 nm光反射动力学同步测量技术在光合作用研究中的应用,以及三者之间的互补与印证作用。     

利用延迟荧光研究DCMU对植物光合的影响

敌草隆（DCMU）阻断植物光合器官类囊体膜上的电子从QA到QB的传递。延迟荧光（DF）是光系统Ⅱ（PSⅡ）反应中心电荷分离效率的内在探针。本文以菠菜叶片作为实验材料,从DF衰减动力学及其总强度的变化研究了DCMU对植物光合作用的影响。DCMU作用后叶片DF衰减动力学曲线表明在快相部分有明显上升趋势,而慢相部分出现下降。不同DCMU浓度处理叶片后延迟荧光强度与叶片光合速率的变化表现出很好的相关性。研究结果表明,植物延迟荧光能很好的表征植物叶片DCMU处理后光合速率的变化。     

水葫芦及菠菜光合作用原初光反应的超快光谱研究

采用相同的分离技术,从水葫芦(Eichhornia crassipes(Mart)Solms.)和菠菜(Spinacia oleracea L.)叶片中提取叶绿体.利用吸收光谱和低温荧光光谱及皮秒荧光单光子计数技术对它们的光谱性质和光系统Ⅱ荧光寿命进行了研究.这两种叶绿体吸收光谱相似,暗示着它们都能高效吸收不同波长的光子.低温荧光光谱显示,水葫芦叶绿体两个光系统之间激发能分配平衡状态差,表明不利于该植物叶绿体高效利用吸收的光子能.采用三指数动力学模型对测定的光系统Ⅱ荧光衰减曲线拟合,水葫芦叶绿体光系统Ⅱ荧光衰减寿命分别是:138,521和1 494 ps;菠菜叶绿体荧光寿命分别是:197,465和1 459ps.并且归属了荧光组分,慢速度荧光衰减是由叶绿素堆积造成的,中等速度荧光衰减源于PSⅡ反应中心重新结合电荷组分,快速度荧光衰减归属于PSⅡ反应中心组分.基于20ps模型计算的水葫芦和菠菜叶绿体PSⅡ反应中心激发能转能效率分别是87%和91%.该结果与转能效率为100%的观点不一致.实验结果支持PSⅡ反应中心电荷分裂20 ps时间常数模型.根据转能效率,水葫芦生长速度不大于菠菜生长速度,但是,水葫芦叶绿体中含有丰富的胡萝卜素成分,其单位质量叶绿体吸收光能大于单位质量菠菜叶绿体吸收的量.实验结果还暗示植物叶绿体体系传能高效,接近于100%.     

外源油菜素内酯对三种杨树在干旱、盐和铜胁迫下光合生理的影响

以胡杨、新疆杨和俄罗斯杨一年生幼苗为材料,采用盆栽实验探讨不同胁迫(干旱,盐和重金属)下,施加外源植物激素油菜素内酯(BRs)对三种杨树叶片的叶绿素含量、叶绿素荧光参数及动力学曲线(OJIP)的影响。结果表明:与对照相比,10μmol/L的BRs可提高三种杨树叶片的光合特性;干旱胁迫下,胡杨的叶绿素含量及叶绿素荧光动力学曲线与对照相比无显著变化,新疆杨和俄罗斯杨的叶绿素a、叶绿素b含量显著降低,荧光动力学曲线J点上升,P点下降;盐胁迫下,胡杨和新疆杨的叶绿素含量变化较小,俄罗斯杨的叶绿素含量降低明显,荧光动力学曲线变化也比较显著;铜胁迫下,三种杨树叶绿素a和b含量与对照相比都显著下降,新疆杨和俄罗斯杨的荧光动力学曲线变化较为显著,荧光参数变化幅度非常大,当施加外源BRs后,三种胁迫下的三种杨树叶绿素含量与对照组相比都有不同程度的升高,其中胡杨、新疆杨和俄罗斯杨的叶绿素a含量分别升高了14%、6.8%和18.8%,新疆杨和俄罗斯杨的荧光动力学曲线及荧光参数接近于对照水平,PSⅡ最大光化学效率(Fv/Fm)和光化学性能指数(PIABS)显著增加。表明BRs能够明显缓解逆境胁迫对杨树PSⅡ反应中心造成的伤害。     

超短脉冲技术测量癌细胞荧光光谱与荧光寿命

研究用于癌症诊断与治疗的光敏剂血卟啉（hematoporphyrin derivative,HPD）的超快光动力学过程,采用超短脉冲激光光谱技术和皮秒时间相关单光子计数系统,测量经血卟啉培养的活体癌细胞与正常细胞的荧光光谱、荧光寿命特性及荧光峰值强度随时间变化曲线,并测量单一细胞内部不同位置的荧光寿命特性,观测到：癌细胞样品在645 nm处具有特有的光谱谱峰;癌细胞样品荧光寿命的快成分约150 ps慢成分约1200 ps,而正常细胞样品快成分约300 ps慢成分约2500 ps;癌细胞样品的荧光峰值强度经12小时衰减约10%,而正常细胞样品衰减约55%;在细胞内部荧光寿命300 ps的快成分十分显著,且中心部位血卟啉浓度最高.癌细胞与正常细胞的荧光光谱、荧光寿命特性及荧光峰值强度随时间变化曲线相差十分明显,反映了癌细胞与正常细胞对血卟啉亲和特性有显著的差异,测量结果确认了荧光光谱技术诊断与治疗癌症的可行性,并对发展超短脉冲激光光谱技术早期诊断与治疗癌症具有重要的指导意义和临床应用价值.     

用470fs时间分辨率荧光光谱法研究光系统：II反应中心原初反应的动 …

以菠菜叶绿体中的ＰＳⅡ颗粒和ＰＳⅡ核心复合物为材料,用４７０ｆｓ时间分辨率的荧光光谱技术研究ＰＳⅡ反应中心原初反应的动力学特性,选择不同的时间测量范围和不同的检测波长,经过解卷积和多指数拟合可以分辨率２￣４个衰减组分,对所得的动力学参数进行分析和讨论,认为其中３ｐｓ的组分与电荷分离有关,而０．８、１２、２５和１００ｐｓ的衰减组分很可能属于能源传递过程,提出了可能的动力学模型。     

叶绿素延迟荧光的发生及其在光合作用研究中的应用

叶绿素延迟荧光主要由绿色植物中光系统Ⅱ的天线色素产生,光系统Ⅱ反应中心色素P680接受天线色素吸收的光能后转变为激发态的P680,P680回到基态时释放出一个电子传给原初电子受体,随后电子沿光合电子传递链向PSI传递。当进入电子传递链的电子发生电荷重组时会使P680再次激发形成P680,P680将激发能传递给天线色素后,激发能以荧光的形式释放出来,即为延迟荧光。延迟荧光的检测和分析技术为无损测定植物光合机构的结构与功能变化提供了新的方法。利用该方法可以获得丰富的光合机构信息,如光系统Ⅱ受体侧及供体侧的伤害程度、跨类囊体膜质子梯度的大小等。本文介绍了延迟荧光的产生原理和测定方法,并且举例说明了延迟荧光测定技术在光合作用研究中的应用。     

去镁叶绿素置换细菌去镁叶绿素对紫细菌反应中心皮秒荧光的影响

选择597 nm作为激发波长,探测范围为600～900 nm的荧光特性,分析了天然反应中心和两种去镁叶绿素置换的紫细菌反应中心的荧光发射光谱.借助细菌叶绿素、细菌去镁叶绿素和植物去镁叶绿素的荧光光谱,对相关组分进行了归类.实验结果表明选择性地置换细菌去镁叶绿素影响了荧光光谱的组成.在天然反应中心、BpheB置换的反应中心和BpheA,B置换的反应中心中可分别解析到4、3和2个荧光发射组分.研究肯定荧光发射组分与去镁叶绿素的结合存在对应关系.实验还分别在686.4、674.1和681.1 nm处测定了不同反应中心内的原初电子供体P的激发态通过荧光衰减的过程,观测到衰减动力学上的差异.说明去镁叶绿素置换影响了细菌反应中心内激发光能传递和原初光化学反应过程.     

用470fs时间分辨率荧光光谱法研究光系统Ⅱ反应中心原初反应的动力学特性

以菠菜（ＳｐｉｎａｃｉａｏｌｅｒａｃｅａＭｉｌ．）叶绿体中的ＰＳⅡ颗粒和ＰＳⅡ核心复合物为材料,用４７０ｆｓ时间分辨率的荧光光谱技术研究ＰＳⅡ反应中心原初反应的动力学特性,选择不同的时间测量范围和不同的检测波长,经过解卷积和多指数拟合可以分辨出２～４个衰减组分,对所得的动力学参数进行分析和讨论,认为其中３ｐｓ的组分与电荷分离有关,而０．８、１２、２５和１００ｐｓ的衰减组分很可能属于能量传递过程,提出了可能的动力学模型。     

红藻条斑紫菜R—藻红蛋白晶体的时间分辩偏振荧光研究

本文在皮秒时域内研究红藻条斑紫菜Ｒ－藻红蛋白单晶在不同晶轴取向下的偏振荧光动力学过程。荧光衰减减表现为二指数过程,即与色素之间的能量传递和激平衡有关的快过程和色素荧光跃迁过程;由于色素间的能量传递,荧光明显退偏振;由于晶体中色素的光学跃迁距取向倾向于沿主晶轴方向分布,在不同的晶轴的取向下各向异性荧光衰减过程明显不同。     

Structure-based kinetic modeling of excited-state transfer and trapping in histidine-tagged photosystem II core complexes from synechocystis

Chlorophyll fluorescence decay kinetics in photosynthesis are dependent on processes of excitation energy transfer, charge separation, and electron transfer in photosystem II (PSII). The interpretation of fluorescence decay kinetics and their accurate simulation by an appropriate kinetic model is highly dependent upon assumptions made concerning the homogeneity and activity of PSII preparations. While relatively simple kinetic models assuming sample heterogeneity have been used to model fluorescence decay in oxygen-evolving PSII core complexes, more complex models have been applied to the electron transport impaired but more highly purified D1-D2-cyt b(559) preparations. To gain more insight into the excited-state dynamics of PSII and to characterize the origins of multicomponent fluorescence decay, we modeled the emission kinetics of purified highly active His-tagged PSII core complexes with structure-based kinetic models. The fluorescence decay kinetics of PSII complexes contained a minimum of three exponential decay components at F(0) and four components at F(m). These kinetics were not described well with the single radical pair energy level model, and the introduction of either static disorder or a dynamic relaxation of the radical pair energy level was required to simulate the fluorescence decay adequately. An unreasonably low yield of charge stabilization and wide distribution of energy levels was required for the static disorder model, and we found the assumption of dynamic relaxation of the primary radical pair to be more suitable. Comparison modeling of the fluorescence decay kinetics from PSII core complexes and D1-D2-cyt b(559) reaction centers indicated that the rates of charge separation and relaxation of the radical pair are likely altered in isolated reaction centers.     

Toxicant Induced Changes on Delayed Fluorescence Decay Kinetics of Cyanobacteria and Green Algae: A Rapid and Sensitive Biotest

Algal tests have developed into routine tools for testing toxicity of pollutants in aquatic environments. Meanwhile, in addition to algal growth rates, an increasing number of fluorescence based methods are used for rapid and sensitive toxicity measures. The present study stresses the suitability of delayed fluorescence (DF) as a promising parameter for biotests. DF is based on the recombination fluorescence at the reaction centre of photosystem II, which is emitted only by photosynthetically active cells. We analyzed the effects of three chemicals (3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), 3,5 Dichlorophenol (3,5 DCP) and copper) on the shape of the DF decay kinetics for potential use in phytoplankton toxicity tests. The short incubation tests were done with four phytoplankton species, with special emphasis on the cyanobacterium Microcystis aeruginosa. All species exhibited a high sensitivity to DCMU, but cyanobacteria were more affected by copper and less by 3,5 DCP than the tested green algae. Analyses of changes in the DF decay curve in response to the added chemicals indicated the feasibility of the DF decay approach as a rapid and sensitive testing tool.     

Delayed fluorescence in photosynthesis

Photosynthesis is a very efficient photochemical process. Nevertheless, plants emit some of the absorbed energy as light quanta. This luminescence is emitted, predominantly, by excited chlorophyll a molecules in the light-harvesting antenna, associated with Photosystem II (PS II) reaction centers. The emission that occurs before the utilization of the excitation energy in the primary photochemical reaction is called prompt fluorescence. Light emission can also be observed from repopulated excited chlorophylls as a result of recombination of the charge pairs. In this case, some time-dependent redox reactions occur before the excitation of the chlorophyll. This delays the light emission and provides the name for this phenomenon—delayed fluorescence (DF), or delayed light emission (DLE). The DF intensity is a decreasing polyphasic function of the time after illumination, which reflects the kinetics of electron transport reactions both on the (electron) donor and the (electron) acceptor sides of PS II. Two main experimental approaches are used for DF measurements: (a) recording of the DF decay in the dark after a single turnover flash or after continuous light excitation and (b) recording of the DF intensity during light adaptation of the photosynthesizing samples (induction curves), following a period of darkness. In this paper we review historical data on DF research and recent advances in the understanding of the relation between the delayed fluorescence and specific reactions in PS II. An experimental method for simultaneous recording of the induction transients of prompt and delayed chlorophyll fluorescence and decay curves of DF in the millisecond time domain is discussed.     

Time-resolved measurements of fluorescence from reaction centres of Rhodopseudomonas viridis and the effect of menaquinone reduction

The kinetics of the fluorescence emitted by the ‘special pair’ of bacteriochlorophyll b molecules in reaction centres from Rhodopseudomonas viridis was recorded in the near infrared, with a time resolution of 1 ns. In nonreduced reaction centres two decay components were resolved with lifetimes of <0.5 and 2.5 ns. Upon reduction of the menaquinone electron acceptor three decay components were detected with lifetimes of < 0.5, 2.5 and 15ns.     

Delayed fluorescence emitted from light harvesting complex II and photosystem II of higher plants in the 100 ns-5 micros time domain

This study presents the first report on delayed fluorescence (DF) emitted from spinach thylakoids, D1/D2/Cytb-559 preparations and solubilized light harvesting complex II (LHCII) in the ns time domain after excitation with saturating laser flashes. The use of a new commercially available multichannel plate with rapid gating permitted a sufficient suppression of detector distortions due to the strong prompt fluorescence. The following results were obtained: (a) in dark-adapted thylakoids, the DF amplitudes at 100 ns and 5 micros after each flash of a train of saturating actinic pulses exhibit characteristic period four oscillations of opposite sign: the DF amplitudes at 100 ns oscillate in the same manner as the quantum yield of prompt fluorescence, whereas those at 5 micros resemble the oscillation of the micros kinetics of P680(.) reduction in samples with an intact water oxidizing complex, (b) the quantum yield of total DF emission in the range up to a few micros is estimated to be <10(-4) for thylakoids, (c) the DF of D1/D2/Cytb-559 exhibits a monophasic decay with tau approximately 50 ns, (d) DF emission is also observed in isolated LHCII with biphasic decay kinetics characterized by tau values of 65 ns and about 800 ns, (e) in contrast to thylakoids, the amplitudes of DF in D1/D2/Cytb-559 preparations and solubilized LHCII do not exhibit any oscillation pattern and (f) all spectra of DF from the different sample types are characteristic for emission from the lowest excited singlet state of chlorophyll a. The implications of these findings and problems to be addressed in future research are briefly discussed.     

Granulocyte kinetics with radioactive diisopropylfluorophosphate (DF32P) and radiochrome (51Cr)]

Determining granulocyte kinetics with DF32P allows various parameters to be gained during the in-vitro marking, such as the total blood granulocyte pool, circulating granulocyte pool, marginal granulocyte pool, daily granulocyte exchange rate and half decay period of granulocytes. The half decay period of granulocytes, bone-marrow reserve in myelocytes, metamyelocytes and band cells as well as polymorphonuclear neutrophils can be determined by in-vitro marking, with DF32P being intravenously injected. The combination of both procedures with DF32P will reveal the half decay period, pool sizes and exchange rates of the proliferating myelocyte compartiment in bone-marrow and mature blood granulocytes. If 51Cr is used for determining granulocyte kinetics the surface activities of various organs, such as heart, liver, spleen, and lungs, can mainly be determined in addition to the half-life of leucocytes, indicating the degradation or storage of cells in certain areas of the body. In addition to normal values those findings are principally presented which were obtained with in-vitro marking by DF32P and 51Cr in chronic myeloid leukaemia, osteomyelofibrosis or osteomyelosclerosis respectively and in hypersplenism.     

Photophysics of ANS. IV. Electron transfer quenching of ANS in alcoholic solvents and mixtures

ANS is observed to decay from the fluorescent state with distributed kinetics in nearly pure ethanol solvent, notwithstanding that in mixed ethanol/water solvents the decay is discrete and biexponential. The origin of this behavior is investigated. In particular, a theory of electron transfer theory in the adiabatic regime is adduced, with specific involvement of solvent cage structure in the form of the solvent-electron polaron wave function. Properties of various polarons for various solvent systems are predicted and, for the case of ethanol and cyclohexanol, employed to generate the observed Arrhenius-type decay parameters in a quantitative fashion.     

Quantitative measurements of NO reaction kinetics with a Clark-type electrode.

Nitric oxide (NO) plays important physiological roles in the body. Knowledge regarding the kinetics of NO catabolism is important for understanding the biological functions of NO. Clark-type NO electrodes have been frequently employed in measuring the kinetics of NO reactions; however, the slow response time of these electrodes can cause measurement errors and limit the application of the electrode in measurements of fast NO reactions. In this study, a simplified diffusion model is given for describing the response process of the NO electrode to the change of NO concentration. The least-square method is used in fitting the currents calculated from the diffusion equation to the experimental curves for determining the diffusion parameters and rate constants. The calculated currents are in excellent accordance with the experimental curves for different NO reaction kinetics. It has been demonstrated that when using an NO electrode with a response time of approximately 6 s to measure fast NO reactions with a half-life of approximately 1s, the response currents of the electrode have large differences compared to the curve of actual NO concentration in the solution; however, the rate constant of NO decay can still be accurately determined by computer simulations with the simplified diffusion model. Theoretical analysis shows that an NO electrode with a response time of 6 s (D/L2=0.06 s-1) and the lowest detection limit of 1 nM NO can be used in measuring kinetics of extremely rapid NO reactions with a half-life below 10 ms.