Water Deficit and Salt Stress Diagnosis Through LED Induced Chlorophyll Fluorescence Analysis in Jatropha curcas L.

LED induced chlorophyll fluorescence analysis is employed to investigate the effect of water deficit and salt stress upon the growth process of Jatropha curcas L.. Red(Fr) and far-red(FFr) chlorophyll fluorescence around 685 nm and 735 nm, respectively, were observed and examined as a function of the stress intensity(salt concentration and water deficit). The fluorescence ratio Fr/FFr which is a valuable nondestructive and nonintrusive indicator of the chlorophyll content of leaves was exploited to monitor the jatropha plants under stress. The data indicated that salinity plays a minor role in the chlorophyll concentration of leaves for NaCl concentrations in the 25 to 200 mM range. The fluorescence ratio also permitted the detection of damage caused by water deficit in the early stages of the plants growing process. A significant variation of the Fr/FFr ratio was observed in the first 10 days of the experiment, and before signs of visual stress became apparent. The results suggest that the Fr/FFr ratio is an early-warning indicator of water deficit stress     

叶绿体延迟荧光中730 nm成分产生机理的光谱学研究

叶绿体685 nm延迟荧光成分被认为源于PSⅡ作用中心的电荷复合。利用多种光谱学测量手段研究了叶绿体延迟荧光光谱中730 nm峰的产生机制。不同浓度下叶绿体延迟荧光光谱实验结果表明：初始随浓度的增加,延迟荧光光谱中685和730 nm成分强度均增强;当浓度增加到7.8 μg·mL-1时,685 nm成分强度达最大,730 nm成分强度继续上升;当浓度增加到31.2 μg·mL-1时,延迟荧光光谱中730 nm成分强度达最大,而685 nm成分已明显下降。吸收光谱实验结果表明：A₆₈₅/A₇₃₀在叶绿体浓度增加的过程中几乎不变。叶绿体730 nm荧光成分的激发光谱实验结果表明：685 nm光对730 nm荧光有较高的激发效率。上述实验结果表明叶绿体延迟荧光光谱中730 nm峰是由PSⅡ所发685 nm成分激发PSⅠ所产生的荧光。同一浓度下叶绿体延迟荧光光谱波形随延迟时间(1～9 s)的不变性进一步证明了这一结论。     

叶绿体不同浓度下光诱导延迟荧光光谱研究

针对《光子学报》文献［1］中实验结果与已有报道相关光谱特征的显著差异,利用多种光谱学手段对不同浓度下叶绿体延迟荧光行进了研究.实验结果表明：PSⅠ反应中心叶绿素P700对PSⅡ所发685 nm成份的吸收随叶绿体浓度的增加而增强,从而导致PSⅠ发出的730 nm成份增强,而使得延迟荧光光谱中730 nm成为主峰,甚至685 nm成份的消失.该研究结果表明：叶绿体延迟荧光光谱中730 nm成份,是由PSⅠ作用中心叶绿素P700对PSⅡ中所发685 nm成份延迟荧光的重吸收,产生激发态所发出的荧光.该结论有助于延迟荧光光谱中各成份产生机理的进一步研究.     

Characterization of Standard Reference Material 2940, Mn-ion-doped glass, spectral correction standard for fluorescence

Standard Reference Material (SRM) 2940 is a cuvette-shaped, Mn-ion-doped glass, recommended for use for relative spectral correction of emission and day-to-day performance validation of steady-state fluorescence spectrometers. Properties of this standard that influence its effective use or contribute to the uncertainty in its certified emission spectrum were explored here. These properties include its photostability, absorbance, dissolution rate in water, anisotropy, temperature coefficient of fluorescence intensity, and fluorescence lifetimes. Long and short lifetime components of the fluorescence displayed different emission spectra, making the certified spectrum useful with fluorescence instruments employing continuous excitation only. The expanded uncertainties in the certified spectrum are about 5% around the peak maximum at 620 nm, using an excitation wavelength of 412 nm. The SRM also exhibits a strong resistance to photodegradation, with no measurable decrease in fluorescence intensity even after 17 h of irradiation with the visible light from a Xe lamp.     

Characterization of Standard Reference Material 2942, Ce-ion-doped glass, spectral correction standard for UV fluorescence

Standard Reference Material (SRM) 2942 is a cuvette-shaped, Ce-ion-doped glass, recommended for use for relative spectral correction of emission from 320 to 430 nm and day-to-day performance verification of steady-state fluorescence spectrometers. Properties of this standard that influence its effective use or contribute to the uncertainty in its certified emission spectrum were explored here. These properties include its photostability, absorbance, dissolution rate in water, anisotropy and temperature coefficient of fluorescence intensity. The expanded uncertainties in the certified spectrum are about 9% around the peak maximum at 330 nm, using an excitation wavelength of 310 nm. The SRM also exhibits a strong resistance to photodegradation, with no measurable decrease in fluorescence intensity even after 25 h of irradiation with UV light>280 nm from a Xe lamp.     

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.     

水稻叶绿素浓度对荧光光谱的影响

为了加强对水稻生长状况的监测,指导水稻的田间施肥,提高施肥利用效率,以增加农作物产量、提高粮食品质,实验室搭建了基于激光诱导荧光技术的荧光探测系统,以研究水稻叶片的叶绿素含量与荧光峰值比之间的相关性。文中测量样本为水稻分蘖期和拔节期的倒二叶,栽培地区位于中国江汉平原。文中先采用凯氏定氮法和相应公式结合测得水稻叶片的叶绿素含量(mg·g-1),再用搭建的荧光探测系统采集了水稻叶片不同叶绿素含量的荧光光谱(激发波长为355 nm)。获得了水稻叶片在不同叶绿素含量下的荧光光谱数据库,定量分析了荧光峰值比F₇₄₀/F₆₈₅(荧光谱峰740 nm、685 nm处的荧光强度比)与叶绿素含量的相关性,发现叶绿素含量的变化对荧光光谱特性影响明显。由实验数据分析可知荧光参数中的峰值比(F₇₄₀/F₆₈₅)与叶绿素含量呈现很好的线性正相关,相关系数(R2)在水稻的分蘖期和拔节期分别达到了0.901 3和0.912 5。实验分析结果表明诱导荧光光谱技术具有方便、快捷、无损等优点,在农作物生长状况的遥感定量监测等方面也具有一定发展潜力。     

Molecular probe FET: Long-wavelength fluorescence excitation effect and proton transfer reaction

We studied the steady-state fluorescence spectra of solutions of FET (4′-(diethylamino)-3-hydroxyflavone) in acetonitrile that were excited at different temperatures by quanta with different energies located in the range of the main absorption band and in its long-wavelength wing. We found that, at room temperature, the emission intensity ratio of the bands of the normal and tautomeric forms, which are located at 505 and 570 nm, respectively, depends on the excitation wavelength. In the range of the main absorption band 300–360 nm, this ratio remains nearly the same, i.e., 1.45, while, upon excitation in the range of the long-wavelength wing 360–380 nm of the main band, it decreases to 1.33 at a wavelength of 460 nm. In this same range, a long-wavelength excitation effect that is unusual for liquid inviscid solvents at room temperature, i.e., a bathochromic shift of the entire short-wavelength emission band by 11 nm, manifests itself. We propose to explain these dependences using energy diagrams, which take into account the dependence of free energy on the orientational polarization of the polar solvent. The observed effect of the long-wavelength shift of the fluorescence spectrum with increasing excitation wavelength is explained in terms of the inhomogeneous broadening of electronic spectra of polar solutions, and it should be described using the scheme of energy states that takes into account sublevels of orientational broadening due to orientational dipole-dipole interactions of the fluorophore with nearest molecules of the polar solvent, as well as the relation between the fluorophore lifetime in the excited state and the dielectric relaxation time of solvent molecules in the field of the fluorophore dipole.     

激光诱导荧光光谱识别动脉粥样硬化斑块的研究

采用氩离子激光为激发光源,分别研究了离体正常动脉壁、动脉粥样硬化斑块和血液等的激光诱导荧光光谱.结果表明,正常动脉壁样品组的荧光光谱强度最大值明显高于动脉粥样硬化斑块样品组;斑块的荧光光谱在516 nm处存在一个小波谷,而正常动脉组织的荧光光谱则无此波谷;斑块组织在598 nm处与578 nm处的荧光强度比值R(I₅₉₈ nm/I₅₇₈ nm)远低于正常动脉壁的比值;血红蛋白含量是引起粥样斑块与正常动脉壁的R(I₅₉₈ nm/I₅₇₈ nm)值不同的主要原因.     

苹果树冠层不同光照区域叶绿素荧光性状计算方法

植物冠层内光照分布决定了植物物理过程与生态环境之间的交互关系,是植物冠层叶绿素荧光动力学研究的重要基础。光谱技术在构建植株冠层组分含量的预测模型中发挥了重要的作用,而针对自由纺锤形苹果树冠层不同光照区域的叶绿素荧光性状研究文献报道较少。因此,以自由纺锤形苹果树为研究对象,将苹果树冠层空间划分为5层,每层划分为边长50 cm的立方体网格,并测定各个网格空间的光照强度,确定其光照分布情况。以此为基础,获取不同光照区域的光谱数据及对应叶绿素荧光性状参数值,通过光谱一阶微分去除系统误差干扰,确定红边区域(660～760 nm)的一阶微分光谱曲线,利用BP神经网络建立红光范围(680～760 nm)内一阶微分光谱最大值与叶绿素荧光性状参数关系模型,创新性地提出了苹果树冠层不同光照区域的叶绿素荧光性状计算方法。并采用决定系数、均方根误差、平均绝对百分比误差等对模型进行有效性评价,试验结果表明：该叶绿素荧光计算方法的计算精度均在80%以上,研究成果可为苹果树的整形修剪,获取充足光照分布提供理论依据。     

Fluorescence Anisotropy of Tyrosinate Anion Using One-, Two- and Three-Photon Excitation

We examined the emission spectra and steady-state anisotropy of tyrosinate anion fluorescence with one-photon (250–310 nm), two-photon (570–620 nm) and three-photon (750–930 nm) excitation. Similar emission spectra of the neutral (pH 7.2) and anionic (pH 13) forms of N-acetyl-L-tyrosinamide (NATyrA) (pK_a 10.6) were observed for all modes of excitation, with the maxima at 302 and 352 nm, respectively. Two-photon excitation (2PE) and three-photon excitation (3PE) spectra of the anionic form were the same as that for one-photon excitation (1PE). In contrast, 2PE spectrum from the neutral form showed ~30-nm shift to shorter wavelengths relative to 1PE spectrum (λ_max 275 nm) at two-photon energy (550 nm), the latter being overlapped with 3PE spectrum, both at two-photon energy (550 nm). Two-photon cross-sections for NATyrA anion at 565–580 nm were 10 % of that for N-acetyl-L-tryptophanamide (NATrpA), and increased to 90 % at 610 nm, while for the neutral form of NATyrA decreased from 2 % of that for NATrpA at 570 nm to near zero at 585 nm. Surprisingly, the fundamental anisotropy of NATyrA anion in vitrified solution at ?60 °C was ~0.05 for 2PE at 610 nm as compared to near 0.3 for 1PE at 305 nm, and wavelength-dependence appears to be a basic feature of its anisotropy. In contrast, the 3PE anisotropy at 900 nm was about 0.5, and 3PE and 1PE anisotropy values appear to be related by the cos⁶ θ to cos² θ photoselection factor (approx. 10/6) independently of excitation wavelength. Attention is drawn to the possible effect of tyrosinate anions in proteins on their multi-photon induced fluorescence emission and excitation spectra as well as excitation anisotropy spectra.     

Fluorescence of bean leaves grown under low-light conditions

The patterns of changes of the spectral shapes of the fluorescence of bean leaves have been identified under low-light conditions of plant cultivation and decrease in chlorophyll concentration in the leaves. Under these experimental conditions, the ratio of peak intensities of fluorescence in the red spectral region, ω = F ₇₄₀/F ₇₆₀, which was recorded after the end of the induction period, was proportional to the chlorophyll content per 1 g of the wet weight of a leaf.     

饱和激发下叶绿素荧光非线性变化及影响分析

叶绿素浓度是海洋初级生产力的重要指标之一,激光诱导荧光技术可以实现海水叶绿素浓度的快速测量。测量叶绿素浓度的传统激光诱导荧光原理,是利用叶绿素荧光与水体Raman散射的强度比值（I_F/R）进行反演,即叶绿素浓度n_chl＝CI_F/R,其中C为系统常量。这是依据叶绿素荧光685 nm、水体Raman散射强度都与激发光强呈线性关系。然而,该理论并没有考虑诱导荧光饱和现象的存在。当诱导激光强度达到一定程度后,685 nm荧光强度随激发光强非线性变化。另外,值得注意的是,水体Raman散射并不存在信号饱和现象。为了探讨饱和激发造成荧光非线性变化的影响,在激光诱导荧光技术测量叶绿素浓度的实验中,设计两种测量方案,即：不同激光功率诱导单一浓度样本的荧光测量,和固定激光功率时不同浓度样本的荧光测量。实验中利用Nd∶YAG三倍频激光355 nm激发获得叶绿素溶液的404 nm处 Raman散射和685 nm荧光。实验结果分为2部分进行讨论：（1）为了分析饱和激发造成荧光变化的非线性特性,通过调节激发光功率测量溶液的受激发射光谱,发现水体Raman散射强度与激发光强呈线性关系,而685 nm荧光强度出现饱和激发下的非线性变化。而且,随叶绿素浓度的增加,685 nm荧光的非线性趋势更为明显,Raman散射强度与激发光强的线性关系中斜率变小。数据分析表明,685 nm荧光数据拟合的4阶多项式和Raman散射效率值,可以定性地表征685 nm荧光的饱和程度。（2）考虑实际海洋激光雷达探测叶绿素浓度应用中存在饱和激发荧光非线性现象,为了分析荧光非线性对传统叶绿素浓度反演理论适用性的影响,在固定激发光强情况下对不同浓度叶绿素溶液的发射光谱进行测量。将激发光功率调节至52.00,80.70,132.10和197.30 mW·cm-2,获取相应激发光强下685 nm荧光与水体Raman散射的强度比值和叶绿素浓度之间的关系。实验表明,激发光强不变的情况下,685 nm荧光与水体Raman散射的强度比值,与叶绿素浓度仍满足线性关系。但是,在较高光强激发时,饱和激发造成的叶绿素荧光非线性变化,导致利用传统激光诱导荧光理论反演的叶绿素浓度值偏小。因此,需要对饱和激发下荧光非线性的影响进行修正,其关系为I_F/R=n_chl/C+C_F,修正值C_F不可忽略。另外,值得一提的是,修正关系中系统常量C随激发光强增加而增大。研究表明,饱和激发造成的荧光非线性,会对激光诱导荧光技术测量叶绿素浓度产生影响,但由于造成荧光非线性因素的复杂性,仅通过荧光数据拟合获得的多项式,无法定量说明其影响权重。然而,当激发光强不变时,可以实验测量获得基于激光诱导荧光原理的修正关系,从而准确反演叶绿素浓度。     

Stress-Induced Alteration of Chlorophyll Fluorescence Polarization and Spectrum in Leaves of <Emphasis Type="Italic">Alocasia macrorrhiza</Emphasis> L. Schott

The value of intrinsic chlorophyll fluorescence polarization, and the intensity in emission spectrum were investigated in leaf segments of Alocasia macrorrhiza under several stress conditions including different temperatures (25–50°C), various concentrations of NaCl (0–250 mM), methyl viologen (MV, 0–25 μM), SDS (0–1.0%) and NaHSO₃ (0–80 μM). Fluorescence emission spectrum of leaves at wavelength regions of 500–800 nm was monitored by excitation at 436 nm. The value of fluorescence polarization (P value), as result of energy transfer and mutual orientation between chlorophyll molecules, was determined by excitation at 436 nm and emission at 685 nm. The results showed that elevated temperature and concentrations of salt (NaCl), photooxidant (MV), surfactant (SDS) and simulated SO₂ (NaHSO₃) treatments all induced a reduction of fluorescence polarization to various degrees. However, alteration of the fluorescence spectrum and emission intensity of F₆₈₅ and F₇₃₁ depended on the individual treatment. Increase in temperature and concentration of NaHSO₃ enhanced fluorescence intensity mainly at F₆₈₅, while an increase in MV concentration led to a decrease at both F₆₈₅ and F₇₃₁. On the contrary, NaCl and SDS did not cause remarkable change in fluorescence spectrum. Among different treatments, the negative correlation between polarization and fluorescence intensity was found with NaHSO₃ treatments only. We concluded that P value being measured with intrinsic chlorophyll fluorescence as probe in leaves is a susceptible indicator responding to changes in environmental conditions. The alteration of P value and fluorescence intensity might not always be shown a functional relation pattern. The possible reasons of differed response to various treatments were discussed.     

Performance Check of Vegetation Fluorescence Imaging Lidar through In Vivo and Remote Estimation of Chlorophyll Concentration Inside Plant Leaves

Laser-induced fluorescence imaging lidar was developed for in vivo plant/vegetation monitoring. Fluorescences of poplar tree leaves growing naturally at a distance of 60 m from the lidar were successfully detected as two spectral images at wavelengths of 685 nm and 740 nm. By comparing chlorophyll concentration quantified with high performance liquid chromatography, it was confirmed that the intensity ratio of the two wavelengths was converted into the chlorophyll concentration inside the leaves. The intensity ratio of the images reflected the status of the poplar tree in the process of senescence so well that it was possible to assess the living status of the tree as a numerical value. The performance characteristics and the potential of the laser-induced fluorescence imaging lidar for monitoring the physiological activities of plants and vegetation are described.     

激光诱导水体频率上转换的荧光发射

用Nd：YAG的二倍频532 nm激光对几种不同水体的激光诱导荧光(LIF)光谱进行了测量,利用特征光谱荧光标记(SFS)技术指认出水体中溶解有机物(DOM)、叶绿素a(Chl a)及类胡萝卜素等物质的特征光谱带。指出在455 nm波长处具有较大强度的荧光峰是附属色素中抗氧化作用的类胡萝卜素(PPC)的贡献。提出了激光诱导PPC荧光频率上转换发射的动力学模型。     

Remote Estimation of the Chlorophyll Concentration of Living Trees Using Laser-induced Fluorescence Imaging Lidar

Monthly variation in chlorophyll concentration of living ginko tree leaves 65 m away from a system was remotely estimated by a laser-induced fluorescence imaging lidar. The combination of a pulsed laser and a short-time gated CCD using an image intensifier made it possible to monitor the weak fluorescence signal from the ginko tree leaves as an image. By applying the experimental idea that a ratio of intensity of the chlorophyll fluorescence at 740 nm to that at 685 nm showed a linear correlation to the chlorophyll concentration, the fluorescence image of the ginko tree obtained by the lidar was converted to the chlorophyll concentration distribution image.     

外周天线LHCⅡ的荧光光谱特性

外周天线LHC在光合作用过程中,担负着吸收和传递光能的作用.我们采用扫描成象荧光光谱技术对菠菜中外周天线LHC的荧光光谱特性进行了研究,在514.5nm的激光激励下获取了积分荧光谱,认为从类胡萝卜素分子到叶绿素分子间存在有能量传递.采用高斯组分光谱解析的方法,解析出LHC的荧光发射有七个谱带:656.7、664.6、671.5、677.2、683.5、689.6、695.3nm,各自所占的比例分别为3.0%、13.1%、13.3%、21.1%、13.2%、33.3%、3.0%,其中658.7nm的发射谱是由叶绿素b分子所发射的,其余的发射谱分别是由吸收峰为662、670/671、676、680nm以及吸收大于690nm的叶绿素a分子所发射.3.0%的叶绿素b分子的荧光发射说明在能量平衡过程中绝大部分能量被叶绿素a分子所禁锢,689.6nm处的荧光所占的比例最大,可能与LHC的一种自保护机制有关     

光系统Ⅱ荧光特性快速扫描成象光谱技术研究

本文以菠菜(Spinica oleracea L.)叶绿体中的PS颗粒复合物、CP47、CP43和LHC为材料,对其结构和功能关系进行了分析,用扫描成象光谱技术对这四种样品的光谱特性进行了研究,并通过对其荧光发射光谱图象的处理,得到它们的荧光发射光谱曲线.分析结果表明,PS颗粒复合物的荧光发射光谱中心波长在680.1nm,波段的范围较宽;CP43荧光光谱的中心波长位于680nm,并在近红外区观察到振动小峰;CP47荧光光谱的中心波长在691.3nm处;CP47和CP43在短波长区640nm和长波长区720nm附近分别出现肩峰,推测可能是样品中游离的叶绿素a和叶绿素b分子引起;LCH荧光发射光谱的峰值位于678nm,光谱范围为576nm～780nm.     

捕光天线LHCⅡ的荧光光谱特性研究

采用稳态荧光光谱技术在低温83 K下用波长为436 nm和507 nm的连续光激发对LHCⅡ进行研究,得到两种波长光激发下LHCⅡ的荧光光谱,并采用高斯组分光谱解析的方法,分别解析出四个谱带,结合吸收光谱和发射光谱分析,认为各自其中两个反映了两种光谱特性：Chl a683.6680/681、Chl a694.0690.0和Chl a/b671.4670.0和Chl a683.8680/681,其余两个长波长组分可能是Chl a分子主发射峰的振动副带.另外还将两种波长光激发下得到的荧光光谱特性做了比较,436 nm光激发下LHCⅡ发出的荧光强度要高于507 nm光的激发,这是由于接收436 nm光的Chl a分子数目多于接收507 nm光的类胡萝卜素分子,且436 nm下Chl a的吸收率也大于507 nm下类胡萝卜素的.从峰值上看,436 nm较507 nm光激励下的荧光光谱峰值产生红移,表明在不同波长光激励下,色素分子之间的能量传递途径是不同的.