Time-resolved fluorescence spectroscopy of spinach chloroplast

Picosecond fluorescent kinetics and time-resolved spectra of spinach chloroplast were measured at room temperature and low temperatures. The measurement is conducted with 530 nm excitation at an average intensity of 2 · 10¹⁴ photons/cm², pulse and at a pulse separation of 6 ns for the 100 pulses used. The 685 nm fluorescent kinetics was found to decay with two components, a fast component with a 56 ps lifetime, and a slow component with a 220 ps lifetime. The 730 nm fluorescent kinetics at room temperature is a single exponential decay with a 100 ps lifetime. The 730 nm fluorescence lifetime was found to increase by a factor of 6 when the temperature was lowered from room temperature to 90 K, while the 685 and 695 nm fluorescent kinetics were unchanged. The time-resolved spectra data obtained within 10 ps after excitation is consistent with the kinetic data reported here. A two-level fluorescence scheme is proposed to explain the kinetics. The effect of excitation with high light intensity and multiple pulses is discussed.     

Measurement of gradients of absorbed light in spinach leaves from chlorophyll fluorescence profiles

Profiles of chlorophyll fluorescence were measured in spinach leaves irradiated with monochromatic light. The characteristics of the profiles within the mesophyll were determined by the optical properties of the leaf tissue and the spectral quality of the actinic light. When leaves were infiltrated with 10^?4M DCMU [3‐(3,4‐dichlorophenyl)‐1, 1‐dimethyl‐urea] or water, treatments that minimized light scattering, irradiation with 2000 μmol m^?2 s^?1 green light produced broad Gaussian‐shaped fluorescence profiles that spanned most of the mesophyll. Profiles for chlorophyll fluorescence in the red (680 ± 16 nm) and far red (λ > 710 nm) were similar except that there was elevated red fluorescence near the adaxial leaf surface relative to far red fluorescence. Fluorescence profiles were narrower in non‐infiltrated leaf samples where light scattering increased the light gradient. The fluorescence profile was broader when the leaf was irradiated on its adaxial versus abaxial surface due to the contrasting optical properties of the palisade and spongy mesophyll. Irradiation with blue, red and green monochromatic light produced profiles that peaked 50, 100 and 150 μm, respectively, beneath the irradiated surface. These results are consistent with previous measurements of the light gradient in spinach and they agree qualitatively with measurements of carbon fixation under monochromatic blue, red and green light. These results suggest that chlorophyll fluorescence profiles may be used to estimate the distribution of quanta that are absorbed within the leaf for photosynthesis.     

Photoinhibition at chilling temperatures and effects of freezing stress on cold acclimated spinach leaves in the field. A fluorescence study

The role of high light stress in a natural environment was studied on spinach plants ( Spinacia oleracea L. cv. Wolter) grown in the field during the winter season. Fluorescence induction (at 293 K and 77 K) of leaves was used to characterize the stress effects. Night frost with minimum temperatures between – 1.5°C and –7.5°C (i.e. above the'frost killing point'at ca. –11.5°C) led to impaired photosynthesis. This was seen as increased initial fluorescence (F_o), decreased ratio of variable to maximum fluorescence (F_V/F_M) and lowered rates of O₂ evolution. The freezing injury was reversible within several frostless days. Exposure to high light (about 900 mol m_–2 s_–1) at chilling temperatures in the field caused photoinhibition, manifested as decreased variable fluorescence (F_V) and F_V/F_M ratio without changes in F_O. The photoinhibitory fluorescence quenching was not stronger after frost than after frostless nights; synergism between light stress and preceding freezing stress was not observed. Fluorescence induction signals at 77 K showed that F_V of photosystems I and II decreased to the same extent, indicating increased thermal deactivation of excited chlorophyll. Photoinhibition was fully reversible at +4°C within 1 h in low light, but only partially in moderate light. Preceding night frosts did not affect the recovery. The photoinhibition observed here is regarded as a protective system of thermal dissipation of excess light energy.     

不同强度的红光和蓝光下菜豆叶片的荧光特性

光质和光强均是影响植物光合作用的重要外部因素,该文以菜豆(Phaseolus vulgaris)为材料,通过叶绿素荧光技术比较研究了菜豆叶片在不同光强的红光和蓝光下叶绿素荧光特性的变化规律。结果表明:随着红光和蓝光光强的增加,菜豆叶片的光适应下的最大光化学效率(Fv'/Fm')呈下降趋势,但与在红光下相比,蓝光下叶片的Fv'/Fm'值较高。随着蓝光光强的增加,菜豆叶片PSⅡ实际光化学效率(Y(Ⅱ))和光化学猝灭系数(q P和q L)先呈上升趋势之后逐渐趋于平稳;而随着红光光强的增加,以上参数呈下降趋势。随着红光和蓝光光强的增加,非光化学猝灭系数(NPQ)、相对电子传递速率(ETR)以及调节性能量耗散的量子产量Y(NPQ)均呈上升趋势,但与在红光下相比,蓝光下叶片NPQ和Y(NPQ)的值较低,而ETR值较高。非调节性能量耗散产量Y(NO)随着红光光强增加而呈上升趋势,而随着蓝光光强增加呈下降趋势。综上可见,随着光强的增加菜豆叶片的光化学效率呈降低趋势,但叶片在蓝光下的光化学吸收和利用效率高于红光。研究结果可为植物对光强和光质的响应提供一定的参考。     

Studies on the temperature effect on bacteriorhodopsin of purple and blue membrane by fluorescence and absorption spectroscopy

Fluorescence and absorption spectra were used to study the temperature effect on theconformation of bacteriorhodopsin (bR) in the blue and purple membranes (termed as bRb and bRprespectively).The maximum emission wavelengths of tryptophan fluorescence in both proteins at roomtemperature are 340 nm,and the fluorescence quantum yield of bRb is about 1.4 fold higher than that of bRp.As temperature increases,the tryptophan fluorescence of bRb decreases,while the tryptophan fluorescenceof bRp increases.The binding study of extrinsic fluorescent probe bis-ANS indicated that the probe can bindonly to bRb,but not to bRp.These results suggest that significant structural difference existed between bRband bRp.It was also found that both kinds of bR are highly thermal stable.The maximum wavelength of theprotein fluorescence emission only shifted from 340 nm to 346 nm at 100℃.More interestingly,as tempera-ture increased,the characteristic absorption peak of bRb at 605 nm decreased and a new absorption peak at380 nm formed.The transition occurred at a narrow temperature range (65℃-70℃).These facts indicatedthat an intermediate can be induced by high temperature.This phenomenon has not been reported before.     

Time-resolved fluorescence spectroscopy of hematoporphyrin-derivative in human lymphocytes

This paper reports on time-resolved microfluorimetric measurements on hematoporphyrin-derivative (HpD)-treated lymphocytes. HpD is at present widely used as a tumor-locating and photosensitizing drug. It is therefore of great importance to study the extent to which the HpD uptake process depends on cell functional and structural properties. Time-resolved fluorescence measurements in single cells are very useful in this respect, since they give information on the content of fluorescent molecules through fluorescence peak-intensity, and, indirectly, on the binding properties through the fluorescence decay times. In particular, we studied the dependence of HpD fluorescence on the cellular functional state. To this end, we performed in-cell fluorescence measurements on human lymphocytes, both in quiescent conditions and in the pre-replicative phase, after stimulation with phytohemagglutinin (PHA). We found a higher HpD content in stimulated lymphocytes. Moreover, we found a spectral band around 575 nm, corresponding to a particular porphyrin species, in which the differences between normal and stimulated lymphocytes are more striking. The porphyrin species emitting in this band seems to play a role in the specific interaction of HpD with tumors, since a similar emission band has also been found in tumor cells containing HpD.     

Profiles of light absorption and chlorophyll within spinach leaves from chlorophyll fluorescence

Chlorophyll fluorescence was used to estimate profiles of absorbed light within chlorophyll solutions and leaves. For chlorophyll solutions, the intensity of the emitted fluorescence declined in a log–linear manner with the distance from the irradiated surface as predicted by Beer's law. The amount of fluorescence was proportional to chlorophyll concentration for chlorophyll solutions given epi‐illumination on a microscope slide. These relationships appeared to hold for more optically complex spinach leaves. The profile of chlorophyll fluorescence emitted by leaf cross sections given epi‐illumination corresponded to chlorophyll content measured in extracts of leaf paradermal sections. Thus epifluorescence was used to estimate relative chlorophyll content through leaf tissues. Fluorescence profiles across leaves depended on wavelength and orientation, reaching a peak at 50–70 µm depth. By infiltrating leaves with water, the pathlengthening due to scattering at the airspace : cell wall interfaces was calculated. Surprisingly, the palisade and spongy mesophyll had similar values for pathlengthening with the value being greatest for green light (550 > 650 > 450 nm). By combining fluorescence profiles with chlorophyll distribution across the leaf, the profile of the apparent extinction coefficient was calculated. The light profiles within spinach leaves could be well approximated by an apparent extinction coefficient and the Beer–Lambert/Bouguer laws. Light was absorbed at greater depths than predicted from fibre optic measurements, with 50% of blue and green light reaching 125 and 240 µm deep, respectively.     

Time-resolved chlorophyll fluorescence studies on photosynthetic mutants of Chlamydomonas reinhardtii: origin of the kinetic decay components

The room temperature chlorophyll fluorescence decay kinetics of photosynthetic mutants of Chlamydomonas reinhardtii have been measured as a function of Photosystem 2 (PS2) trap closure, DNB-induced quenching at F_M, and time-resolved emission spectra. The overall decays have been analyzed in terms of three or four kinetic components where necessary. A comparison of the characteristics of the decay components exhibited by the mutants with the wild-type has been carried out to elucidate the precise origins of the different emissions in relation to the observed pigment-protein complexes. It is shown that a) charge recombination in PS2 is not necessary for the presence of long-lived decay components, b) there are two rapid PS1-associated emissions (=30 and 150–200 ps), c) a slow PS1 decay is observed (=1.73 ns) in the absence of PS1 reaction centres, d) the two variable components (=0.25–1.2 and 0.5–2.2 ns) observed in the wild-type arise from LHC2 and e) a rapid (=50–250 ps) decay is associated with the PS2 core antenna (CP3 and CP4). These results show that the intact thylakoid membrane system is too complex to distinguish all of the individual kinetic components.Abbreviations Aexp preexponential factor (Amplitude) - chl chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - DNB m, dinitrobenzene - F_M maximum chl fluorescence level - F₀ initial chl fluorescence level - F_v variable chl fluorescence (F_M–F₀) - LHC light harvesting chl a/b protein complex - PS photosystem - Q_A primary stable electron acceptor of PS2     

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%.     

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     

Fluorescence correlation spectroscopy in the nanosecond time range: Photon antibunching in dye fluorescence

Possibilities for the use of fluorescence correlation spectroscopy in the nanosecond time range are demonstrated. The experiment is based on a cw argon ion laser, a microfluorimeter, two photon detectors, and a time-to-analog converting system. Experiments using solutions of rhodamine 6G and pyronine G in water at concentrations of about 20 molecules per sample volume are reported. The photon anticorrelation component decaying with a time constant close to the excited state lifetime was observed.     

Evidence that the variable chlorophyll fluorescence in Chlamydomonas reinhardtii is not recombination luminescence

Room temperature single photon timing measurements on intact, Chlamydomonas reinhardtii cells at low excitation energies have been analysed using a four exponential kinetic model. Closing the PSII reaction centres produced two major variable lifetime and two minor constant lifetime components. The yield of each component mirrored the changes in lifetime. Such observations indicate the presence of well-connected PSII centres favoring excitation energy transfer. A Chlamydomonas mutant lacking PSII reaction centre proteins exhibited decay components equivalent to those seen at F_M in the wild-type. A titration of in vivo fluorescence, in both the mutant and wild-type algae, using DNB, produced decay components similar to those seen on opening PSII reaction centres. Such observations indicate that the luminescence hypothesis for the origin of the long-lived lifetime component is not the case.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1, 1-dimethyl urea - DNB m,Dinitrobenzene - PSII photosystem II - RCII PSII recation centre - I^- reduced pheophytin - Q_A primary stable electron ecceptor of PSII - Ch1 chlorophyl1 - LHCII light harvesting Ch1a/b protein complex of PSII - F_O initial fluorescence level - F_M maximum fluorescence level - F_V variable fluorescence (F_M-F_O) - ps picosecond - ns nanosecond     

Synchrotron-excited time-resolved fluorescence spectroscopy of adenosine,protonated adenosine and 6N, 6N-dimethyladenosine in aqueous solution at room temperature

The fluorescence behavior of adenosine in neutral solution has been studied by time-resolved spectroscopy using synchrotron excitation and timecorrelated single photon counting, and by decay time measurements. Three emissions have been identified and correlated with three excitation spectra. The assignment of these transitions has been made by comparison with similar measurements on ⁶N, ⁶N-dimethyladenosine (6 DMA), and on adenosine in acid solution (ADO H⁺). It is proposed that two of the transitions of adenosine which correlate with 6DMA originate from coplanar and orthogonal rotational conformers of the amino group. The other transition, correlating with ADO H⁺ may originate either from the ³H-imino tautomer, or from a differently solvated rotational conformer.A partial presentation of this work has been made at the Second Congress of the European Society for Photobiology Padova, Italy, 6–10 September 1987     

Light scattering,chlorophyll fluorescence and state of the adenylate system in illuminated spinach leaves

Scattering of green light and chlorophyll fluorescence by spinach leaves kept in a stream of air or nitrogen were compared with leaf adenylate levels during illumination with blue, red or far-red light. Energy charge and ATP-ADP ratios exhibited considerable variability in different leaves both in the dark and in the light. Variability is explained by different possible states of the reaction oxidizing triose phosphate or reducing 3-phosphoglycerate. Except when oxygen levels were low, there was an inverse relationship between light scattering and chlorophyll fluorescence during illumination with blue or red light. When CO₂ was added to a stream of CO₂-free air, chlorophyll fluorescence increased, sometimes after a transient decrease, and both light scattering and leaf $\text{ATP}\text{ADP}$ ratios decreased. Similar observations were made when air was replaced by nitrogen under blue or high-intensity red light. Under these conditions, over-reduction caused inhibition of electron transport and phosphorylation in chloroplasts. However, when air was replaced by nitrogen during illumination with low-intensity red light or far-red light, light scattering increased instead of decreasing. Under these light conditions, $\text{ATP}\text{ADP}$ ratios were maintained in the light. They decreased drastically only after darkening. Although $\text{ATP}\text{ADP}$ ratios responded faster than light scattering or the slow secondary decline of chlorophyll fluorescence due to illumination, it appeared that in the steady state, light scattering and chlorophyll fluorescence are useful indicators of the phosphorylation state of the leaf adenylate system at least under aerobic conditions, when chloroplast and extrachloroplast adenylate systems can effectively communicate.     

Exploring the conformational equilibrium of E. coli thioredoxin reductase: characterization of two catalytically important states by ultrafast flavin fluorescence spectroscopy

The conformational dynamics of wild-type Escherichia coli thioredoxin reductase (TrxR) and the mutant enzyme C138S were studied by ultrafast time-resolved fluorescence of the flavin cofactor in combination with circular dichroism (both in the flavin fingerprint and far-UV regions) and steady-state fluorescence and absorption spectroscopy. The spectroscopic data show two conformational states of the enzyme (named FO and FR), of which the physical characteristics differ considerably. Ultrafast fluorescence lifetime measurements make it possible to distinguish between the two different populations: Dominant picosecond lifetimes of approximately 1 ps (contribution 75%) and 7 ps (8%) are associated with the FO species in TrxR C138S. Long-lived fluorescence with two time constants in the range of 0.2-1 ns (total contribution 17%) originates from enzyme molecules in the FR conformation. The near absence of fast lifetime components in oxidized wild-type TrxR supports the idea of this enzyme being predominantly in the FR conformation. The emission spectrum of the FO conformation is blue-shifted with respect to that of the FR conformation. Because of the large difference in fluorescence characteristics, fluorescence measurements on time scales longer than 100 ps are fully determined by the fraction of enzyme molecules in the FR conformation. Binding of the thiol reagent phenyl mercuric acetate to wild-type enzyme and TrxR C138S stabilizes the enzymes in the FR conformation. Specific binding of the NADPH-analog, AADP(+), to the FR conformation resulted in dynamic fluorescence quenching in support of the multiple quenching sites model. Raising the temperature from 277K-323K resulted in a moderate shift to the FR conformation for TrxR C138S. High concentrations of the cosolvent glycerol triggered the domain rotation from the FO to the FR conformation.     

Effect of wounding on ethylene biosynthesis and senescence of detached spinach leaves

Parameters of senescence and ethylene biosynthesis pathway were screened simultaneously in detached spinach leaves and leaf discs. Senescence was enhanced by application of 1-aminocyclopropane-1-carboxylic acid (ACC) and was retarded by amino-ethoxyvinylglycine (AVG). Evidence is presented showing that the bursts of both wound- and climacteric-like ethylene promoted senescence of detached leaves and leaf discs. This ethylene-enhanced leaf senescence was dependent on: (a) ethylene production rates in the tissue; (b) the degree of wounding. Wounding resulted in elevated levels of 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC), which declined in advanced stages of senescence. The results suggest that wounding might be regarded as one of the primary events in the induction of the senescence syndrome in detached leaves and leaf discs, while ethylene is implicated as a regulator of the rate of the process.     

Time-resolved fluorescence spectroscopy of lumazine protein from Photobacterium phosphoreum using synchrotron radiation

Time-resolved fluorescence on lumazine protein from Photobacterium phosphoreum was performed with synchrotron radiation as a source of continuously tunable excitation. The experiments yielded structural and dynamic details from which two aspects became apparent. From fluorescence anisotropy decay monitoring of lumazine fluorescence with different excitation wavelengths, the average correlation times were shown to change, which must indicate the presence of anisotropic motion of the protein. A similar study with 7-oxolumazine as the fluorescent ligand led to comparable results. The other remarkable observation dealt with the buildup of acceptor fluorescence, also observed with 7-oxolumazine although much less pronounced, which is caused by the finite energy transfer process between the single donor tryptophan and the energy accepting lumazine derivatives. Global analytical approaches in data analysis were used to yield realistic correlation times and reciprocal transfer rate constants. It was found that the tryptophan residue has a large motional freedom as also reported previously for this protein and for the related protein from P. leiognathi (Lee et al. 1985; Kulinski et al. 1987). The average distance between the tryptophan residue and the ligand donor-acceptor couple has been determined to be 2.7 nm for the same donor and two different acceptors.     

Quantitation of protein on gels and blots by infrared fluorescence of Coomassie blue and Fast Green

Coomassie blue staining of gels and blots is commonly employed for detection and quantitation of proteins by densitometry. We found that Coomassie blue or Fast Green FCF bound to protein fluoresces in the near infrared. We took advantage of this property to develop a rapid and sensitive method for detection and quantitation of proteins in gels and on blots. The fluorescence response is quantitative for protein content between 10 ng and 20 microg per band or spot. Staining and destaining require only 30 min, and the method is compatible with subsequent immunodetection.     

Secondary fluorescence kinetics of spinach leaves in relation to the onset of photosynthetic carbon assimilation

When spinach leaves are re-illuminated, after dark periods of 90 s or less, an initial fluorescence peak is observed which rapidly gives way to a much lower terminal value. After 2 min or more in the dark, however, there is a secondary rise, at about 50–70 s, which then gives way, more slowly, to approximately the same low terminal value as before. The secondary rise is eliminated or disguised by feeding D,L-glyceraldehyde (a specific inhibitor of photosynthetic carbon assimilation) and by manose, 2-deoxyglucose and glucosamine, all of which are believed to sequester cytoplasmic orthophosphate. This secondary rise in fluorescence is discussed in relation to photosynthetic induction and the manner in which these compounds may modulate fluorescence by their effect on the availability of orthophosphate and their consequent impact on the adenylate status of the stroma.Abbreviations DCMU 3(3,4-dichlorophenyl)-1,1-dimethylurea - CCCP carbonylcyanidchlorophenylhydrazon     

Blue-green fluorescence excited by UV laser on leaves of different species originates from cutin and is sensitive to leaf temperature

Under ultra-violet excitation, intact leaves generate a strong blue-green fluorescence emission with several bands. Their integrated energy is 6 to 11 times the energy released by chlorophyll a bands (Chappelle et al. 1984, Applied Optics 23, 134–138). This paper provides evidence that the blue-green fluorescence emission comes mainly from outer epidermal layers of the leaves and can be transferred on a quartz lamina by quickly dipping the leaves in organic solvents with subsequent solvent evaporation. Blue-green fluorescence displays a diffusion-controlled quenching of fluorescence intensity between 4°C (high fluorescence) and 37°C (low fluorescence). The blue-green fluorescence emissivity is not linked to short-term metabolic effects other than leaf temperature, but epidermis adaptations both to drought and to excessive radiation increase emissivity.