A SINGLE PULSE PICOSECOND LASER STUDY OF EXCITON DYNAMICS IN CHLOROPLASTS

Abstract. Using single picosecond laser pulses at 610 nm, the fluorescence yield (φ) of spinach chloroplasts as a function of intensity ( I ) (10¹²-10¹⁶ photons/pulse/cm²) was studied in the range of 21–300 K. The quantum yield decreases with increasing intensity and the φ vs I curves are identical at the emission maxima of 685 and 735 nm. This result is interpreted in terms of singlet exciton-exciton annihilation on the level of the light-harvesting pigments which occurs before energy is transferred to the Photosystem I pigments which emit at 735 nm.
The yield φ is decreased by factors of 12 and 43 at 300 and 21 K, respectively. The shapes of the φ vs I curves are not well accounted for in terms of a model which is based on a Poisson distribution of photon hits in separate photosynthetic units, but can be satisfactorily described using a one-parameter fit and an exciton-exciton annihilation model. The bimolecular annihilation rate constant is found to be γ= (5–15) times 10^-9cm³s^-1 and to exhibit only a minor temperature dependence. Lower bound values of the singlet exciton diffusion coefficient (≥ 10^-3cm²s^-1), diffusion length (≥ 2 times 10^-6cm) and Förster energy transfer rates (≥ 3 ≥ 10¹⁰s^-1) are estimated from γ using the appropriate theoretical relationships.     

PICOSECOND FLUORESCENCE STUDIES OF P700-ENRICHED PARTICLES OF SPINACH CHLOROPLASTS

Dynamic properties of the picosecond fluorescence of highly enriched reaction-center particles of photosystem I (8 - 10 chlorophylls/P700) prepared from spinach have been investigated. The number (N) of photons used to excite chlorophyll molecules per reaction center was controlled between 0.06 and 80. The 1/e lifetime was ca. 25 ps for N 1. which is much shorter than previously measured lifetimes of photosystem I particles. The initial fluorescence intensity saturated at higher excitation intensities (N ≲ 1). This was interpreted in terms of interaction and annihilation among excited chlorophyll molecules which occur almost entirely within the duration of a laser flash. The spectrum-resolved fluorescence decay was faster at 690 than at 680 nm. This implies that two kinds of antenna chlorophylls, apart from and in close proximity to P700, have different lifetimes. Upon heat treatment a component with a much longer fluorescence decay time was observed. The growth of this component upon heat treatment at increasing temperatures showed a correlation with a decrease in the amount of P700 that could be photooxidized.     

THE FLUORESCENCE EXCITATION SPECTRUM OF QUININE BISULFATE

Abstract— A new measurement of the fluorescence excitation spectrum of chromatographically homogeneous quinine bisulfate has been made using a spectrometer offering high resolution and high sensitivity. Comparison of this spectrum with the absorption spectrum of the compound indicates that its quantum yield is independent of the wave number of the exciting light within ± 5 per cent for 2.57 μ^-l v 5.0μ^-1.     

EXCITATION OF CHLOROPLASTS INDUCED BY PHENYLACETALDEHYDE

Abstract Chloroplasts promote a slow oxygen consumption by phenylacetaldehyde. The latter elicits a sustained red emission and induces reduction of tetrazolium blue. Addition of horseradish peroxidase greatly increases both O₂ uptake and the initial light emission, but has little or no effect upon the reduction of tetrazolium blue. These results indicate that chlorophylls in chloroplasts can be efficiently excited in the absence of light.     

EXCITATION OF CHLOROPLASTS IN Euglena gracilis IN THE ABSENCE OF LIGHT

Abstract— Challenging Euglena gracilis —a unicellular microorganism that contains chloroplasts—with phenylacetaldehyde induces malondialdehyde formation, sustained red emission and Hill activity. In chloroplasts, phenylacetaldehyde appears to undergo peroxidase catalyzed oxidation to formic acid and triplet benzaldehyde; the latter or, less likely, a precursor thereof promotes lipid peroxidation. Triplet benzaldehyde and/or the excited species formed in lipid peroxidation transfer energy to the chlorophylls. This explanation also applies to spinach chloroplasts preparations, thus accounting for the previous unexplained observation that phenylacetaldehyde induced sustained red emission and Hill activity. A homogeneous picture is now available regarding the intracellular generation of excited states and concomitant excitation of built-in structures.     

THE FLUORESCENCE DECAY OF REDUCED NICOTINAMIDES IN AQUEOUS SOLUTION AFTER EXCITATION WITH A UV-MODE LOCKED Ar ION LASER

Abstract— The fluorescence decay kinetics of the reduced nicotinamides NMNH, NADH and NADPH in aqueous solution were investigated using an Ar ion laser, mode locked in the UV, as source of excitation and single photon counting electronics in the detection system allowing for a time resolution in the picosecond range. Analysis of the experimental fluorescence decay showed that the dinucleotides did not follow a single exponential decay law. Good fitting was accomplished with a sum of two exponentials. The mononucleotide fluorescence decay was a single exponential for at least 95% of its amplitude.
The heterogeneity in lifetimes of the fluorescence of the dinucleotides was interpreted in terms of an exciplex mechanism.     

PICOSECOND FLUORESCENCE STUDY OF PHOTOSYNTHETIC MUTANTS OF Chlamydomonas reinhardii: ORIGIN OF THE FLUORESCENCE DECAY KINETICS OF CHLOROPLASTS

Abstract— The fluorescence decay kinetics of photosynthetic mutants of Chlamydomonas reinhardii which lack photosystem II (PS II), photosystem I (PS I), and both PS II and PS I have been measured. The PS II mutant strain8–36C exhibits fluorescence decay lifetime components of 53, 424 and 2197 ps. The fluorescence decay of a PS I mutant strain12–7 contains two major fluorescence decay components with lifetimes of 152 and 424 ps. The fluorescence decay of mutant strain C2, which lacks both PS II and PS I, is nearly single exponential with a lifetime of 2561 ± 222 ps. In simulations in which it is assumed that wild-type decays are a simple sum of the major decay components of the isolated parts of the photosynthetic unit as measured in the mutants, curves are obtained that fit the wild-type C. reinhardii fluorescence decay data when the absorption cross-sections of PS II and PS I are weighted approximately equally. The 89 ps lifetime component in the wild-type is an average of 53 and 152 ps components arising from excitation transfer to and trapping in PS I and PS II. The single step transfer time in PS I is estimated to be between 100 and 700 fs depending on assumptions about array size. We find that between two and four visits to the PS I reaction center are required before final trapping.     

三元若丹明激光染料荧光寿命研究

利用单光子计数技术测试了新型三元若丹明激光染料在不同溶剂中的荧光寿命、荧光光谱及其寿命的实验数据.实验表明,所研究的三元若丹明染料存在着有效的分子内能量传递过程,这些过程使得激光染料的荧光量子效率及光稳定性明显改善.     

OXYGEN QUENCHING OF CHLOROPHYLL FLUORESCENCE IN CHLOROPLASTS

Abstract— Three phases of chlorophyll a fluorescence quenching by O₂ are observed in green plants. The effects of various inhibitors on photosynthetic partial processes in chloroplasts were investigated in attempts to (1) localize the O₂-quenching sites and (2) assess possible physiological significance of O₂-quenching. Our results localize the most sensitive (and presumably functionally important) phase to a site between plastoquinone and the photosystem I acceptor, chlorophyll (P₇₀₀), possibly plastocyanin. It is suggested that PC may transfer electrons to oxygen in addition to P₇₀₀.     

RESOLUTION OF THE FLUORESCENCE EXCITATION SPECTRUM OF INDOLE INTO THE 1La AND 1Lb EXCITATION BANDS*

Abstract— The fluorescence excitation spectrum and the excitation polarization spectrum of indole in propylene glycol were measured at — 58°C, after selecting by optical filters the emission originating from the ¹L_a electronic level. From the analysis of these spectra, the excitation spectrum was resolved into the ¹L_a and ¹L_a excitation bands. A similar resolution of the excitation spectrum of tryptophan is given. This method can also be applied to the resolution of the emission spectrum in cases of dual emission.     

EXCITATION OF MESOPHYLL AND BUNDLE SHEATH CHLOROPLASTS OF Atriplex repanda IN THE ABSENCE OF LIGHT

Abstract— Mesophyll and bundle sheath chloroplasts isolated from Atriplex repanda cells promote oxygen consumption by isobutyraldehyde or phenylacetaldehyde. In all cases, a red emission and reduction of tetrazolium blue was observed. Addition of horseradish peroxidase greatly increases the reduction of the dye. In the presence of 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea, the reduction of the Hill acceptor was fully suppressed. This suppression was abolished when 2, 6-dichlorophenolindophenol and ascorbate were added to the systems. These results indicate that, in mesophyll and bundle sheath chloroplasts, chlorophylls can be efficiently excited in the absence of light and an electron flow through the photosystems can be promoted.     

INFLUENCE OF THE ENVIRONMENT ON THE EXCITATION WAVELENGTH DEPENDENCE OF THE FLUORESCENCE QUANTUM YIELD OF INDOLE

Abstract— The influence of excitation wavelength, pH, oxygen and solvents, upon fluorescence quantum yields, were measured for insole Indole in neutral aqueous solution exhibits the same wavelength dependence as tryptophan, which indicates that COO- absorption is not responsible for the effect. Parameters such as pH and oxygen influence only the absolute fluorescence quantum yields but not their relative variation with wavelength, indicating competition with fluorescence emission for S1 deactivation. without any influence upon deactivation of higher excited states. In contrast, solvents exhibit a specific influence upon the wavelength dependence; for indole, the decrease of fluorescence yield excited around 215 nm, compared with the yield in the first absorption band is about 40% in water, 10% in acetonitrile, 70% in n-hexane and cyclohexane, whereas no appreciable decrease occurs in methanol, ethanol or n-butanol. These observations, together with the Stokes shifts of the emission spectra, may be well correlated with Kosower's Z-values, expressing microscopic solvent-solute interactions.     

FLUORESCENCE OF BACTERIORHODOPSIN UNDER SUBPICOSECOND LIGHT EXCITATION

Abstract— A streak camera detection technique has been used to record the fluorescence of bacteriorhodopsin at room temperature induced by single subpicosecond light pulses. The fluorescence lifetime of bacteriorhodopsin has been found to be less than 2 ps.     

FLUORESCENCE AND LUMINESCENCE STUDIES OF IN VIVO CHLOROPHYLL WITH A LASER PHOSPHOROSCOPE*

Abstract— A simple apparatus using a He-Ne laser and a rotating sector gives repetitive light pulses with rise and fall times of a few μsec and intensities of up to 0.65 W/cm². Fluorescence during the actinic pulse and luminescence following it (after 20 μsec) may be recorded and compared. Sampling of the suspension of photosynthetic cells or chloroplasts by intermittent flow permits the averaging of 25 such measurements. With Chlorella, the fast (30 μsec) and the medium (30 msec) components of luminescence do not saturate at the same light intensity. The former saturates in the same range as the photochemical fluorescence rise, whereas the latter saturates at higher intensities. Under a variety of conditions, the decay of luminescence intensity is always strongly polyphasic. Analysis of the kinetics discloses a dependency with the log concentration of the hypothetical precursor, suggesting a connection of luminescence with electrochemical phenomena. The shape of the luminescence integral, as well as the light saturation pattern, suggests another kind of interpretation, in terms of several pools, possibly two of equal size. In the presence of hydroxylamine, only one pool is seen. The stimulating effect of preillumination is only observed for the medium component. However, a stimulation of short duration also exists for the fast component. The problems of number of pools, mechanism, and stimulation are briefly discussed.     

PHOTOCHEMISTRY OF THE REACTION CENTERS OF SYSTEM II UNDER REPETITIVE FLASH GROUP EXCITATION IN ISOLATED CHLOROPLASTS

Abstract— Absorption changes induced in isolated chloroplasts by excitation with repetitive flash groups have been measured at 690 nm, indicating the photochemical turnover of chlorophyll-a_II (Chl-αn), and at 480 nm and 513 nm respectively, reflecting via electrochromic effect the formation of a transmembrane electric field. The data are compared with measurements of oxygen evolution. In chloroplasts with practically fully intact oxygen evolving capacity it was found: 1. The initial amplitude of the 690 nm absorption change induced by the second flash as a function of the time t_v between the first and second flash of a group increases with a half life of about 35 µs. On the other hand, the average oxygen yield due to the second flash as a function of the time t_v rises with a half life of about 600 µs (and a kinetics in the ms-range of minor extent), confirming the data of Vater et al. (1968). 2. Under far red background illumination, where contributions due to PS I in the µs-range can be excluded, the difference spectrum in the red of the absorption changes induced by the first flash corresponds with that of the absorption changes induced by the second flash fired 200 µ after the first flash. 3. The pattern of the absorption changes at 690 nm induced by repetitive double flash groups at t_v= 200 µs does not markedly change in normal chloroplasts by the presence of DBMIB?. Similar 690 nm absorption changes occur in trypsin treated chloroplasts, independent of the presence of DCMU. 4. The fast regeneration in the µs-range of Chl-a_n is also observed in the third flash of a triple flash group at a time t_v= 200 µs between the flashes of the group. 5. The initial amplitudes of the absorption changes with a decay kinetics slower than 100 µs induced by the second flash at 480 nm and 513 nm, respectively, as a function of the time t, between the first and second flash of a group, are characterized by a recovery half-time of about 600 µs, confirming earlier measurements at 520 nm (Witt and Zickler, 1974). On the basis of these results it is inferred that there does exist a photoreaction of Chl-α_n., with an electron acceptor, referred to as X_a, other than the ‘primary’ plastoquinone acceptor X320, if X320 persists in its reduced state. Under conditions of X320 being in the reduced state, this photochemical reaction was shown to be highly dissipative with respect to charging up the watersplitting enzyme system Y. Furthermore, this Chl-a_n-photoreaction with X_a does not lead to a vectorial transmembrane charge separation, which is stable for more than a few microseconds. Different models for the functional and structural organization of PS II are discussed.     

CALCIUM-DEPENDENT FLUORESCENCE LIFETIMES OF INDO-1 FOR ONE- AND TWO-PHOTON EXCITATION OF FLUORESCENCE

Abstract— We characterized the fluorescence intensity decays of Indo-1, which is commonly used as an emission wavelength-ratiometric calcium probe. The apparent lifetime of the long-wavelength side of the emission of Indo-1 is dependent on Ca²⁺. This long-wavelength emission displays the characteristics of an excited-state reaction, that is, a negative preexponential component in thc multiexponential analysis. The emission spectra and lifetime of Indo-1 appear to be identical for one-photon and two-photon excitation at 351 and 702 mn, respectively, suggesting that the relative one- and two-photon cross sections are similar for the calcium-free and calcium-bound forms of Indo-1. Also, the two-photon cross section of Indo-1 is relatively high, about 4 × 10⁻⁴⁹ cm⁴ s/photon molecule at 690 nm for both the calcium-free and calcium-bound forms. Hence, Indo-1 can be used for calcium imaging based on one- or two-photon excitation, using either emission wavelength ratios or lifetime imaging methods.     

激光诱导荧光衰减探测光化学反应

激光荧光光谱为重要光谱分析手段之一,广泛用于物理、化学、生物学等研究领域。 但对于一些含有发色基团的大有机分子,由于分子振动自由度很大,振动能级连续分布, 其荧光谱成为宽展的光谱带,振动带结构不复存在,使荧光光谱难于用作样品的成份鉴定。对于溶液样品由于分子间的碰撞及长程力的作用,也可能造成荧光猝灭和分子间电 子能量转移,特别是对那些吸收光子后易发生光分解的物质,所产生的新分子或碎片往 往也有荧光特征,叠加在原有分子的荧光带上,使得所要检测的任何一种成份的荧光强 度对所有其它荧光猝灭物和敏化物的浓度及其荧光特征有很复杂的依赖关系,这就增加 了对实验所得到的荧光光谱解释的难度。     

FLUORESCENCE OF MELANIN-DEPENDENCE UPON EXCITATION WAVELENGTH AND CONCENTRATION

Abstract— An introduction to the fundamental characteristics of synthetic melanin fluorescence is presented. The particular difficulties associated with the detection and reduction of the relatively weak signal are discussed and a technique is described for correcting the fluorescence spectra for attenuation of the excitation and emission beams. Spectra are reported for the excitation wavelength range 340–400 nm and an emission range of 360–560 nm. The concentration dependence of the corrected fluorescence signal is examined and is shown to be linear. The variation of the fluorescence spectra with excitation wavelength suggests a two-component fluorescence, for the wavelength range studied. The presence of an isosbestic point in the spectra is used to identify the fluorophores as components of a reaction equilibrium. The possible relationship of this equilibrium to that associated with the melanin photo ESR is discussed     

FLUORESCENCE INDUCTION IN ALGAE AND CHLOROPLASTS

Abstract —The first phases of the fluorescence transient elicited by illumination of dark-adapted algae or isolated chloroplasts (biphasic rise φ_***v - φ_I - _P) have previously been shown to be controlled by two quenchers: Q , the primary acceptor of Photosystem 2 interacting with the secondary acceptor pool A ; and R , a non-photochemical quencher which goes into a non-quenching state as A is reduced.
The dependence of the kinetics of φ decay after illumination upon the redox state of A was studied. It is suggested that some of the centres are in a disconnected state, where electron transfer between Q and A is hindered, the amount of such centres being correlated to the reduction state of A . The implications of this hypothesis on the problem of the variation of the Q-A 'equilibrium constant' under different experimental conditions, and on Murata's 'weak light effect' are discussed.
The effect of 3-(3,4 dichlorophenyl)-1,1 dimethylurea (DCMU) on R is shown to depend upon the redox state of A . A DCMU-induced shift of the midpoint potential of R may account for this dependence.
Evidence is given suggesting that the transient reduction of A which occurs in algae during the φ_I-φ_P rise is controlled by an induction process on the acceptor side of Photosystem 1.