Biological weighting functions as a tool for evaluating two ways to measure UVB radiation inhibition on photosynthesis

To estimate the inhibitory effect of the changing UVB radiation (UVBR, 280-315nm) on earth's ecosystems, an understanding of its wavelength dependency is needed. The tool used for these estimations is the biological weighting function (BWF), whereby the inhibition of different wavelengths is calculated. BWFs were determined for three algae species from different classes, Phaeodactylum tricornutum (Bacillariophyceae), Dunaliella tertiolecta (Chlorophyceae) and Rhodomonas sp. (Cryptophyceae), using polychromatic irradiation, where the UVBR spectra were varied with cut-off filters. For each alga, BWFs were determined for two photosynthetic parameters; the quantum yield measured as fluorescence from Photo System II in a pulse-amplitude-modulation (PAM) fluorometer, and the fixation of (14)C-labelled carbon dioxide. The BWFs were calculated with the Rundel method, using the radiation data between 270 and 360nm with 1nm resolution. The results show that the UVBR damages were generally higher when using the carbon fixation measurements than when measuring with the PAM technique. When using PAM, P. tricornutum in particular had a sensitivity intermediate between the sensitive Rhodomonas sp. and the more tolerant D. tertiolecta, but was as sensitive as, or even more sensitive, than Rhodomonas sp. when using carbon fixation. D. tertiolecta was shown to be less sensitive when using both techniques and the inhibition of its photosynthesis was almost as high when using PAM as when using carbon fixation. We concluded that, although the PAM technique has advantages such as being cleaner and easier to use, it is unable to substitute the carbon fixation measurements. Not only are the algae less sensitive when measured with PAM than they are when measured as carbon fixation, the relationship between the effects on the algae measured with the two techniques also differs. As fixation of carbon dioxide integrates a larger part of the photosynthetic machinery, it should be favoured as a measure of photosynthesis.     

Reduction in growth rate in Phaeodactylum tricornutum (Bacillariophyceae) and Dunaliella tertiolecta (Chlorophyceae) induced by UV-B radiation

The effect of UV-B radiation (UVBR, 280-315 nm) on growth rate during 72 h of incubation, was measured for two marine microalgae -Dunaliella tertiolecta (Chlorophyceae) and Phaeodactylum tricornutum (Bacillariophyceae). The resulting inhibition of growth rate was analysed by calculating biological weighting functions (BWFs). The growth rate of D. tertiolecta was slightly more inhibited by UVBR (over the whole range of the spectrum) than was the growth rate of P. tricornutum, but the wavelength dependencies were the same. Our results were compared with results from photosynthesis experiments of Andreasson and W?ngberg [1] , where two methods, pulse amplitude modulation (PAM) fluorescence and carbon fixation, were measured for these same algae. The BWF for the growth rate, here, showed more wavelength dependency than the BWF for the previous two photosynthesis measurements - except for the carbon fixation BWF in P. tricornutum, which was closer to the BWF for growth rate. The wavelength dependency of the growth rate inhibition showed less variation between the species than the inhibition of the photosynthesis.     

Mechanism study on the origin of delayed fluorescence by an analytic modeling of the electronic reflux for photosynthetic electron transport chain

A mathematical-physical analysis model, which describes individually the electronic reflux of several significant components in the photosynthesis electron transport chain, was firstly developed. The process of electrons flowing back to the oxidized reaction center P(680)(+) was simulated by a series of photochemical reaction equations, resulting in getting the linked differential equations of delayed fluorescence (DF) intensity. MATLAB provided a computationally efficient method to solve these linked equations. Simulations based on this model showed that the decay kinetics of DF accord with double exponential. DF components decaying in the millisecond range (fast phase) are related to the charge recombination of P(680)(+) and Q(A)(-). The components decaying in the seconds range are associated with the recombination of P(680)(+) with Q(B)(2-). The developed model was tested in maize leaves treated with different electron blockers to induce changes in photosynthesis electron transport chain. The experimental results demonstrated that the developed model can accurately determine the regulatory effects of electron blockers on photosynthesis electron transport chain. Therefore, the model presented here could be potentially useful for studying the electron transfer in plant. It also provides an experimental workbench for testing hypotheses as to the underlying mechanism controlling the change for different phases of DF.     

EXCITED STATE pKa BEHAVIOUR OF DAPI. A RATIONALIZATION OF THE FLUORESCENCE ENHANCEMENT OF DAPI IN DAPI-NUCLEIC ACID COMPLEXES*

Abstract— The steady state and time resolved fluorescence of the drug and chromosomal staining agent, 4′,6-diamidino-2-phenylindole dihydrochloride, DAPI, was examined under different solvent conditions. In solutions between pH 3 and pH 9 the fluorescence spectral maximum of DAPI was found at 460 nm. The fluorescence decayed with double exponential kinetics, with decay times of 2.86 and 0.144 ns, at all wavelengths below 550 nm. At 550 nm single exponential decay kinetics with a lifetime of 0.153 ns was observed. The fluorescence spectrum could be resolved into two components, the 2.86 ns component having a spectral maximum near 450 nm and the 0.144 ns component having a spectral maximum near 490 nm. The results are rationalized in terms of there being two different configurations of DAPI, one of which undergoes a rapid protonation of the indole ring by proton transfer from the 6-amidinium group in the excited singlet state. The 0.144 ns component is assigned as the fluorescence from the excited state of the protonated indole ring. The results provide an explanation of the fluorescence enhancement in DAPI-nucleic acid complexes.     

Fluorescence imaging of Foscan and Foslip in the plasma membrane and in whole cells

A fluorescence microscope equipped with a condenser for total internal reflection (TIR) illumination was combined with a pulsed laser diode and a time-gated image intensifying camera for fluorescence lifetime measurements of single cells. In particular, fluorescence patterns, decay kinetics, and lifetime images of the lipophilic photosensitizers Foscan and Foslip were studied in whole cells as well as in close vicinity to their plasma membranes. Fluorescence lifetimes of both photosensitizers in cultivated HeLa cells decreased from about 8 ns at an incubation time of 3 h to about 5 ns at an incubation time of 24 h. This seems to result from an increase in aggregation (or self-quenching) of the photosensitizers during incubation. Selective measurements within or in close proximity to the plasma membrane indicate that Foscan and Foslip are taken up by the cells in a similar way, but may be located in different cellular sites after an incubation time of 24 h. A combination of TIR and fluorescence lifetime imaging microscopy (FLIM), described for the first time, appears to be promising for understanding some key mechanisms of photodynamic therapy (PDT).     

Laser picosecond microspectrofluorometry of haematoporphyrin in cells and liposomes.

The results of a laser picosecond microspectrofluorometric study of the spectral and kinetic characteristics of haematoporphyrin (Hp) fluorescence at various sites in cultured SPEV cells and phosphatidylcholine liposomes are presented. The computer-controlled detection system is based on the single-photon counting method with picosecond time resolution. In aqueous medium, the Hp fluorescence spectrum is characterized by two bands at 615 and 675 nm. In living cells and liposomes, Hp fluorescence is red shifted to 630 and 690 nm. In addition a new band at 665 nm is detected. The dependence of this band on the incubation time and Hp concentration was investigated. The fluorescence decay kinetics of Hp in a culture medium, liposome and a cell nuclear membrane were measured. Possible Hp aggregate formation in the lipid bilayer and its implications are discussed.     

CCl2(A 1B 1,a3B1)自由基被烷烃类分子猝灭动力学

用放电 LIF实验装置,对CCl4/Ar混合气体放电产生CCl2自由基,再用541.52 nm激光将电子基态CCl2激励到激发态A 1B1(0,4,0)振动能级上,通过检测激发态CCl2时间分辨荧光信号,测得室温下CCl2(A 1B1)被烷烃类分子猝灭的实验结果,用我们提出的三能级模型分析处理实验数据,获得CCl2(A 1B1)态和CCl2(a 3B1)态的碰撞猝灭速率常数kA和ka值.     

CCl2自由基被无机小分子猝灭的速率常数

对CCl4/Ar混合气体脉冲直流高压放电产生CCl2自由基,用541.52 nm激光将电子基态CCl2激励到A 1B1(0,4,0)振动能级上. 通过检测激发态CCl2时间分辨荧光信号,测得室温下CCl2(A 1B1和a 3B1)被O2、N2、NO、 CO2、 CS2、H2O、SO2,和SF6分子猝灭的实验结果.用我们提出的三能级模型分析处理实验数据,获得了CCl2(A 1B1)态和CCl2(a 3B1)态的碰撞猝灭速率常数kA和ka值.     

Refractive index modulation in polymeric photochromic films

Photochromic properties of methylacrylate monomers and polymers containing azobenzene groups with heterocyclic sulfonamide functionalities viz sulfisomidyne (4-amino-N-[2,6-dimethylpyrimidyn-4-yl]benzenesulfonamide) and sulfamethoxazole (4-amino-N-[5-methylisoxazol-3-yl]benzenesulfonamide) substituents were investigated. On illumination with light the azobenzene group underwent trans-cis isomerisation, which was manifested by a drop in the absorbance of the maximum absorption peak at ca. 450 nm and by decrease in refractive index. Quantum chemical calculations showed significant differences in UV-VIS spectra, dipole moments, polarizability and refractive index between both cis and trans form of the chromophoric monomers. The illumination of spin-coated polymer films during ellipsometry measurements resulted in a change in refractive index within the range of 0.014 to 0.025. The dynamics of growth and decay of refractive index changes, was described by biexponential functions approach.     

Phototransformations of 5-aminolevulinic acid-induced protoporphyrin IX in vitro: a spectroscopic study

Human adenocarcinoma cells of the line WiDr were incubated with 5-aminolevulinic acid to induce protoporphyrin IX (PpIX) and then exposed to laser light of wavelength 635 nm. The PpIX fluorescence decreased with increasing exposure. The decay rate was slightly dependent on the initial PpIX concentration. The PpIX fluorescence was halved by a fluence of about 40 J/cm2. Several fluorescing photoproducts were formed. The main one, supposedly the chlorine-type photoprotoporphyrin (Ppp), had a fluorescence excitation spectrum stretching out to about 680 nm with a maximum at around 668 nm. The formation kinetics of this product was dependent on the initial PpIX concentration. Moreover, it was selectively bleached by exposure to light at 670 nm. A photoproduct with an emission maximum at 652 nm, different from Ppp, remained after this exposure. Traces of a photoproduct(s) with fluorescence emission slightly blue-shifted compared with that of PpIX, supposedly water-soluble porphyrins, were also detected after light exposure.     

Combinational Biosynthesis of a Fluorescent Cyanobacterial Holo-α-Phycocyanin in <Emphasis Type="Italic">Escherichia coli</Emphasis> by Using One Expression Vector

The phycobiliproteins (PBSs) are large pigment proteins found in certain algae that play a central role in harvesting light energy for photosynthesis. Phycocyanin (PC) is one type of PBSs that gains increasing attention owing to its various biological and pharmacological properties. In this paper, an expression vector containing five essential genes in charge of biosynthesis of cyanobacterial C-phycocyanin (C-PC) holo-α subunit (holo-CpcA) was successfully constructed resulting in over-expression of a fluorescent holo-CpcA in E. coli BL21. The vector harbored two cassettes: one cassette carried genes hox1 and pcyA required for conversion of heme to phycocyanobilin (PCB), and the other cassette carried cpcA encoding CpcA along with cpcE and cpcF both of which were necessary and sufficient for the correct addition of PCB to CpcA. The vector system contained a His-tag for protein purification. The purified protein showed correct molecular weight on SDS-PAGE gel and emitted orange fluorescence by UV excitation. The maximum peak of absorbance spectrum was at 623 nm, and the maximum peak of fluorescence emission and excitation were at 648 and 633 nm, respectively, which were similar to those of native C-PC. This study provides an efficient method for large-scale production of the fluorescent holo-CpcA in biotechnological applications. Guan and Qin contributed equally to this study.     

EVIDENCE FOR THE FORMATION OF A CHLOROPHYLL a/ZEAXANTHIN COMPLEX IN LECITHIN LIPOSOMES FROM FLUORESCENCE DECAY KINETICS

The interaction of Chi a with zeaxanthin (Zea), which is an analogue of lutein, has been studied in soya bean lecithin liposomes using the fluorescence of Chi as monitor. The fluorescence emission spectrum at 4.2 K of Chi a showed characteristic changes in the presence of Zea: the emission maximum shifted from 688 nm to 680 nm, and a peak at 731 nm appeared. The fluorescence decay kinetics of Chi a alone could be described by the sum of two exponential components (T₁,≅0.8 ns, T₂≅2.5 ns). In the presence of Zea a component with a long lifetime, T≅5 ns, appeared with a large relative amplitude (40%). This indicated the formation of a Chl a /Zea complex, in which Chl a /Chl a interaction is negligible, presumably because of strong interaction between Chl a and Zea. The fluorescence anisotropy decay kinetics supported the hypothesis of the formation of a large Chl a containing complex in the presence of Zea. A rotational correlation time, φ≅14 ns at 4°C and φ≅21 ns at 30°C, was found, which is distinctly larger than for samples containing Chl a only. We interpret these results as further evidence for a strong interaction between Chl a and Zea in the hydrophobic environment of the lecithin liposomes. This interaction may also occur in the Chl-proteins of the Chi alb light-harvesting complex of plant photosynthesis.     

FLUORESCENCE DECAY KINETICS and CHARACTERISTICS OF BIMOLECULAR EXCITON ANNIHILATION IN CHLOROPLASTS

Abstract— The decay profiles of the fluorescence of dark-adapted spinach chloroplasts (0^A°C) excited with single 30 ps 532 nm laser pulses of varying intensities were measured with a low-jitter streak camera system. By comparing the decay profiles of the fluorescence at low and high laser fluences, i.e. in the absence and presence, respectively, of dynamic bimolecular exciton-exciton annihilation effects, the duration of such dynamic annihilation events can be estimated. A simple model suggests that the influence of bimolecular annihilation events on the fluorescence decay kinetics should disappear within a time interval corresponding to the low intensity, unimolecular lifetime of the exciton population which is subject to exciton-exciton annihilation. The low intensity fluorescence decay profiles are characterized by three to four lifetimes (Reviewed by A. R. Holzwarth, Photochem. Photobiol. 43,707–725, 1986); it is shown here that only the shortest fluorescence components are subject to exciton annihilation, since the kinetics of the fluorescence decay are influenced by annihilations only within the initial 150–200 ps time interval after the excitation pulse. The amplitudes (but not the decay kinetics) of the longer-lived fluorescence components are decreased at high levels of laser pulse excitations, suggesting that these components are derived from the shorter-lived fluorescence decay components. The implications of these results are*discussed within the contexts of current models of the fluorescence in chloroplasts.     

Reduction of the IgE-binding ability and maintenance of immunogenicity of gamma-irradiated Dermatophagoides farinae

House dust mites, Dermatophagoides farinae (DF) and Dermatophagoides pteronyssinus, are major allergens in the most common indoor allergen and are important risk factor for asthma. The modified antigen has been studied to treat allergic disorder. This study was carried out to measure possibility of modified allergen using gamma irradiation to treat allergy such as asthma. DF solutions (2 mg/ml) as target allergen were irradiated with Co-60 at 50 and 100 kGy. Conformational alternation of irradiated DF was observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Levels of anti-irradiated DF mouse IgGs (sub-isotypes) against intact DF were measured similar to that of anti-intact DF IgGs. The binding abilities of house dust mite-allergic patients’ IgE were reduced depending on radiation dose, and irradiation could inhibit the binding ability of patients’ IgE more than 40%. This study has shown that the binding ability of IgE was reduced by conformational alteration by irradiation and the irradiated DF had epitopes capable to induce immunogeniciy.     

Temperature effects on femtosecond transient absorption kinetics of semiconducting single-walled carbon nanotubes

We report femtosecond transient absorption kinetics measured for selected semiconducting single-walled carbon nanotubes at different temperatures between 77 and 290 K. The nanotubes are embedded in a thin polymethylmethacrylate film, and the dominance of individual species enabled to probe selectively the kinetics associated with two desired tube types, the (6,5) and (7,5) tubes. A strikingly similar temperature dependence is found between the maximum change in the amplitude of transient absorption kinetics, the overall decay time and steady-state fluorescence emission intensity. The simplest explanation for our data is that the temperature dependence of the fluorescence intensity and the exciton decay kinetics are dominated by nonradiative decay and that the radiative decay rate is weakly temperature dependent.     

Fluorescence and photodissociation of rhodamine 575 cations in a quadrupole ion trap

The fluorescence and photodissociation of rhodamine 575 cations confined to a quadrupole ion trap are observed during laser irradiation at 488 nm. The kinetics of photodissociation is measured by time-dependent mass spectra and time-dependent fluorescence. The rhodamine ion signal and fluorescence decay are studied as functions of buffer gas pressure, laser fluence, and irradiation time. The decay rates of the ions in the mass spectra agree with decay rates of the fluorescence. Some of the fragment ions also fluoresce and further dissociate. The photodissociation rate is found to depend on the incident laser fluence and buffer gas pressure. The implications of rapid absorption/fluorescence cycling for photodissociation of dye-labeled biomolecular ions under continuous irradiation are discussed.     

ENERGY TRANSFER AMONG THE CHROMOPHORES OF C-PHYCOCYANIN FROM Anabaena variabilis USING STEADY STATE AND TIME-RESOLVED FLUORESCENCE SPECTROSCOPY

Abstract We have investigated the model of energy transfer between sensitizing (s) and fluorescing (f) chromophores for the αβ monomer and for the separated α and β subunits of C-phycocyanin from Anabaena variabilis using fluorescence emission spectroscopy, fluorescence excitation polarization, and picosecond-resolved fluorescence decay kinetics. The fluorescence emission maximum occurs at 640 nm for all samples. The fluorescence excitation polarization is constant ( P = 0.40) across the absorption hand for the α subunit, but it increases across the absorption band towards longer wavelength for the β subunit and the αβ monomer. The fluorescence decay kinetics exhibit two exponential lifetimes of 1.3-1.5 ns and 340-500 ps for the αβ monomer and for the α and β subunit preparations.
We attribute the change in polarization across the absorption band to energy transfer among the three chromophores in the αβ monomer and among the two chromophores in the separated β subunit. The constant, relatively high polarization in the separated a subunit, having only one chromophore, is consistent with the absence of both energy transfer and chromophore rotation. The concentration of the α subunit did not affect the decay kinetics, suggesting that the short lifetime component does not arise from aggregation of the α subunits. The biexponential decay kinetics of the α subunit cannot be explained using the sensitizing-fluorescing model. The possibility of conformational interactions is under investigation.     

Fluorescence studies of lens epithelial cells and their constituents

Steady-state and time-resolved fluorescence measurements have been made of human and rabbit lens epithelial cells and their total soluble protein. Excitation at 350 nm results in broad fluorescence spectra peaking at 450 nm and stretching into the visible past 650 nm. The fluorescence excitation spectra peak around 350 nm. We assign the species responsible for this absorption and fluorescence as NADPH. Because the absorption of near-UV light (300-400 nm) is responsible for cell damage and death, we postulate that excited states of NADPH are implicated in the mechanisms of cell damage. Preirradiation with 355 nm light leads to a loss of NADPH fluorescence but with no change in decay kinetics. Possible mechanisms for cell damage are explored.     

The Number of Accumulated Photons and the Quality of Stimulated Emission Depletion Lifetime Images

Time binning is used to increase the number of photon counts in the peak channel of stimulated emission depletion fluorescence lifetime decay curves to determine how it affects the resulting lifetime image. The fluorescence lifetime of the fluorophore, Alexa Fluor 594 phalloidin, bound to F‐actin is probed in cultured S2 cells at a spatial resolution of ~40 nm. This corresponds to a 10‐fold smaller probe volume compared to confocal imaging, and a reduced number of photons contributing to the signal. Pixel‐by‐pixel fluorescence lifetime measurements and error analysis show that an average of 40 ± 30 photon counts in the peak channel with a signal‐to‐noise ratio of 20 is enough to calculate a reliable fluorescence lifetime from a single exponential fluorescence decay. No heterogeneity in the actin cytoskeleton in different regions of the cultured cells was measured in the 40–400 nm spatial regime.     

Effects of slight variations in nutrient loadings on pore plugging in soil columns

The high nutrient concentrations that would exist near the nutrient injection well during the application of cometabolicin situ bioremediation may lead to the development of significant quantities of biomass at this point in the subsurface. This biomass can decrease the porosity of the soil to such an extent that nutrient injection is no longer possible. In this work, experiments were conducted using a porous media biofilm reactor, operated under constant substrate loading conditions, such that the pressure drop across the reactor was allowed to increase to maintain a constant volumetric flow rate through the reactor. Results suggest that biomass production, and hence biofilm thickness, near the injection feed port is highly sensitive to substrate loading. In addition, these variations in biofilm thickness produce dramatic differences in the pressure drop that is attained across the reactor. Use of the Kozeny-Carman equation can be used to predict that once a critical depth has been exceeded, the pressure drop across the bed will increase exponentially within biofilm depth. This result means that pressure is not a reliable indicator of the onset of pore plugging.