Exciton quenching by oxidized chlorophyll Z across the two adjacent monomers in a photosystem II core dimer.

This study aimed to clarify (1) which pigment in a photosystem II (PSII) core complex is responsible for the 695-nm emission at 77 K and (2) the molecular basis for the oxidation-induced fluorescence quenching in PSII. Picosecond time-resolved fluorescence dynamics was compared between the dimeric and monomeric PSII with and without addition of an oxidant. The results indicated that the excitation-energy flow to the 695-nm-emitting chlorophyll (Chl) at 36 K and 77 K was hindered upon monomerization, clearly demonstrating significant exciton migration from the Chls on one monomer to the 695-nm-emitting pigment on the adjacent monomer. Oxidation of the redox-active Chl, which is named ChlZ caused almost equal quenching of the 684-nm and 695-nm emission bands in the dimer, and lower quenching of the 695-nm band in the monomer. These results suggested two possible scenarios responsible for the 695-nm emission band: (A) Chl11-13 pair and the oxidized ChlZD1 work as the 695-nm emitting Chl and the quenching site, respectively, and (B) Chl29 and the oxidized ChlZD2 work as the 695-nm emitting Chl and the quenching site, respectively.

Red shift in the spectrum of a chlorophyll species is essential for the drought-induced dissipation of excess light energy in a poikilohydric moss, Bryum argenteum.

Some mosses are extremely tolerant of drought stress. Their high drought tolerance relies on their ability to effectively dissipate absorbed light energy to heat under dry conditions. The energy dissipation mechanism in a drought-tolerant moss, Bryum argenteum, has been investigated using low-temperature picosecond time-resolved fluorescence spectroscopy. The results are compared between moss thalli samples harvested in Antarctica and in Japan. Both samples show almost the same quenching properties, suggesting an identical drought tolerance mechanism for the same species with two completely different habitats. A global target analysis was applied to a large set of data on the fluorescence-quenching dynamics for the 430-nm (chlorophyll-a selective) and 460-nm (chlorophyll-b and carotenoid selective) excitations in the temperature region from 5 to 77 K. This analysis strongly suggested that the quencher is formed in the major peripheral antenna of photosystem II, whose emission spectrum is significantly broadened and red-shifted in its quenched form. Two emission components at around 717 and 725 nm were assigned to photosystem I (PS I). The former component at around 717 nm is mildly quenched and probably bound to the PS I core complex, while the latter at around 725 nm is probably bound to the light-harvesting complex. The dehydration treatment caused a blue shift of the PS I emission peak via reduction of the exciton energy flow to the pigment responsible for the 725 nm band.

Fluorescence property of photosystem II protein complexes bound to a gold nanoparticle.

Development of an efficient photo-anode system for water oxidation is key to the success of artificial photosynthesis. We previously assembled photosystem II (PSII) proteins, which are an efficient natural photocatalyst for water oxidation, on a gold nanoparticle (GNP) to prepare a PSII-GNP conjugate as an anode system in a light-driven water-splitting nano-device (Noji et al., J. Phys. Chem. Lett., 2011, 2, 2448-2452). In the current study, we characterized the fluorescence property of the PSII-GNP conjugate by static and time-resolved fluorescence measurements, and compared with that of free PSII proteins. It was shown that in a static fluorescence spectrum measured at 77 K, the amplitude of a major peak at 683 nm was significantly reduced and a red shoulder at 693 nm disappeared in PSII-GNP. Time-resolved fluorescence measurements showed that picosecond components at 683 nm decayed faster by factors of 1.4-2.1 in PSII-GNP than in free PSII, explaining the observed quenching of the major fluorescence peak. In addition, a nanosecond-decay component arising from a 'red chlorophyll' at 693 nm was lost in time-resolved fluorescence of PSII-GNP, probably due to a structural perturbation of this chlorophyll by interaction with GNP. Consistently with these fluorescence properties, degradation of PSII during strong-light illumination was two times slower in PSII-GNP than in free PSII. The enhanced durability of PSII is an advantageous property of the PSII-GNP conjugate in the development of an artificial photosynthesis device.

Development of a novel cryogenic microscope with numerical aperture of 0.9 and its application to photosynthesis research.

A novel cryogenic optical-microscope system was developed in which the objective lens is set inside of the cryostat adiabatic vacuum space. Being isolated from the sample when it was cooled, the objective lens was maintained at room temperature during the cryogenic measurement. Therefore, the authors were able to use a color-aberration corrected objective lens with a numerical aperture of 0.9. The lens is equipped with an air vent for compatibility to the vacuum. The theoretically expected spatial resolutions of 0.39µm along the lateral direction and 1.3µm along the axial direction were achieved by the developed system. The system was applied to the observations of non-uniform distributions of the photosystems in the cells of a green alga, Chlamydomonas reinhardtii, at 94K. Gaussian decomposition analysis of the fluorescence spectra at all the pixels clearly demonstrated a non-uniform distribution of the two photosystems, as reflected in the variable ratios of the fluorescence intensities assigned to photosystem II and to those assigned to photosystem I. The system was also applied to the fluorescence spectroscopy of single isolated photosystem I complexes at 90K. The fluorescence, assigned to be emitted from a single photosystem I trimer, showed an intermittent fluctuation called blinking, which is typical for a fluorescence signal from a single molecule. The vibronic fluorescence bands at around 790nm were observed for single photosystem I trimers, suggesting that the color aberration is not serious up to the 800nm spectral region.

Observation of unusual molecular diffusion behaviour below the lower critical solution temperature of water/2-butoxyethanol mixtures by using fluorescence correlation spectroscopy.

The effect of solute affinity on solute diffusion in binary liquids well below the lower critical solution temperature (LCST) was studied by using fluorescence correlation spectroscopy. We measured the hydrodynamic radii of a hydrophobic and an amphiphilic fluorescent dye under systematic variation of the relative molar fractions of water/2-butoxyethanol and, for comparison, of water/methanol mixtures, which do not show phase separation. We found that the apparent hydrodynamic radius of the hydrophobic dye almost doubled in water/2-butoxyethanol, whereas it remained largely unchanged for the amphiphilic dye and in water/methanol mixtures. Our results indicate that the translational diffusion of solutes is influenced by transient local solution structures, even at temperatures well below the LCST. We conclude that, even far below LCST, different solutes can experience different environments in binary liquid mixtures depending on both the solute and solvent properties, all of which impact their reactivity.

Picosecond dynamics of hydrogen bond rearrangements during phase separation of a triethylamine and water mixture.

The earliest stages of phase separation in a liquid triethylamine (TEA)-water mixture were observed using a picosecond IR laser pulse to produce a temperature jump and ultrafast Raman spectroscopy. Raman spectral changes in the water OH stretching region showed that the temperature rise induced by IR pulses equilibrated within a few tens of picoseconds. Amplitude changes in the TEA CH-stretching region of difference Raman spectra consisted of an initial faster and a subsequent slower process. The faster process within 100 ps is attributed to hydrogen bond weakening caused by the temperature rise. The slower process attributed to phase separation was observed for several nanoseconds, showing the number of hydrogen bond between TEA and water gradually decreased with time. The kinetics of hydrogen bond scission during phase separation indicated a linear growth of the phase-separated component, as observed previously on the nanosecond time scale, rather than the more usual exponential growth. A peak blueshift was observed in the difference Raman spectra during phase separation. This shift implies that hydrogen bond scission of TEA-water aggregates involving very few water molecules took place in the initial stage of phase separation (up to 2 ns), and then was followed by the breaking of TEA-water pairs surrounded by water molecules. This effect may be a result from spatial inhomogeneities associated with the phase separation process: aggregates or clusters existing naturally in solution even below the lower critical soluble temperature.

Additive-free size-controlled synthesis of gold square nanoplates using photochemical reaction in dynamic phase-separating media.

Ultrafast phase separation of water and 2-butoxyethanol mixture was induced by nanosecond IR laser pulse irradiation. After a certain delay time, a UV laser pulse was introduced to induce photoreduction of aurate ions, which led to the formation of gold nanoparticles in dynamic phase-separating media. The structure and size of the nanoparticles varied depending on the delay time between the IR and UV pulses. For a delay time of 5 and 6 µs, gold square plates having edge lengths of 150 and 100 nm were selectively obtained, respectively. With a delay time of 3 µs, on the other hand, the size of the square plates varied widely from 100 nm to a few micrometers. The size of the gold square plates was also varied by varying the total irradiation time of the IR and UV pulses. The size distribution of the square plates obtained under different conditions suggests that the growth process of the square plates was affected by the size of the nanophases during phase separation. Electron diffraction patterns of the synthesized square plates showed that the square plates were highly crystalline with a Au(100) surface. These results showed that the nanophases formed during laser-induced phase separation can provide detergent-free reaction fields for size-controlled nanomaterial synthesis.

Li@C60 thin films: characterization and nonlinear optical properties.

Organic materials have attracted considerable attention in nonlinear optical (NLO) applications as they have several advantages over inorganic materials, including high NLO response, and fast response time as well as low-cost and easy fabrication. Lithium-containing C60 (Li@C60) is promising for NLO over other organic materials because of its strong NLO response proven by theoretical and experimental studies. However, the low purity of Li@C60 has been a bottleneck for applications in the fields of solar cells, electronics and optics. In 2010, highly purified Li@C60 was finally obtained, encouraging further studies. In this study, we demonstrate a facile method to fabricate thin films of Li@C60 and their strong NLO potential for high harmonic generation by showing its comparatively strong emission of degenerate-six-wave mixing, a fifth-order NLO effect.

Transactivation activity of LBP-1 proteins and their dimerization in living cells.

LBP-1 proteins form dimers and act as transcription factors that activate a number of genes related to cell growth and differentiation. LBP-1a and LBP-1c are localized in the cytoplasm when transiently expressed in cultured cells, but translocated into the nucleus after forming heterodimers with LBP-1b, which is a splicing variant of LBP-1a with an intrinsic nuclear localization signal (NLS). Here, we report that LBP-1b showed potent transactivation activity, and that forcibly expressed LBP-1a and LBP-1c in the nucleus essentially exhibited very little or no transactivation activity. Mutations in the NLS that abolished the NLS activity of LBP-1b also abrogated the transactivation activity. We have found that LBP-1 proteins contain a putative sterile alpha motif domain indispensable for their dimerization capability in the C-terminal region. To demonstrate whether homo- and heterodimers composed of LBP-1a and/or LBP-1c are generated in the nucleus, we applied the FLIM-based fluorescence resonance energy transfer imaging technique to living cells. It revealed that dimers composed of LBP-1a and LBP-1c were re-formed probably by a partner-exchange of LBP-1b-containing heterodimers.

Photo-controlled phase separation and mixing of a mixture of water and 2-butoxyethanol caused by photochromic isomerisation of spiropyran.

Photo induced phase separation of a mixture of water and 2-butoxyethanol, in which spiropyran was dissolved as a photoresponsive molecule, was investigated. It was found that the phase separation temperature of the merocyanine (MC) form solution was higher than that of the spiropyrane (SP) form; therefore phase separation was induced by visible light irradiation to the solution which caused the photoisomerization from the MC form to the SP form. The system also exhibits reversible photoinduced phase mixing by irradiation of UV light. The photo-chemical phase separation was also induced by the nanosecond laser pulse irradiation and the dynamics of the phase domain growth were studied.

Data on synthesis and characterization of new p-nitro stilbene Schiff bases derivatives as an electrochemical DNA potential spacer.

The structural investigation of synthesized compounds can be carried out by various spectroscopic techniques. It is an important prospect in order to elucidate the structure of the desired products before being further utilized. The preparation of new p-nitro stilbene Schiff base derivatives as an electrochemical DNA potential spacer was synthesized using (E)-4-(4-nitrostyryl)aniline from Heck reaction with aldehydes in ethanolic solution. The data presented here in this article contains FTIR, UV-Vis and 1H and 13C NMR of (E)-4-(4-nitrostyryl)aniline and nitrostyryl aniline derivatives.

Time-resolved brewster angle microscopy for photochemical and photothermal studies on thin-films and monolayers.

Transient events in thin films and interfaces have been studied using the technique of time resolved pump-probe nanosecond Brewster angle microscopy. For p-polarized light there is a minimum reflectivity at the Brewster angle. When the interface is viewed with light that is both incident and reflected at the Brewster angle the resulting image is dark. Subsequent small changes is refractive index will then cause an increase in the reflectivity in affected regions providing high contrast images of an altered interface with a dark background level. This is the basis of Brewster angle microscopy. In the present work two synchronized nanosecond pulsed lasers were used in the pump-probe configuration in order to induce changes at an air-liquid interface and to monitor the resulting morphology changes over a range of time delays from nanosecond to milliseconds after laser-excitation. This method can be used to observe morphological changes in phase altering thin-films and molecular monolayers. Further it can be used to obtain information about transient photochemistry even in optically thin materials and nano-films. In the current work the method is used to monitor laser induced processes in phase separating binary liquid mixtures as well as in monolayers of photo-responsive amphiphilic molecules derived from spiropyran on water. The two systems are quite different but provide valuable comparisons.

Real-time observation of X-ray-induced intramolecular and interatomic electronic decay in CH2I2.

The increasing availability of X-ray free-electron lasers (XFELs) has catalyzed the development of single-object structural determination and of structural dynamics tracking in real-time. Disentangling the molecular-level reactions triggered by the interaction with an XFEL pulse is a fundamental step towards developing such applications. Here we report real-time observations of XFEL-induced electronic decay via short-lived transient electronic states in the diiodomethane molecule, using a femtosecond near-infrared probe laser. We determine the lifetimes of the transient states populated during the XFEL-induced Auger cascades and find that multiply charged iodine ions are issued from short-lived (∼20 fs) transient states, whereas the singly charged ones originate from significantly longer-lived states (∼100 fs). We identify the mechanisms behind these different time scales: contrary to the short-lived transient states which relax by molecular Auger decay, the long-lived ones decay by an interatomic Coulombic decay between two iodine atoms, during the molecular fragmentation.

Chirp effect in hard X-ray generation from liquid target when irradiated by femtosecond pulses.

The chirp effect on a X-ray emission intensity from a CsCl aqueous solution jet irradiated by femtosecond pulses was systematically studied. The p-polarized chirped pulses were more efficient as compared with the shortest pulses determined by the spectral bandwidth. The negatively-chirped pulses of approximately 240 fs duration produced up to 10 times larger X-ray intensity as compared with the transform-limited 160 fs pulses. The angular dependence of X-ray generation can be explained by the resonant absorption. Numerical simulations of electron density evolution due to the avalanche and multi-photon absorption supports qualitatively well the experimental observations.

What silicon nanocluster is most likely formed in etching experiments? Theoretical DFT study.

Density functional calculations at the B3LYP/6-31G* level were performed for Si nanoclusters of ca. 1 nm in size. The structural, energetic, electronic as well as the estimated absorption spectra by the time-dependent DFT (TDDFT) calculations using varied functionals and basis sets for the representative cluster models are all in favor of the formation of most probable Si35H36 nanocluster in recent electrochemical etching experiments. The nanostructure has a complete H-termination at the borderline regions and lacks from the presence of any defects like surface Si-Si dimer units formed via self-healing of dangling Si-Si bonds or from any relatively short H...H contacts.

Time-Resolved Structured Illumination Microscopy for Phase Separation Dynamics of Water and 2-Butoxyethanol Mixtures: Interpretation of "Early Stage" Involving Micelle-Like Structures.

Phase separation dynamics of a water/2-butoxyethanol (2BE) mixture was studied with newly developed time-resolved structured illumination microscopy (SIM). Interestingly, an employed hydrophobic fluorescent probe for SIM showed spectral shifts up to 500 ns after a laser-induced temperature jump, which suggests 2BE micellar-like aggregates become more hydrophobic at the initial stage of phase separation. This hydrophobic environment in 2BE aggregates, probably due to the ejection of water molecules, continued up to at least 10 µs. Time-resolved SIM and previously reported light scattering data clearly showed that the size of a periodic structure remained constant (ca. 300 nm) from 3 to 10 µs, and then the growth of periodic structures having the self-similarity started. We think that the former and the latter processes correspond to "early stage" (concentration growth) and "late stage" (size growth), respectively, in phase separation dynamics. Here we suggest that, in the early stage, the entity to bear 2BE phase be water-poor 2BE aggregates, and the number density of these aggregates would simply increase in time.

Ternary complex formation of pVHL, elongin B and elongin C visualized in living cells by a fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy technique.

The tumor suppressor von Hippel-Lindau (VHL) gene product forms a complex with elongin B and elongin C, and acts as a recognition subunit of a ubiquitin E3 ligase. Interactions between components in the complex were investigated in living cells by fluorescence resonance energy transfer (FRET)-fluorescence lifetime imaging microscopy (FLIM). Elongin B-cerulean or cerulean-elongin B was coexpressed with elongin C-citrine or citrine-elongin C in CHO-K1 cells. FRET signals were examined by measuring a change in the fluorescence lifetime of donors and by monitoring a corresponding fluorescence rise of acceptors. Clear FRET signals between elongin B and elongin C were observed in all combinations, except for the combination of elongin B-cerulean and citrine-elongin C. Although similar experiments to examine interaction between pVHL30 and elongin C linked to cerulean or citrine were performed, FRET signals were rarely observed among all the combinations. However, the signal was greatly increased by coexpression of elongin B. These results, together with results of coimmunoprecipitation experiment using pVHL, elongin C and elongin B, suggest that a conformational change of elongin C and/or pVHL was induced by binding of elongin B. The conformational change of elongin C was investigated by measuring changes in the intramolecular FRET signal of elongin C linked to cerulean and citrine at its N- and C-terminus, respectively. A strong FRET signal was observed in the absence of elongin B, and this signal was modestly increased by coexpression of elongin B, demonstrating that a conformation change of elongin C was induced by the binding of elongin B.

Conformational changes in inhibitory PAS domain protein associated with binding of HIF-1α and Bcl-xL in living cells.

Inhibitory PAS domain protein (IPAS) is a dual function protein acting as a transcriptional repressor and as a pro-apoptotic protein. Simultaneous dual-color single-molecule imaging of EGFP-IPAS coexpressed with Mit-TagRFP-T in living HeLa cells revealed that fraction of EGFP-IPAS was arrested in the nucleus and on mitochondria. Transiently expressed Cerulean-IPAS in HEK293T cells was present in nuclear speckles when coexpressed with Citrine-HIF-1α or Citrine-HLF. Fluorescence lifetime imaging microscopy (FLIM) analysis of Citrine-IPAS-Cerulean in living CHO-K1 cells clarified the presence of intramolecular FRET. Reduced lifetimes of the donor were partially restored by coexpression of HIF-1α or Bcl-xL, binding proteins of IPAS in the nucleus and mitochondria, respectively. This alteration in lifetimes demonstrates that conformational changes occurred in IPAS by their binding.

Experimentally determined growth exponents during the late stage of spinodal demixing in binary liquid mixtures.

Spinodal demixing was initiated in two systems, with critical and off-critical compositions, using nanosecond pulsed laser-induced temperature jumps (T-jumps) of various magnitude. In this way, deep quenches could be imposed on the systems. One system was the simple triethylamine (TEA)/water mixture and the other was the ionic mixture of 2-butoxyethanol (2BE)/water/KCl. The demixing process was followed using the technique of nanosecond time-resolved microscopic shadowgraphy. The growth of the evolving phase-separated domains followed a simple power law with respect to time in every case. For a given composition, the magnitude of the T-jump had little effect on the growth exponent, however the composition was found to influence the rate of domain growth. At off-critical mole fractions of 0.2 with respect to TEA, the domains grew according to the following expression: L(t)=t(0.70) (where L(t)= the domain size) whereas at the critical TEA mole fraction of 0.08 the domains grew as L(t)=t(0.52). 2BE/water/KCl mixtures quenched at the just off-critical composition of fraction with respect to 2BE evolved as L(t)=t(0.63). These results will be compared to theoretical models and simulations and discussed in terms of estimated Reynolds numbers as well as the consumption and conversion of the available surface energy that fuels the demixing process.

Structure-Based Modeling of Fluorescence Kinetics of Photosystem II: Relation between Its Dimeric Form and Photoregulation.

A photosystem II-enriched membrane (PSII-em) consists of the PSII core complex (PSII-cc) which is surrounded by peripheral antenna complexes. PSII-cc consists of two core antenna (CP43 and CP47) and the reaction center (RC) complex. Time-resolved fluorescence spectra of a PSII-em were measured at 77 K. The data were globally analyzed with a new compartment model, which has a minimum number of compartments and is consistent with the structure of PSII-cc. The reliability of the model was investigated by fitting the data of different experimental conditions. From the analysis, the energy-transfer time constants from the peripheral antenna to CP47 and CP43 were estimated to be 20 and 35 ps, respectively. With an exponential time constant of 320 ps, the excitation energy was estimated to accumulate in the reddest chlorophyll (Red Chl), giving a 692 nm fluorescence peak. The excited state on the Red Chl was confirmed to be quenched upon the addition of an oxidant, as reported previously. The calculations based on the Förster theory predicted that the excitation energy on Chl29 is quenched by ChlZD1(+), which is a redox active but not involved in the electron-transfer chain, located in the D1 subunit of RC, in the other monomer with an exponential time constant of 75 ps. This quenching pathway is consistent with our structure-based simulation of PSII-cc, which assigned Chl29 as the Red Chl. On the other hand, the alternative interpretation assigning Chl26 as the Red Chl was not excluded. The excited Chl26 was predicted to be quenched by another redox active ChlZD2(+) in the D2 subunit of RC in the same monomer unit with an exponential time constant of 88 ps.