Modulation of H-NS transcriptional silencing by magnesium.

The bacterial histone-like protein H-NS silences AT-rich DNA, binding DNA as 'stiffened' filaments or 'bridged' intrastrand loops. The switch between these modes has been suggested to depend on the concentration of divalent cations, in particular Mg2+, with elevated Mg2+ concentrations associated with a transition to bridging. Here we demonstrate that the observed binding mode is a function of the local concentration of H-NS and its cognate binding sites, as well as the affinity of the interactions between them. Mg2+ does not control a binary switch between these two modes but rather modulates the affinity of this interaction, inhibiting the DNA-binding and silencing activity of H-NS in a continuous linear fashion. The direct relationship between conditions that favor stiffening and transcriptional silencing activity suggests that although both modes can occur in the cell, stiffening is the predominant mode of binding at silenced genes.

Strong Dependence of Quantum-Dot Delayed Luminescence on Excitation Pulse Width.

Delayed luminescence involving charge-carrier trapping and detrapping has recently been identified as a widespread and possibly universal phenomenon in colloidal quantum dots. Its near-power-law decay suggests a relationship with blinking. Here, using colloidal CuInS2 and CdSe quantum dots as model systems, we show that short (nanosecond) excitation pulses yield less delayed luminescence intensity and faster delayed luminescence decay than observed with long (millisecond) square-wave excitation pulses. Increasing the excitation power also affects the delayed luminescence intensity, but the delayed luminescence decay kinetics appear much less sensitive to excitation power than to excitation pulse width. An idealized four-state kinetic model reproduces the major experimental trends and highlights the very slow approach to steady state during photoexcitation, stemming from extremely slow detrapping of the metastable charge-separated state responsible for delayed luminescence. The impacts of these findings on proposed relationships between delayed luminescence and blinking are discussed.

Photoluminescence Blinking and Reversible Electron Trapping in Copper-Doped CdSe Nanocrystals.

Single-particle photoluminescence blinking is observed in the copper-centered deep-trap luminescence of copper-doped CdSe (Cu(+):CdSe) nanocrystals. Blinking dynamics for Cu(+):CdSe and undoped CdSe nanocrystals are analyzed to identify the effect of Cu(+), which selectively traps photogenerated holes. Analysis of the blinking data reveals that the Cu(+):CdSe and CdSe nanocrystal "off"-state dynamics are statistically identical, but the Cu(+):CdSe nanocrystal "on" state is shorter lived. Additionally, a new and pronounced temperature-dependent delayed luminescence is observed in the Cu(+):CdSe nanocrystals that persists long beyond the radiative lifetime of the luminescent excited state. This delayed luminescence is analogous to the well-known donor-acceptor pair luminescence of bulk copper-doped phosphors and is interpreted as revealing metastable charge-separated excited states formed by reversible electron trapping at the nanocrystal surfaces. A mechanistic link between this delayed luminescence and the luminescence blinking is proposed. Collectively, these data suggest that electron (rather than hole) trapping/detrapping is responsible for photoluminescence intermittency in these nanocrystals.

Imaging the effects of annealing on the polymorphic phases of poly(vinylidene fluoride).

The effect of annealing on the phase transformation and the dielectric properties of poly(vinylidene fluoride) (PVDF) is explored using quasi-single molecule (quasi-SM) microscopy. The solvatochromic properties of nile red (NR) are employed to measure the spatial distribution of the local dielectric constant (ε) in ∼30 µm thick PVDF films before and after annealing at 90 °C. The results presented here demonstrate that nonannealed films exhibit much larger ε distributions, both in terms of magnitude and distribution, when compared to annealed films. The polymorphic phase of PVDF before and after annealing is also confirmed using X-ray diffraction. Nonannealed films are found to be in the γ-phase with annealing promoting the transition to primarily ß-phase. Combining these results, we conclude that the decrease in ε with annealing time is due to the phase transformation from γ- to ß-phase. Using quasi-SM imaging techniques one can readily visualize the ε environments present within different polymorphic phases of PVDF.

Evolutionary expansion of a regulatory network by counter-silencing.

Horizontal gene transfer plays a major role in bacterial evolution. Successful acquisition of new genes requires their incorporation into existing regulatory networks. This study compares the regulation of conserved genes in the PhoPQ regulon of Salmonella enterica serovar Typhimurium with that of PhoPQ-regulated horizontally acquired genes, which are silenced by the histone-like protein H-NS. We demonstrate that PhoP upregulates conserved and horizontally acquired genes by distinct mechanisms. Conserved genes are regulated by classical PhoP-mediated activation and are invariant in promoter architecture, whereas horizontally acquired genes exhibit variable promoter architecture and are regulated by PhoP-mediated counter-silencing. Biochemical analyses show that a horizontally acquired promoter adopts different structures in the silenced and counter-silenced states, implicating the remodelling of the H-NS nucleoprotein filament and the subsequent restoration of open-complex formation as the central mechanism of counter-silencing. Our results indicate that counter-silencing is favoured in the regulatory integration of newly acquired genes because it is able to accommodate multiple promoter architectures.

Dielectric dependence of single-molecule photoluminescence intermittency: nile red in poly(vinylidene fluoride).

The dependence of single-molecule photoluminescence intermittency (PI) or "blinking" on the local dielectric constant (ε) is examined for nile red (NR) in thin films of poly(vinylidene fluoride) (PVDF). In previous studies, variation of the local dielectric constant was accomplished by studying luminophores in chemically and structurally different hosts. In contrast, the NR/PVDF guest-host pair allows for the investigation of PI as a function of ε while keeping the chemical composition of both the luminophore and host unchanged. The solvatochromic properties of NR are used to measure the local ε, while fluctuations in NR emission intensity over time provide a measure of the PI. PVDF is an ideal host for this study because it provides submicron-sized dielectric domains that vary from nonpolar (ε ≈ 2) to very polar (ε ≈ 70). The results presented here demonstrate that the local dielectric environment can have a pronounced effect on PI. We find that the NR emissive events increase 5-fold with an increase in ε from 2.2 to 74. A complex dependence on ε is also observed for NR nonemissive event durations, initially increasing as ε increases from 2.2 to 3.4 but decreasing in duration with further increase in ε. The variation in emissive event durations with ε is reproduced using a photoinduced electron-transfer model involving electron transfer from NR to PVDF. In addition, an increase in NR photostability with an increase in ε is observed, suggesting that the dielectric environment plays an important role in defining the photostability of NR in PVDF.

Ultrafast charge transfer in nickel phthalocyanine probed by femtosecond Raman-induced Kerr effect spectroscopy.

The recently developed technique of femtosecond stimulated Raman spectroscopy, and its variant, femtosecond Raman-induced Kerr effect spectroscopy (FRIKES), offer access to ultrafast excited-state dynamics via structurally specific vibrational spectra. We have used FRIKES to study the photoexcitation dynamics of nickel(II) phthalocyanine with eight butoxy substituents, NiPc(OBu)8. NiPc(OBu)8 is reported to have a relatively long-lived ligand-to-metal charge-transfer (LMCT) state, an essential characteristic for efficient electron transfer in photocatalysis. Following photoexcitation, vibrational transitions in the FRIKES spectra, assignable to phthalocyanine ring modes, evolve on the femtosecond to picosecond time scales. Correlation of ring core size with the frequency of the ν10 (asymmetric C-N stretching) mode confirms the identity of the LMCT state, which has a ∼500 ps lifetime, as well as that of a precursor d-d excited state. An even earlier (∼0.2 ps) transient is observed and tentatively assigned to a higher-lying Jahn-Teller-active LMCT state. This study illustrates the power of FRIKES spectroscopy in elucidating ultrafast molecular dynamics.

Measuring the spatial distribution of dielectric constants in polymers through quasi-single molecule microscopy.

The variation in dielectric constant is measured for thin films of poly(methyl methacrylate) (PMMA) and poly(vinylidene fluoride) (PVDF) using confocal fluorescence microscopy. Spatial variation in the local dielectric constant of the polymer films on the ~250 nm length scale is measured using the solvochromatic emission from incorporated nile red (NR) at "quasi-single molecule" (10(-7) M) and true single molecule (SM) concentrations (10(-9) M). Correlation of the NR fluorescence wavelength maximum with dielectric constant is used to transform images of NR's emission maxima to spatial variation in local dielectric constant. We demonstrate that the distributions of dielectric environments measured in the quasi- and true SM approaches are equivalent; however, the enhanced signal rates present in the quasi-SM approach result in this technique being more efficient. In addition, the quasi-SM technique reports directly on the continuous spatial variation in dielectric constant, information that is difficult to obtain in true SM studies. With regards to the polymers of interest, the results presented here demonstrate that a limited distribution of dielectric environments is present in PMMA; however, a broad distribution of environments exists in PVDF consistent with this polymer existing as a distribution of structural phases.

Proton transfer and photoluminescence intermittency of single emitters in dyed crystals.

The role of proton transfer in the photoluminescence intermittency (PI) of single molecules of violamine R (VR) overgrown in potassium acid phthalate (KAP) crystals is evaluated in comparisons of protonated (KAP) and deuterated (DKAP) mixed crystals between 23 and 60 °C. The PI is analyzed by the construction of cumulative distribution functions that are statistically compared. We find that the on- and off-interval duration distributions change with isotopic substitution consistent with proton transfer contributing to the PI of VR. The on- and off-interval duration distributions have distinct temperature dependencies consistent with different mechanisms for dark state production and decay. Additional evidence for proton-transfer is provided by distributions of single molecule emission-energy maxima that reflect emission from protonated and deprotonated VR. A mechanism for the PI of KAP is presented, where the dark state is assigned to formation of the colorless, leuco form of VR, formed by proton transfer from VR to the KAP lattice, and decay of the dark state involves ring-opening promoted by proton transfer from KAP to VR. The distributed kinetics for dark-state production and decay are modeled using a log-normal distribution for the PI data in preference to a power-law previously assumed. A discussion of the log-normal distribution with regards to PI and proton transfer is presented.

Photoluminescence intermittency from single quantum dots to organic molecules: emerging themes.

Recent experimental and theoretical studies of photoluminescence intermittency (PI) or "blinking" exhibited by single core/shell quantum dots and single organic luminophores are reviewed. For quantum dots, a discussion of early models describing the origin of PI in these materials and recent challenges to these models are presented. For organic luminophores the role of electron transfer, proton transfer and other photophysical processes in PI are discussed. Finally, new experimental and data analysis methods are outlined that promise to be instrumental in future discoveries regarding the origin(s) of PI exhibited by single emitters.

Time-resolved infrared absorption studies of the solvent-dependent photochemistry of ClNO.

The photochemistry of nitrosyl chloride (ClNO) dissolved in cyclohexane is investigated using ultrafast time-resolved infrared (TRIR) spectroscopy. Following 266 nm photolysis, the photochemistry is measured by following changes in optical density at frequencies spanning the N═O stretch fundamental transition. A photoinduced depletion in optical density is observed consistent with the depletion of ground-state ClNO. The depletion in optical density remains constant out to ∼50 ps demonstrating that ClNO photodissociation is not followed by recombination of the Cl and NO photofragments. In addition, no evidence for the formation of the ClON photoisomer is observed. These results stand in contrast to previous studies in acetonitrile where ClNO photolysis is followed by geminate recombination of Cl and NO, and by the production of ClON. These differences in ClNO photochemistry are proposed to arise from the population of different excited-states caused by solvent dependence of the ground-state potential energy surface minimum along the Cl-N stretch coordinate. Solvent-dependent vibrational relaxation and differences in strength of the solvent cage are also proposed to contribute to the solvent-dependent photochemistry. Finally, these results are placed in the context of recent models of ClNO photochemistry and role of this compound in tropospheric ozone production.

Dielectric dependence of the first molecular hyperpolarizability for electro-optic chromophores.

Experimental and computational studies of the solvent dependence of the first molecular hyperpolarizability (ß) for two donor-bridge-acceptor chromophores (CLD-1 and YLD156) are presented. Hyper-Rayleigh scattering (HRS) measurements are performed with 1907 nm excitation in a series of solvents with dielectric constants ranging from ~2 (toluene) to ~36 (acetonitrile). For both chromophores an approximately 2-fold increase in ß is observed by HRS over this range of dielectric constants. Computational studies employing a polarized continuum model to represent the solvent are capable of reproducing this experimental result. The experimental and computational results are compared to the predictions of the widely employed two-state model (TSM) for ß. Surprisingly, for the chromophores studied here the TSM predicts that ß should decrease with increasing dielectric constant over the range investigated. The results presented here demonstrate that the TSM provides neither a quantitative nor qualitative description of the solvent dependence of ß for CLD-1 and YLD156. The enhancement of ß with increased dielectric constant suggests that modification of the dielectric surrounding the chromophore is one path by which the performance of nonlinear optical devices employing these chromophores may be significantly enhanced.

Two mechanisms for fluorescence intermittency of single violamine R molecules.

The environment and temperature-dependent photoluminescence (PL) intermittency or "blinking" demonstrated by single violamine R (VR) molecules is investigated in two environments: poly(vinyl alcohol) (PVOH) and single crystals of potassium acid phthalate (KAP). In addition, temperatures ranging from 23 °C to 85 °C are studied, spanning the glass-transition temperature of PVOH (T(g) = 72 °C). The PL intermittency exhibited by VR is analyzed using probability histograms of emissive and non-emissive periods. In both PVOH and KAP, these histograms are best fit by a power law, consistent with the kinetics for dark state production and decay being dispersed as observed in previous studies. However, these systems have different temperature dependences, signifying two different blinking mechanisms for VR. In PVOH, the on- and off-event probability histograms do not vary with temperature, consistent with electron transfer via tunneling between VR and the polymer. In KAP the same histograms are temperature dependent, and show that blinking slows down at higher temperatures. This result is inconsistent with an electron-transfer process being responsible for blinking. Instead, a non-adiabatic proton-transfer between VR and KAP is presented as a model consistent with this temperature dependence. In summary, the results presented here demonstrate that for a given luminophore, the photochemical processes responsible for PL intermittency can change with environment.

Measuring order in contact-poled organic electrooptic materials with variable-angle polarization-referenced absorption spectroscopy (VAPRAS).

Organic nonlinear electrooptical (ONLO) chromophores must be acentrically ordered for the ONLO material to have electrooptic (EO) activity. The magnitude of the order is characterized by the acentric order parameter, , where ß is the major Euler angle between the main axis of the chromophore and the poling field which imposes the acentric order. The acentric order parameter, which is difficult to measure directly, is related to the centrosymmetric order parameter, defined as = ½(3-1), through the underlying statistical distribution. We have developed a method to determine centrosymmetric order of the ONLO chromophores when the order is low (i.e., < 0.1). We have extended the method (begun by Graf et al. J. Appl. Phys. 1994, 75, 3335.) based on the absorption of light to determine the centrosymmetric order parameter induced by a poling field on a thin film sample of ONLO material. We find that the order parameters, analyzed by two different methods, are similar and also consistent with theoretical estimates from modeling of the system using coarse-grained Monte Carlo statistical mechanical methods.

Reduced dimensionality in organic electro-optic materials: theory and defined order.

Identification of electronic intermolecular electrostatic interactions that can significantly enhance poling-induced order is important to the advancement of the field of organic electro-optics. Here, we demonstrate an example of such improvement achieved through exploitation of the interaction of coumarin pendant groups in chromophore-containing macromolecules. Acentric order enhancement is explained in terms of lattice-symmetry effects, where constraint of orientational degrees of freedom alters the relationship between centrosymmetric and acentric order. We demonstrate both experimentally and theoretically that lattice dimensionality can be defined using the relationship between centrosymmetric order and acentric order. Experimentally: Acentric order is determined by attenuated total reflection measurement of electro-optic activity coupled with hyper-Rayleigh scattering measurement of molecular first hyperpolarizability, and centrosymmetric order is determined by the variable angle polarization referenced absorption spectroscopy method. Theoretically: Order is determined from statistical mechanical models that predict the properties of soft condensed matter.

Unraveling the dispersed kinetics of dichlorofluorescein in potassium hydrogen phthalate crystals.

The connection between photoluminescence (PL) intermittency and excited-state kinetics is explored for 2',7'-dichlorofluorescein (DCF) isolated in crystals of potassium acid phthalate (KAP) using time-tagged, time-resolved, time-correlated single-photon counting (T3R-TCSPC). In this technique, PL intermittency or "blinking" is measured in conjunction with the time of photon arrival relative to photoexcitation, allowing for the correlation of emissive intensities and excited-state decay kinetics of single molecules. The blinking trace is parsed into emissive and nonemissive segments using change-point-detection analysis, and the duration of these segments are used to quantify PL intermittency. The results presented here demonstrate that two populations of DCF exist in KAP, with one population demonstrating single-exponential excited state decay over the course of the blinking trace, and the other demonstrating stretched-exponential decay. Molecules demonstrating single-exponential decay also demonstrate modest intensity variation in the blinking trace. Correlation of the emission intensity and excited-state lifetimes demonstrates that for these molecules spectral diffusion is largely responsible for the evolution in emission intensity. In contrast, molecules demonstrating nonexponential excited-state decay vary in emission intensity. Correlation of the emissive intensities with the excited-state lifetimes demonstrates that these molecules undergo changes in both radiative and nonradiative decay rate constants as well as spectral diffusion. These observations suggest that DCF exists in two environments in KAP differentiated by the propensity for proton-transfer with the surrounding KAP matrix. The results presented here provide further insight into the origin of PL intermittency demonstrated by DCF in KAP and related systems.

Modeling the optical behavior of complex organic media: from molecules to materials.

For the past three decades, a full understanding of the electro-optic (EO) effect in amorphous organic media has remained elusive. Calculating a bulk material property from fundamental molecular properties, intermolecular electrostatic forces, and field-induced net acentric dipolar order has proven to be very challenging. Moreover, there has been a gap between ab initio quantum-mechanical (QM) predictions of molecular properties and their experimental verification at the level of bulk materials and devices. This report unifies QM-based estimates of molecular properties with the statistical mechanical interpretation of the order in solid phases of electric-field-poled, amorphous, organic dipolar chromophore-containing materials. By combining interdependent statistical and quantum mechanical methods, bulk material EO properties are predicted. Dipolar order in bulk, amorphous phases of EO materials can be understood in terms of simple coarse-grained force field models when the dielectric properties of the media are taken into account. Parameters used in the statistical mechanical modeling are not adjusted from the QM-based values, yet the agreement with the experimentally determined electro-optic coefficient is excellent.

Proton-transfer mechanism for dispersed decay kinetics of single molecules isolated in potassium hydrogen phthalate.

The excited-state decay kinetics of single 2',7'-dichlorofluorescein (DCF) molecules oriented and overgrown within crystals of potassium acid phthalate (KAP) are reported. Time-correlated single-photon counting measurements (TCSPC) of 56 DCF molecules in KAP reveal that single-exponential decay is exhibited by roughly half of the molecules. The remainder demonstrates complex excited-state decay kinetics that are well fit by a stretched exponential function consistent with dispersed kinetics. Histograms of single-molecule luminescence energies revealed environmental fluctuations and distinct chemical species. The TCSPC results are compared to Monte Carlo simulations employing a first-passage model for excited-state decay. Agreement between experiment and theory, on both bulk and single-molecule levels, suggests that a subset of the DCF molecules in KAP experience fluctuations in the surrounding environment that modify the energy barrier to proton transfer leading to dispersed kinetics.

Triplet states of the nonlinear optical chromophore DCM in single crystals of potassium hydrogen phthalate and their relationship to single-molecule dark states.

Single-molecule dark states are often attributed to photoexcited triplets with scant evidence of the participation of paramagnetic molecules. The photodynamics of blinking single molecules of 4-dicyanomethylene-2-methyl-6-p-dimethylaminostyryl-4H-pyran (DCM) in crystals of potassium hydrogen phthalate (KAP) were compared with the lifetimes of DCM triplet states, likewise in KAP, whose zero-field splitting (ZFS) tensors were fully characterized by time-resolved electron paramagnetic resonance (TR-EPR) spectroscopy. Luminescent mixed crystals of KAP were grown from solutions containing 10(-4) -10(-9) M DCM, a model optically nonlinear chromophore. The luminescent dye was localized in the {111} crystalline growth sectors. The photoexcited triplets states of DCM in the heavily dyed (10(-4) M) crystals were analyzed by TR-EPR spectroscopy. The photoexcited singlet states of DCM in lightly dyed crystals (10(-9) M) were analyzed by single-molecule microscopy. Large blue shifts in the absorption and emission spectra of DCM in KAP were interpreted as a consequence of protonation at the dimethylamino nitrogen atom, an assignment supported by calculations of the zero-field splitting (ZFS) tensors of molecules in their triplet states. Experimental ZFS tensors with eigenvalues comparable to those of the computed tensors were determined from the angular dependence of the EPR spectra of DCMH(+) triplets within KAP single crystals with respect to the applied magnetic field. Data from individual growth sectors failed to show magnetically equivalent site occupancies, evidence of the kinetic ordering during growth. The intermittent fluorescence of individual chromophores was analyzed. The distributions of on(off) times were characterized by distributed rates fit to power laws. The lifetime of the triplet states was analyzed from the time decay of the EPR signals between 100 and 165 K. The data were well fit with a single time constant for the signal decay, a result wholly inconsistent with the blinking of single molecules with off times commonly of tens of seconds. Triplet decay was extrapolated to approximately 25 micros at room temperature. Therefore, the assumption that single-molecule dark states originate with triplet excited states is not sustainable for single DCM molecules in KAP.

Femtosecond pump-probe studies of actinic-wavelength dependence in aqueous chlorine dioxide photochemistry.

The actinic or photolysis-wavelength dependence of aqueous chlorine dioxide (OClO) photochemistry is investigated using femtosecond pump-probe spectroscopy. Following photoexcitation at 310, 335, and 410 nm the photoinduced evolution in optical density is measured from the UV to the near IR. Analysis of the optical-density evolution illustrates that the quantum yield for atomic chlorine production (Phi(Cl)) increases with actinic energy, with Phi(Cl)=0.16+/-0.02 for 410 nm excitation and increasing to 0.25+/-0.01 and 0.54+/-0.10 for 335 and 310 nm excitations, respectively. Consistent with previous studies, the production of Cl occurs through two channels, with one channel corresponding to prompt (<5 ps) Cl formation and the other corresponding to the thermal decomposition of ClOO formed by OClO photoisomerization. The partitioning between Cl production channels is dependent on actinic energy, with prompt Cl production enhanced with an increase in actinic energy. Limited evidence is found for enhanced ClO production with an increase in actinic energy. Stimulated emission and excited-state absorption features associated with OClO populating the optically prepared (2)A(2) surface decrease with an increase in actinic energy suggesting that the excited-state decay dynamics are also actinic energy dependent. The studies presented here provide detailed information on the actinic-wavelength dependence of OClO photochemistry in aqueous solution.