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.

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.

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.

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.

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.

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.

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 TRIR studies of ClNO photochemistry in solution: evidence for photoisomerization and geminate recombination.

The photochemistry of nitrosyl chloride (ClNO) in the solution phase is investigated using Fourier transform infrared (FTIR) and ultrafast time-resolved infrared (TRIR) spectroscopies. The NO-stretch fundamental transition for ClNO dissolved in cyclohexane, carbon tetrachloride, chloroform, dichloromethane, and acetonitrile is measured, with the frequency and line width of this transition demonstrating a strong dependence on solvent polarity. Following the photolysis of ClNO dissolved in acetonitrile at 266 nm, the subsequent optical-density evolution across the entire width of the NO-stretch fundamental is measured. Analysis of the optical-density evolution demonstrates that geminate recombination of the primary photofragments resulting in the reformation of ground state ClNO occurs with a quantum yield of 0.54 +/- 0.06. In addition, an increase in optical density is observed at 1860 cm(-1) that is assigned to the NO-stretch fundamental transition of the photoisomer, ClON, having a formation quantum yield of 0.07 +/- 0.02. This work represents the first definitive observation of ClNO photoisomerization in solution. Finally, essentially no evidence is observed for significant vibrational excitation of the NO fragment following photodissociation, in marked contrast to the behavior observed in the gas phase. An environment-dependent dissociation scheme is proposed in which the interplay between solvent polarity and the location of the ground state potential-energy-surface minimum along the Cl-N coordinate provides for the optical preparation of different excited states thereby affecting the extent of NO vibrational excitation following photolysis.

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.

Rational enhancement of second-order nonlinearity: bis-(4-methoxyphenyl)hetero-aryl-amino donor-based chromophores: design, synthesis, and electrooptic activity.

Two new highly hyperpolarizable chromophores, based on N,N- bis-(4-methoxyphenyl) aryl-amino donors and phenyl-trifluoromethyl-tricyanofuran (CF3-Ph-TCF) acceptor linked together via pi-conjugation through 2,5-divinylenethienyl moieties as the bridge, have been designed and synthesized successfully for the first time. The aryl moieties on the donor side of the chromophore molecules were varied as to be thiophene and 1-n-hexylpyrrole. The linear and nonlinear optical (NLO) properties of all compounds were evaluated in addition to recording relevant thermal and electrochemical data. The properties of the two new molecules were comparatively studied. These results are critically analyzed along with two other compounds, reported earlier from our laboratories and our collaborator's, that contain (i) aliphatic chain-bearing aniline and (ii) dianisylaniline as donors, keeping the bridge (2,5-divinylenethienyl-), and the acceptor (CF3-Ph-TCF), constant. Trends in theoretically (density functional theory, DFT) predicted, zero-frequency gas-phase hyperpolarizability [beta(0;0,0)] values are shown to be consistent with the trends in beta HRS(-2omega;omega,omega), as measured by Hyper-Rayleigh Scattering (HRS), when corrected to zero-frequency using the two-level model (TLM) approximation. Similarly, trends in poling efficiency data (r33/E(p)) and wavelength dispersion measured by reflection ellipsometry (using a Teng-Man apparatus) and attenuated total reflection (ATR) are found to fit the TLM and DFT predictions. A 3-fold enhancement in bulk nonlinearity (r33) is realized as the donor subunits are changed from alkylaniline to dianisylaminopyrrole donors. The results of these studies provide insight into the complicated effects on molecular hyperpolarizability of substituting heteroaromatic subunits into the donor group structures. These studies also demonstrate that, when frequency dependence and electric-field-induced ordering behavior are correctly accounted for, ab initio DFT generated beta(0;0,0) is effective as a predictor of changes in r33 behavior based on chromophore structure modification. Thus DFT can provide valuable insight into the electronic structure origin of complex optical phenomena in organic media.

Spectrally tunable uncaging of biological stimuli from nanocapsules.

This communication describes the first uncaging of stimuli in the far red, wavelengths that have much less of an adverse affect on cellular systems, via photolysis of photosensitized nanocapsules.

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.

Single-molecule microscopy studies of electric-field poling in chromophore-polymer composite materials.

One strategy for increasing the efficiency of organic electrooptic devices based on chromophore-polymer composite materials is to improve chromophore ordering. In these materials, ordering is induced through the interaction of the chromophore dipole moment with an external electric field, applied at temperatures near the Tg of the polymer host, a process referred to as "poling". To provide insight into the molecular details of the poling process under conditions representative of device construction, the rotational dynamics of single 4-dicyano-methylene-2-methyl-6-(p-(dimethylamino)styryl)-4H-pyran (DCM) molecules in poly(methyl acrylate) at T = Tg + 11 degrees C in the presence and absence of an electric field are investigated using single-molecule confocal fluorescence microscopy. Single-molecule rotational dynamics are monitored through the time evolution of the fluorescence anisotropy. The anisotropy correlation function demonstrates nonexponential decay, with beta values derived from fits using the Kohlrausch-Williams-Watts law ranging from 0.7 to 1 with beta(KWW) = 0.83. This observation is consistent with previous studies of molecular rotation dynamics in polymer melts and reflects the dynamical heterogeneity provided by the polymer host. The rotational dynamics of DCM are weakly perturbed in the presence of a 50 V/microm electric field, typical of the field strength employed in device construction. The expected perturbation of the rotational dynamics is determined and found to be consistent with the alignment potential created by the electric field relative to the amount of thermal energy available. The relevance of these findings with respect to current models of the poling process is discussed. This work demonstrates the utility of polarization-sensitive single-molecule microscopy in elucidating the details of molecular reorientation during poling.

Linear and nonlinear optical properties of a macrocyclic trichromophore bundle with parallel-aligned dipole moments.

A macrocyclic trichromophore bundle 1 with parallel-aligned dipole moments has been synthesized to study the influence of aggregation and orientation of a nonlinear optical (NLO) chromophore on its optical properties. The linear and nonlinear optical properties of 1 and a single chromophore standard 2 have been studied by UV-vis absorption, fluorescence, solvatochromic spectrometry, and hyper-Rayleigh scattering (HRS). Reduced first-order hyperpolarizability beta, hypsochromic shift, enhanced solvatochromic shifts, and fluorescence quenching for individual chromophores were observed when 1 was compared with 2. Analysis of the data showed that the transition dipole moment changes only slightly when the chromophores are parallel aligned in the bundle architecture. However, the apparent hyperpolarizability of the individual chromophores decreased significantly by about 20%. The reduction in beta for the individual chromophores in 1 is largely due to the hypsochromic shift, i.e., excitation energy increase of the interband (charge-transfer) energy gap and the reduced difference between the ground-state and excited-state dipole moments. The hypsochromic shift and fluorescence quenching are consistent with exciton theory. Possible reasons for the enhanced solvatochromic shift are discussed.

Single-molecule orientations in dyed salt crystals.

We present single-molecule confocal microscopy studies of orientational distributions for luminophores isolated in potassium hydrogen phthalate (KAP) crystals. The incorporation of dye molecules that bear no size or shape similarity to the host ions is observed, demonstrating that single-molecule studies on mixed crystals need not be restricted to isomorphous host/guest pairs. Violamine R is oriented and overgrown by the fast vicinal slopes of growth hillocks within the symmetry-related {010} growth sectors and DCM deposits in the {11} growth sectors of KAP. Both mixed crystals exhibit modest absorption dichroism relative to basic pyranine-doped K(2)SO(4). The latter was studied to ensure that a range of orientations was sampled in our experiments. Average orientations determined at the single-molecule level were in close agreement to ensemble-averaged measurements for all three systems, and the chromophore orientational distributions were broader than anticipated, indicating that the crystals incorporate guest molecules in a range of orientations outside the measured ensemble average.

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.

The phase-dependent photochemical reaction dynamics of halooxides and nitrosyl halides.

Recent progress in understanding the phase-dependent reactivity of halooxides and nitrosyl halides is outlined. Halooxide reactivity is represented by the photochemistry of chlorine dioxide (OClO) and dichlorine monoxide (ClOCl). The gas phase photochemical dynamics of OClO are contrasted with the dynamics in condensed environments. The role of excited-state symmetry in defining the reaction dynamics and the observation of photoisomerization resulting in the production of ClOO are discussed. The current understanding of the excited-state reaction dynamics of ClOCl and evidence for photoisomerization of this species resulting in the production of ClClO are outlined. Finally, the photochemical reaction dynamics of the nitrosyl halide ClNO are presented. The main difference between the gas and condensed phase reaction dynamics of this species is that whereas photodissociation to form Cl and NO dominates the gas phase reaction dynamics, photoisomerization resulting in ClON production occurs to an appreciable extent in condensed environments. The observation of photoisomerization for OClO, ClOCl and ClNO suggests that this process is a general feature of the condensed phase reaction dynamics for smaller halooxides and nitrosyl halides. Finally, future areas for study in both halooxide and nitrosyl halide photoreactivity are outlined.

Vibrational analysis of isopropyl nitrate and isobutyl nitrate.

Raman and infrared spectra of isopropyl nitrate and isobutyl nitrate are reported. These spectra are used in combination with computational studies employing density functional theory at the B3-LYP/6-31G* level to assign the vibrational transitions to their corresponding normal coordinates. Similar to other alkyl nitrates, the frequency of the NO2 symmetric stretch remains relatively unchanged while the asymmetric stretch shifts to lower frequency with increasing alpha-carbon substitution. The mode assignments involving the photochemically relevant -ONO2 chromophore agree well with those from previous infrared work. Raman depolarization ratios are also presented, and provide evidence that the condensed phase, ground-state molecular structure of isobutyl nitrate is of Cs symmetry. In contrast, the minimum energy structure of isopropyl nitrate is predicted to contain a pronounced twist around the C-O bond relative to the Cs-symmetry structure that lies 2.6 kcal/mol higher in energy. Infrared intensities of isopropyl nitrate are consistent with the twisted geometry, demonstrating that this conformer is favored in solution.

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.

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.