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Single fluorophores and single-pair fluorescence resonance energy transfer are studied with a new confocal fluorescence microscope that allows, the wavelength and emission time of each detected photon to be simultaneously measured with single molecule sensitivity. The initial results from a simultaneous time- and wavelength-resolved single molecule confocal microscope system are presented.

Photoelectron Photoion Coincidence (PEPICO) spectroscopy holds the promise of a universal, isomer-selective, and sensitive analytical technique for time-resolved quantitative analysis of bimolecular chemical reactions. Unfortunately, its low dynamic range of ~103 has largely precluded its use for this purpose, where a dynamic range of at least 105 is generally required. This limitation is due to the false coincidence background common to all coincidence experiments, especially at high count rates. Electron/ion pairs emanating from separate ionization events but arriving within the ion time of flight (TOF) range of interest constitute the false coincidence background. Although this background has uniform intensity at every m/z value, the Poisson scatter in the false coincidence background obscures small signals. In this paper, temporal ion deflection coupled with a position-sensitive ion detector enables suppression of the false coincidence background, increasing the dynamic range in the PEPICO TOF mass spectrum by 2-3 orders of magnitude. The ions experience a time-dependent electric deflection field at a well-defined fraction of their time of flight. This deflection defines an m/zand ionization-time dependent ion impact position for true coincidences, whereas false coincidences appear randomly outside this region and can be efficiently suppressed. When cold argon clusters are ionized, false coincidence suppression allows us to observe species up to Ar9+, whereas Ar9+ is the largest observable cluster under traditional operation. This advance provides mass-selected photoelectron spectra for fast, high sensitivity quantitative analysis of reacting systems. [ABSTRACT FROM AUTHOR]

The origin of the resonant structures in the spectrum of the predissociative part of the A state in the NO–Ar van der Waals cluster has been investigated. We have employed direct excitation to the predissociative part of the NO–Ar A state followed by rotational state selective ionization of the NO fragment. Velocity map imaging of the NO ion yields the recoil energy of the rotational state-selected fragment. A substantial contribution of rotational hotbands to the resonant structures is observed. Our data indicate that a centrifugal barrier as the origin of these resonances can be ruled out. We hypothesize that after the NO–Ar cluster is excited to the A state sufficient mixing within the rotating cluster takes place as it changes geometry from being T shaped in the NO(X)–Ar state to linear in the NO(A)–Ar state. This mixing allows the low energy and high angular momentum (J≈4.5) tumbling motion of the initially populated hotbands in the ground state NO(X)–Ar complex to be converted into NO(A,n=2) spinning rotation in the A state of the complex. The electronically excited spinning complex falls apart adiabatically producing rotationally excited NO(A,n=2) at the energetic threshold. This interpretation indicates that the resonances can be attributed to some type of vibrational Feshbach resonance. The appearance energy for the formation of NO(A,n=0)+Ar is found to be 44294.3±1.4 cm-1. [ABSTRACT FROM AUTHOR]

Lipid membranes composed of an iminodiacetic acid functionalized lipid, DSIDA, in a POPC matrix has displayed switchable properties by [Cu.sup.2+] recognition to assemble microdomains that has acted as high affinity sets for His-tagged proteins. The microdomains have shown an order of magnitude enhanced affinity for the proteins when compared to homogeneously functionalized POPC membranes with [Ni.sup.2+]-DOIDA, while a rapid release and restoration of the original membrane is accomplished with micromolar concentrations of EDTA.

The potential uses of multichannel joint statistical analysis of multispectral time series in a single quantum dot are presented. Intensity change points of the donor and acceptor channels are determined.

Multiparameter fluorescence spectroscopy and model-free statistical analysis methods are used for analyzing the complex quantum dot (QD)-dye hybrid Forster resonance energy transfer (FRET) systems at the single-particle level and also excited-state dynamics in multichromophore systems. The analysis has suggested an approach for using QD-dye hybrid FRET-based sensors in single particle applications.

Ultrafast photodynamics in a 1,3,5-hexatriene are studied using femtosecond time-resolved photoionization and photoelectron spectroscopy. The trans and cis isomers have distinctly different dynamics following excitation at the S2 origin near 250 nm. An intermediate, presumably the S1 state, is observed for both trans and cis isomers with lifetimes of 270 fs and 730 fs, respectively. Time-delayed photoelectron spectra of cis-hexatriene determine a 300 fs time scale for vibrational energy redistribution within the intermediate S1 state. © 1996 American Institute of Physics. [ABSTRACT FROM AUTHOR]

Results are presented from femtosecond time-resolved coherent Raman experiments in which we excite and monitor vibrational coherence in gas-phase samples of benzene and 1,3,5-hexatriene. Different physical mechanisms for coherence decay are seen in these two molecules. In benzene, where the Raman polarizability is largely isotropic, the Q branch of the vibrational Raman spectrum is the primary feature excited. Molecules in different rotational states have different Q-branch transition frequencies due to vibration–rotation interaction. Thus, the macroscopic polarization that is observed in these experiments decays because it has many frequency components from molecules in different rotational states, and these frequency components go out of phase with each other. In 1,3,5-hexatriene, the Raman excitation produces molecules in a coherent superposition of rotational states, through (O, P, R, and S branch) transitions that are strong due to the large anisotropy of the Raman polarizability. The coherent superposition of rotational states corresponds to initially spatially oriented, vibrationally excited, molecules that are freely rotating. The rotation of molecules away from the initial orientation is primarily responsible for the coherence decay in this case. These experiments produce large (∼10% efficiency) Raman shifted signals with modest excitation pulse energies (10 μJ) demonstrating the feasibility of this approach for a variety of gas phase studies. © 1995 American Institute of Physics. [ABSTRACT FROM AUTHOR]

In this paper we describe a method, based on the laser-induced grating technique, for studying the spectroscopy of vibrational overtone-excited gas-phase water. Two phase-coherent visible laser beams whose frequencies are in the range of the third overtone of the OH stretch in water are crossed in the gas-phase sample. As the wavelength of these excitation beams is scanned through individual rovibrational OH overtone transitions, vibrational energy is deposited into the water in a spatially sinusoidal pattern. A fixed-frequency 266 nm probe laser beam is diffracted from the resultant transmission diffraction grating in water. We show that under collision-free conditions, probe laser diffraction is observed from the initially excited grating, which is a necessary condition for using this technique to study the absorption spectroscopy of the vibrationally excited molecules. Under multiple collision conditions, a probe laser wavelength-independent refractive index grating is formed within the bulk sample. In addition, we observe temporal oscillations in the grating diffraction efficiency arising from excitation of standing acoustic waves. © 1995 American Institute of Physics. [ABSTRACT FROM AUTHOR]

A new excited-state spectroscopic method is reported.It is a two-color laser-induced grating tecnique for detecting optical transitions of rovibronically excited molecules in the gas phase. (AIP) [ABSTRACT FROM AUTHOR]

Curved membranes are an essential feature of dynamic cellular structures, including endocytic pits, filopodia protrusions and most organelles. It has been proposed that specialized proteins induce curvature by binding to membranes through two primary mechanisms: membrane scaffolding by curved proteins or complexes; and insertion of wedge-like amphipathic helices into the membrane. Recent computational studies have raised questions about the efficiency of the helix-insertion mechanism, predicting that proteins must cover nearly 100% of the membrane surface to generate high curvature, an improbable physiological situation. Thus, at present, we lack a sufficient physical explanation of how protein attachment bends membranes efficiently. On the basis of studies of epsin1 and AP180, proteins involved in clathrin-mediated endocytosis, we propose a third general mechanism for bending fluid cellular membranes: protein-protein crowding. By correlating membrane tubulation with measurements of protein densities on membrane surfaces, we demonstrate that lateral pressure generated by collisions between bound proteins drives bending. Whether proteins attach by inserting a helix or by binding lipid heads with an engineered tag, protein coverage above ~20% is sufficient to bend membranes. Consistent with this crowding mechanism, we find that even proteins unrelated to membrane curvature, such as green fluorescent protein (GFP), can bend membranes when sufficiently concentrated. These findings demonstrate a highly efficient mechanism by which the crowded protein environment on the surface of cellular membranes can contribute to membrane shape change. [ABSTRACT FROM AUTHOR]

We have developed a multiplexed time- and photon-energy–resolved photoionization mass spectrometer for the study of the kinetics and isomeric product branching of gas phase, neutral chemical reactions. The instrument utilizes a side-sampled flow tube reactor, continuously tunable synchrotron radiation for photoionization, a multimass double-focusing mass spectrometer with 100% duty cycle, and a time- and position-sensitive detector for single ion counting. This approach enables multiplexed, universal detection of molecules with high sensitivity and selectivity. In addition to measurement of rate coefficients as a function of temperature and pressure, different structural isomers can be distinguished based on their photoionization efficiency curves, providing a more detailed probe of reaction mechanisms. The multiplexed three-dimensional data structure (intensity as a function of molecular mass, reaction time, and photoionization energy) provides insights that might not be available in serial acquisition, as well as additional constraints on data interpretation. [ABSTRACT FROM AUTHOR]

Directing the orientation of molecular assemblies is a key step toward creating complex hierarchical structures that yield higher order functional materials. Here, we demonstrate the directed orientation of functionalized lipid domains and protein–membrane assemblies, using an electric field. [ABSTRACT FROM AUTHOR]

The article shows the capabilities of combining multiparameter spectroscopy and model-free statistical method in the study of complex quantum dot (QD)-dye Förster resonance energy transfer (FRET) systems at the single-particle level. According to the authors, multiparameter measurements that combine spectral and temporal information provide correlated signals to identify the origin of measured fluorescence changes.

Endocytic recycling of transmembrane proteins is essential to cell signaling, ligand uptake, protein traffic and degradation. The intracellular domains of many transmembrane proteins are ubiquitylated, which promotes their internalization by clathrin-mediated endocytosis. How might this enhanced internalization impact endocytic uptake of transmembrane proteins that lack ubiquitylation? Recent work demonstrates that diverse transmembrane proteins compete for space within highly crowded endocytic structures, suggesting that enhanced internalization of one group of transmembrane proteins may come at the expense of other groups. Here we show that preferential internalization of poly-ubiquitylated transmembrane proteins results in reduced endocytosis of mono-ubiquitylated and non-ubiquitylated proteins. Using a combination of live-cell imaging and ligand uptake assays, we confirmed that increased ubiquitylation correlates with increased internalization by clathrin-coated vesicles. Further, poly-ubiquitylated receptors significantly outcompeted their mono-ubiquitylated and non-ubiquitylated counterparts for localization to endocytic sites and uptake of extracellular ligands. These findings demonstrate the inherent interdependence of transmembrane protein recycling, suggesting that clathrin-coated vesicles act as selective filters, prioritizing highly ubiquitylated transmembrane proteins for uptake while leaving proteins with little or no ubiquitylation behind. Given that poly-ubiquitylation is thought to signal protein aging and damage, our findings suggest a mechanism for selective internalization of high priority cargo proteins, with simultaneously exclusion and protection of functional proteins that lack poly-ubiquitylation., Significance: Ubiquitylation is essential for maintaining cellular homeostasis by regulating transmembrane protein trafficking, degradation, and signaling. Traditionally, poly-ubiquitylation has been viewed primarily as a signal for protein degradation, while mono-ubiquitylation is considered sufficient to trigger endocytosis. However, our work reveals a previously unrecognized role for poly-ubiquitylation in clathrin-mediated endocytosis, demonstrating that poly-ubiquitylated transmembrane proteins outcompete their non-ubiquitylated counterparts for incorporation into clathrin-coated vesicles, thereby establishing a competitive framework for endocytic cargo sorting. This mechanism reveals a selective sorting mechanism driven by the extent of ubiquitylation, which could regulate the removal of damaged proteins while protecting functional proteins at the plasma membrane., Competing Interests: Competing interests The authors declare no competing interests.