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Intermittent Single-Molecule Interfacial Electron Transfer Dynamics  

SciTech Connect

We report on single molecule studies of photosensitized interfacial electron transfer (ET) processes in Coumarin 343 (C343)-TiO2 nanoparticle (NP) and Cresyl Violet (CV+)-TiO2 NP systems, using time-correlated single photon counting coupled with scanning confocal fluorescence microscopy. Fluorescence intensity trajectories of individual dye molecules adsorbed on a semiconductor NP surface showed fluorescence fluctuations and blinking, with time constrants distributed from sub-milliseconds to several seconds.

Biju, Vasudevan P.; Micic, Miodrag; Hu, Dehong; Lu, H. Peter



Single Molecule Electron Paramagnetic Resonance  

NASA Astrophysics Data System (ADS)

Electron paramagnetic resonance (EPR) is a powerful spectroscopic tool for studying the dynamics of biomolecular systems. EPR measurements on bulk samples using a commercial X-band spectrometer provide insight into atomic-scale structure and dynamics of ensembles of biomolecules. Separately, single molecule measurements of biomolecular systems allow researchers to capture heterogeneous behaviors that have revealed the molecular mechanisms behind many biological processes. We are merging these two powerful techniques to perform single molecule EPR. In this experiment, we selectively label double-stranded DNA molecules with nitrogen-vacancy (NV) center nanodiamonds and optically detect the magnetic resonance of the NV probe. Shifts and broadening of our EPR peaks indicate the changing position of the attached DNA relative to the applied magnetic field. Using this new technique, we have successfully measured the first EPR spectrum of a single biomolecule. By controlling the geometry of the diamond and the applied magnetic field, we will quantitatively determine the rotational and translational dynamics of single biomolecules. This research provides the foundation for an advanced single molecule magnetic resonance approach to studies of complex biomolecular systems.

Teeling-Smith, Richelle M.; Johnston-Halperin, Ezekiel; Poirier, Michael G.; Hammel, P. Chris



Identifying Mechanisms of Interfacial Dynamics Using Single-Molecule Tracking  

PubMed Central

The “soft” (i.e. non-covalent) interactions between molecules and surfaces are complex and highly-varied (e.g. hydrophobic, hydrogen bonding, ionic) often leading to heterogeneous interfacial behavior. Heterogeneity can arise either from spatial variation of the surface/interface itself or from molecular configurations (i.e. conformation, orientation, aggregation state, etc.). By observing adsorption, diffusion, and desorption of individual fluorescent molecules, single-molecule tracking can characterize these types of heterogeneous interfacial behavior in ways that are inaccessible to traditional ensemble-averaged methods. Moreover, the fluorescence intensity or emission wavelength (in resonance energy transfer experiments) can be used to simultaneously track molecular configuration and directly relate this to the resulting interfacial mobility or affinity. In this feature article, we review recent advances involving the use of single-molecule tracking to characterize heterogeneous molecule-surface interactions including: multiple modes of diffusion and desorption associated with both internal and external molecular configuration, Arrhenius activated interfacial transport, spatially dependent interactions, and many more.

Kastantin, Mark; Walder, Robert; Schwartz, Daniel K.



Single Molecule Spectroscopy of Electron Transfer  

SciTech Connect

The objectives of this research are threefold: (1) to develop methods for the study electron transfer processes at the single molecule level, (2) to develop a series of modifiable and structurally well defined molecular and nanoparticle systems suitable for detailed single molecule/particle and bulk spectroscopic investigation, (3) to relate experiment to theory in order to elucidate the dependence of electron transfer processes on molecular and electronic structure, coupling and reorganization energies. We have begun the systematic development of single molecule spectroscopy (SMS) of electron transfer and summaries of recent studies are shown. There is a tremendous need for experiments designed to probe the discrete electronic and molecular dynamic fluctuations of single molecules near electrodes and at nanoparticle surfaces. Single molecule spectroscopy (SMS) has emerged as a powerful method to measure properties of individual molecules which would normally be obscured in ensemble-averaged measurement. Fluctuations in the fluorescence time trajectories contain detailed molecular level statistical and dynamical information of the system. The full distribution of a molecular property is revealed in the stochastic fluctuations, giving information about the range of possible behaviors that lead to the ensemble average. In the case of electron transfer, this level of understanding is particularly important to the field of molecular and nanoscale electronics: from a device-design standpoint, understanding and controlling this picture of the overall range of possible behaviors will likely prove to be as important as designing ia the ideal behavior of any given molecule.

Michael Holman; Ling Zang; Ruchuan Liu; David M. Adams



Single-Molecule Spectroscopy and Dynamics at Nanoscale Interfacial Processes  

NASA Astrophysics Data System (ADS)

Surface Enhanced Raman Scattering (SERS) fluctuation observed at confined "hot spots" at nanometer scale has been studied using combined AFM-enhanced multi-channel Raman microscopy, theoretical modeling, and computational simulation. We report our laser intensity dependent studies of the nano-SERS fluctuation to demonstrate and characterize that the photoinduced and spontaneous nature of the nano-SERS fluctuation. The origin of the nano-SERS fluctuation has been attributed to the fluctuation of the interaction of the molecules with the local electromagnetic field that depends on the substrate nano-metallic structures. Finite element method (FEM) simulation of the field distribution for the tip-enhanced Raman scanning microscopy has provided quantitative characterization of the near-field enhancement. Interfacial electron transfer typically involves intrinsic inhomogeneous and complex mechanism. Single-molecular spectroscopy studies of electron-cation recombination dynamics at the dye-sensitized TiO2 semiconductor interface have begin to shed light on the molecular-level understanding of the inhomogeneous interfacial electron transfer dynamics at this system.

Lu, H. Peter



Single-molecule electron transfer reactions in nanomaterials  

SciTech Connect

Here we report the study of single molecule electron transfer dynamics by coupling fluorescence microscopy at a conventional electrochemical cell. The single-molecule fluorescence spectroelectrochemistry of cresyl violet in aqueous solution and on nanoparticle surface were studied. We observed that the single-molecule fluorescence intensity of cresyl violet is modulated synchronously with the cyclic voltammetric potential scanning. We attribute the fluorescence intensity change of single cresyl violet molecules to the electron transfer reaction driven by the electrochemical potential.

Hu, Dehong; Lei, Chenghong; Ackerman, Eric J.



Quantum interference in single molecule electronic systems  

NASA Astrophysics Data System (ADS)

We present a general analytical formula and an ab initio study of quantum interference in multibranch molecules. Ab initio calculations are used to investigate quantum interference in a benzene-1,2-dithiolate (BDT) molecule sandwiched between gold electrodes and through oligoynes of various lengths. We show that when a point charge is located in the plane of a BDT molecule and its position varied, the electrical conductance exhibits a clear interference effect, whereas when the charge approaches a BDT molecule along a line normal to the plane of the molecule and passing through the center of the phenyl ring, interference effects are negligible. In the case of oligoynes, quantum interference leads to the appearance of a critical energy Ec at which the electron transmission coefficient T(E) of chains with even or odd numbers of atoms is independent of length. To illustrate the underlying physics, we derive a general analytical formula for electron transport through multibranch structures and demonstrate the versatility of the formula by comparing it with the above ab initio simulations. We also employ the analytical formula to investigate the current inside the molecule and demonstrate that large countercurrents can occur within a ringlike molecule such as BDT, when the point charge is located in the plane of the molecule. The formula can be used to describe quantum interference and Fano resonances in structures with branches containing arbitrary elastic scattering regions connecting nodal sites.

Sparks, R. E.; García-Suárez, V. M.; Manrique, D. Zs.; Lambert, C. J.



Single-Molecule Electronic Measurements with Metal Electrodes  

ERIC Educational Resources Information Center

|A review of concepts like tunneling through a metal-molecule-metal-junction, contrast with electrochemical and optical-charge injection, strong-coupling limit, calculations of tunnel transport, electron transfer through Redox-active molecules is presented. This is followed by a discussion of experimental approaches for single-molecule

Lindsay, Stuart



Electronic control inside a molecule : towards single molecule devices  

NASA Astrophysics Data System (ADS)

The chimerical single molecule engineering has been proven to be accessible through the use of scanning tunnelling microscopy (STM) [1]. In this field, one particularly attractive area is the study of single molecules adsorbed on semiconductor surfaces. It has been recently demonstrated that a spatial fine control of the molecular dynamics is possible through the use of tunnelling current [2]. In order to improve the electronic control of a single molecule, we are currently investigating a promising system: CaF2 on Si(111). This system has been extensively studied as a model system to deposit insulator on silicon. Here we are using this system to electronically decouple the molecule from the substrate. I will present LT STM experiments on atomically thick CaF islands on Si(111). The measured electronic properties of these islands demonstrate their potential as ideal templates to study single molecules. Finally I will present some preliminary results on N-HBC [3] adsorbed on a CaF island. [1] G. Binnig and H. Rohrer, ``In touch with atoms'', Rev. Mod. Phys. 71, S324-S330 (1999) [2] M. Lastapis et al, Science, 308, 1000 (2005) [3] S.Draper et al, JACS, 126, 8694 (2004)

Lastapis, Mathieu; Fukuma, Yurie; Boland, John



Tools for Studying Electron and Spin Transport in Single Molecules  

NASA Astrophysics Data System (ADS)

Experiments in the field of single-molecule electronics are challenging in part because it can be very difficult to control and characterize the device structure. Molecules contacted by metal electrodes cannot easily be imaged by microscopy techniques. Moreover, if one attempts to characterize the device structure simply by measuring a current-voltage curve, it is easy to mistake nonlinear transport across a bare tunnel junction or a metallic short for a molecular signal. I will discuss the development of a set of experimental test structures that enable the properties of a molecular device to be tuned controllably in-situ, so that the transport mechanisms can be studied more systematically and compared with theoretical predictions. My collaborators and I are developing the means to use several different types of such experimental "knobs" in coordination: electrostatic gating to shift the energy levels in a molecule, mechanical motion to adjust the molecular configuration or the molecule-electrode coupling strength, illumination with light to promote electrons to excited states or to make and break chemical bonds, and the use of ferromagnetic electrodes to study spin-polarized transport. Our work so far has provided new insights into Kondo physics, the coupling between a molecule's electronic and mechanical degrees of freedom, and spin transport through a molecule between magnetic electrodes. Collaborators: Radek Bialczak, Alex Champagne, Luke Donev, Jonas Goldsmith, Jacob Grose, Janice Guikema, Jiwoong Park, Josh Parks, Abhay Pasupathy, Jason Petta, Sara Slater, Burak Ulgut, Alexander Soldatov, H'ector Abruña, and Paul McEuen.

Ralph, Daniel C.



Inelastic electron tunneling spectroscopy on single-molecule magnets  

NASA Astrophysics Data System (ADS)

Single molecule magnets (SMMs) containing a fixed number of transition metal ions have promise in applications requiring mono-disperse nanomagnets. A well-studied example is the polynuclear metal Mn_12 complex having the general formula Mn_12O_12(O_2CR)_16(H_2O)_4. We report on the fabrication and characterization of Al-Al_2O_3/Mn_12-Pb tunnel junctions that have been prepared by spin coating oxidized Al surfaces with a methylene chloride solution containing dissolved Mn_12 molecules and then depositing Pb counterelectrodes. Inelastic electron tunneling spectroscopy (IETS) measurements were performed as a function of temperature and magnetic field on samples that showed a well-developed superconducting Pb gap at low temperatures. We compare results on three types of samples: clean unaltered samples, samples exposed to pure methylene chloride, and samples doped with Mn_12 SMMs. Progress toward the goal of identifying the vibrational modes of the Mn12 molecules and detecting the effect of magnetic field on the large spin (S=10) ground state will be described.

Nesbitt, J. R.; Arnason, S. B.; Hebard, A. F.; Christou, G.



Nanoscopic heterogeneities in adsorption and electron transfer processes of perylene diimide dye on TiO 2 nanoparticles studied by single-molecule fluorescence spectroscopy  

Microsoft Academic Search

The interfacial electron transfer processes between a fluorescent water-soluble perylene diimide dye (WS-PDI) and TiO2 nanoparticles were investigated using single-molecule fluorescence spectroscopy. Based on the single-molecule fluorescence spectral measurements, it was suggested that the local environment and\\/or the structural conformation of single WS-PDI molecules play important roles in the efficiency of the electron injection from WS-PDI in the singlet excited

Takashi Tachikawa; Shi-Cong Cui; Sachiko Tojo; Mamoru Fujitsuka; Tetsuro Majima



Electron transfer behaviour of biological macromolecules towards the single-molecule level  

NASA Astrophysics Data System (ADS)

Redox metalloproteins immobilized on metallic surfaces in contact with aqueous biological media are important in many areas of pure and applied sciences. Redox metalloprotein films are currently being addressed by new approaches where biotechnology including modified and synthetic proteins is combined with state-of-the-art physical electrochemistry with emphasis on single-crystal, atomically planar electrode surfaces, in situ scanning tunnelling microscopy (STM) and other surface techniques. These approaches have brought bioelectrochemistry important steps forward towards the nanoscale and single-molecule levels. We discuss here these advances with reference to two specific redox metalloproteins, the blue single-copper protein Pseudomonas aeruginosa azurin and the single-haem protein Saccharomyces cerevisiae yeast cytochrome c, and a short oligonucleotide. Both proteins can be immobilized on Au(111) by chemisorption via exposed sulfur-containing residues. Voltammetric, interfacial capacitance, x-ray photoelectron spectroscopy and microcantilever sensor data, together with in situ STM with single-molecule resolution, all point to a coherent view of monolayer organization with protein electron transfer (ET) function retained. In situ STM can also address the microscopic mechanisms for electron tunnelling through the biomolecules and offers novel notions such as coherent multi-ET between the substrate and tip via the molecular redox levels. This differs in important respects from electrochemical ET at a single metal/electrolyte interface. Similar data for a short oligonucleotide immobilized on Au(111) show that oligonucleotides can be characterized with comparable detail, with novel perspectives for addressing DNA electronic conduction mechanisms and for biological screening towards the single-molecule level.

Zhang, Jingdong; Grubb, Mikala; Hansen, Allan G.; Kuznetsov, Alexander M.; Boisen, Anja; Wackerbarth, Hainer; Ulstrup, Jens



Direct electrical single-molecule detection of DNA through electron transfer induced by hybridization.  


Single-stranded DNA was utilized as a probe tip for single-molecule DNA detection. Hybridization of the DNA tip and target DNA induces electron tunneling through the resulting DNA duplex. It is demonstrated that the DNA tip allows not only genetic detection but also discovery of single-nucleotide polymorphisms at the single-molecule level. PMID:23508214

Nishino, Tomoaki; Bui, Phuc Tan



Nanoscopic heterogeneities in adsorption and electron transfer processes of perylene diimide dye on TiO 2 nanoparticles studied by single-molecule fluorescence spectroscopy  

NASA Astrophysics Data System (ADS)

The interfacial electron transfer processes between a fluorescent water-soluble perylene diimide dye (WS-PDI) and TiO 2 nanoparticles were investigated using single-molecule fluorescence spectroscopy. Based on the single-molecule fluorescence spectral measurements, it was suggested that the local environment and/or the structural conformation of single WS-PDI molecules play important roles in the efficiency of the electron injection from WS-PDI in the singlet excited state to TiO 2. The observed single-molecule fluorescence blinking behavior was interpreted in terms of the interfacial electron-transfer dynamics between the single WS-PDI molecules and TiO 2 nanoparticles.

Tachikawa, Takashi; Cui, Shi-Cong; Tojo, Sachiko; Fujitsuka, Mamoru; Majima, Tetsuro



Photo-Induced Single Molecule Electron Transfer at the Molecule-Nanoparticle Interface  

NASA Astrophysics Data System (ADS)

Single molecule fluorescence spectroscopy was used to study photoinduced electron transfer (ET) dynamics across single donor-bridge-acceptor junctions consisting of perylene-3,4:9,10-bis(dicarboximide) (PDI), n-phenylene bridge with COOH anchoring group, and antimony doped Tin Oxide(ATO) nanoparticles. Photo-excitation of PDI initiates electron transfer from its excited state into ATO nanoparticles. Electron transfer was confirmed and ensemble average rate was measured by transient infrared absorption spectroscopy, in which injected electrons in ATO were directly monitored. Single molecule fluorescence from donor molecule was confirmed by the observed blinking behavior, fluorescence spectrum, and excitation polarization dependence. Single molecule fluorescence lifetime was measured by time-correlated single photon counting, from which forward electron transfer rate from adsorbate excited state to nanoparticle was determined. The dependence of these single molecule ET rates and their fluctuation on the length of phenylene bridge and the nature of semiconductors are being investigated.

Lian, Tianquan; Goh, Wanhee; Guo, Jianchang; Liu, Xi; Ahrens, Michael; Schierloh, Emilie; Wasielewski, Michael



REVIEWS OF TOPICAL PROBLEMS Electron-vibration interaction in tunneling processes through single molecules  

NASA Astrophysics Data System (ADS)

It is shown how effective Hamiltonians are constructed in the framework of the adiabatic approach to the electron-vibration interaction in electron tunneling through single molecules. Methods for calculating tunneling characteristics are discussed and possible features resulting from the electron-vibration coupling are described. The intensity of vibrations excited by a tunneling current in various systems is examined.

Arseev, Petr I.; Maslova, N. S.



Controlling electronic States and transport properties at the level of single molecules.  


Since molecular electronics has been rapidly growing as a promising alternative to conventional electronics towards the ultimate miniaturization of electronic devices through the bottom-up strategy, it has become a long-term desire to understand and control the transport properties at the level of single molecules. In this Research News article it is shown that one may modify the electronic states of single molecules and thus control their transport properties through designing and fabrication of functional molecules or manipulating molecules with scanning tunneling microscopy. The rectifying effect of single molecules can be realized by designing a donor-barrier-acceptor architecture of Pyridine-sigma-C(60) molecules to achieve the Aviram-Ratner rectifier and by modifying electronic states through azafullerene C(59)N molecules. The effect of the negative differential resistances can be realized by appropriately matching the molecular orbital symmetries between a cobalt phthalocyanine (CoPc) molecule and a Ni electrode. The electronic states and transport properties of single molecules, such as CoPc and melamine molecules, can be altered through manipulation or modifying molecular structures, leading to functionalized molecular devices. PMID:20301130

Pan, Shuan; Zhao, Aidi; Wang, Bing; Yang, Jinlong; Hou, Jianguo



Single-Molecule Imaging with X-Ray Free-Electron Lasers: Dream or Reality?  

SciTech Connect

X-ray free-electron lasers (XFEL) are revolutionary photon sources, whose ultrashort, brilliant pulses are expected to allow single-molecule diffraction experiments providing structural information on the atomic length scale of nonperiodic objects. This ultimate goal, however, is currently hampered by several challenging questions basically concerning sample damage, Coulomb explosion, and the role of nonlinearity. By employing an original ab initio approach, we address these issues showing that XFEL-based single-molecule imaging will be only possible with a few-hundred long attosecond pulses, due to significant radiation damage and the formation of preferred multisoliton clusters which reshape the overall electronic density of the molecular system at the femtosecond scale.

Fratalocchi, A. [PRIMALIGHT, Faculty of Electrical Engineering, Applied Mathematics and Computational Science, King Abdullah University of Science and Technology (KAUST), Thuwal 23955-6900 (Saudi Arabia)] [Department of Physics, Sapienza University of Rome, P.le A. Moro 2, 00185, Rome (Italy); Ruocco, G. [Department of Physics, Sapienza University of Rome, P.le A. Moro 2, 00185, Rome (Italy)] [IPCF-CNR, c/o Department of Physics, Sapienza University, P.le Aldo Moro 2, 00185, Rome (Italy)



Single Molecules  

NSDL National Science Digital Library

A new molecular science journal, Single Molecules, from Wiley Interscience, "will provide researchers with a broad overview of current methods and techniques, recent applications and shortcomings of present techniques in the field of single molecules." With temporary free access, the journal's latest issue contains a few full-text articles, with more articles being regularly added. This journal is currently calling for papers.


Conformation-controlled electron transport in single-molecule junctions containing oligo(phenylene ethynylene) derivatives.  


Understanding the relationships between the molecular structure and electronic transport characteristics of single-molecule junctions is of fundamental and technological importance for future molecular electronics. Herein, we report a combined experimental and theoretical study on the single-molecule conductance of a series of oligo(phenylene ethynylene) (OPE) molecular wires, which consist of two phenyl-ethynyl-phenyl ? units with different dihedral angles. The molecular conductance was studied by scanning tunneling microscopy (STM)-based break-junction techniques under different conditions, including variable temperature and bias potential, which suggested that a coherent tunneling mechanism takes place in the OPE molecular wires with a length of 2.5 nm. The conductance of OPE molecular junctions are strongly affected by the coupling strength between the two ? systems, which can be tuned by controlling their intramolecular conformation. A cos(2)? dependence was revealed between the molecular conductance and dihedral angles between the two conjugated units. Theoretical investigations on the basis of density functional theory and nonequilibrium Green's functions (NEGF) gave consistent results with the experimental observations and provided insights into the conformation-dominated molecular conductance. PMID:23729379

Wang, Le-Jia; Yong, Ai; Zhou, Kai-Ge; Tan, Lin; Ye, Jian; Wu, Guo-Ping; Xu, Zhu-Guo; Zhang, Hao-Li



Fast electron transfer through a single molecule natively structured redox protein  

NASA Astrophysics Data System (ADS)

The electron transfer properties of proteins are normally measured as molecularly averaged ensembles. Through these and related measurements, proteins are widely regarded as macroscopically insulating materials. Using scanning tunnelling microscopy (STM), we present new measurements of the conductance through single-molecules of the electron transfer protein cytochrome b562 in its native conformation, under pseudo-physiological conditions. This is achieved by thiol (SH) linker pairs at opposite ends of the molecule through protein engineering, resulting in defined covalent contact between a gold surface and a platinum-iridium STM tip. Two different orientations of the linkers were examined: a long-axis configuration (SH-LA) and a short-axis configuration (SH-SA). In each case, the molecular conductance could be `gated' through electrochemical control of the heme redox state. Reproducible and remarkably high conductance was observed in this relatively complex electron transfer system, with single-molecule conductance values peaking around 18 nS and 12 nS for the SH-SA and SH-LA cytochrome b562 molecules near zero electrochemical overpotential. This strongly points to the important role of the heme co-factor bound to the natively structured protein. We suggest that the two-step model of protein electron transfer in the STM geometry requires a multi-electron transfer to explain such a high conductance. The model also yields a low value for the reorganisation energy, implying that solvent reorganisation is largely absent.The electron transfer properties of proteins are normally measured as molecularly averaged ensembles. Through these and related measurements, proteins are widely regarded as macroscopically insulating materials. Using scanning tunnelling microscopy (STM), we present new measurements of the conductance through single-molecules of the electron transfer protein cytochrome b562 in its native conformation, under pseudo-physiological conditions. This is achieved by thiol (SH) linker pairs at opposite ends of the molecule through protein engineering, resulting in defined covalent contact between a gold surface and a platinum-iridium STM tip. Two different orientations of the linkers were examined: a long-axis configuration (SH-LA) and a short-axis configuration (SH-SA). In each case, the molecular conductance could be `gated' through electrochemical control of the heme redox state. Reproducible and remarkably high conductance was observed in this relatively complex electron transfer system, with single-molecule conductance values peaking around 18 nS and 12 nS for the SH-SA and SH-LA cytochrome b562 molecules near zero electrochemical overpotential. This strongly points to the important role of the heme co-factor bound to the natively structured protein. We suggest that the two-step model of protein electron transfer in the STM geometry requires a multi-electron transfer to explain such a high conductance. The model also yields a low value for the reorganisation energy, implying that solvent reorganisation is largely absent. Electronic supplementary information (ESI) available: Experimental methods, DNA and protein sequences, additional STM statistical analysis and images, electrochemical data and It-z data analysis. See DOI: 10.1039/c2nr32131a

Della Pia, Eduardo Antonio; Chi, Qijin; MacDonald, J. Emyr; Ulstrup, Jens; Jones, D. Dafydd; Elliott, Martin



Resolving Single Molecule Lysozyme Dynamics with a Carbon Nanotube Electronic Circuit  

NASA Astrophysics Data System (ADS)

High resolution, real-time monitoring of a single lysozyme molecule is demonstrated by fabricating nanoscale electronic devices based on single-walled carbon nanotubes (SWCNT). In this sensor platform, a biomolecule of interest is attached to a single SWCNT device. The electrical conductance transduces chemical events with single molecule sensitivity and 10 microsecond resolution. In this work, enzymatic turnover by lysozyme is investigated, because the mechanistic details for its processivity and dynamics remain incompletely understood. Stochastically distributed binding events between a lysozyme and its binding substrate, peptidoglycan, are monitored via the sensor conductance. Furthermore, the magnitude and repetition rate of these events varies with pH and the presence of inhibitors or denaturation agents. Changes in the conductance signal are analyzed in terms of lysozyme's internal hinge motion, binding events, and enzymatic processing.

Choi, Yongki; Moody, Issa S.; Perez, Israel; Sheps, Tatyana; Weiss, Gregory A.; Collins, Philip G.



High Quality Nanogap Electrodes for Electronic Transport Measurements of Single Molecules  

NASA Astrophysics Data System (ADS)

Electromigrated metal electrodes and resulting devices have shown great promise in moving towards the realization of single molecule-based electronic devices holding the potential for a wide range of electronic applications. At present, a major concern is that the electronic behavior of such devices may be greatly influenced by residual nanoscale metal particles. We have developed a computer controlled electromigration (CCE) process for creating nanogaps at room temperature which allows us to characterize a bare nanogap before putting a molecule into the nanogap.^1 This is very different from other approaches used in the field where nanogaps are formed at low temperature with molecules already attached to the nanowire by employing a simple ramp up in voltage. Among the bare nanogaps we produced using CCE, tunneling behavior is observed with no indication of transport signatures associated with metal particle formation. Details of molecular measurements utilizing these clean gaps will be discussed. This work was supported by the National Science Foundation (NIRT Grant No. 0304531 and MRSEC award DMR05-20020). ^1D. R. Strachan, D. E. Smith, D. E. Johnston et al., Appl. Phys. Lett. 86 043109 (2005).

Johnston, Danvers E.; Strachan, Douglas R.; Guiton, Beth S.; Davies, Peter K.; Park, Tae Hong; Therien, Michael J.; Johnson, A. T. Charlie



Manipulation and characterization of thin-film interfacial chemistry: Sol-gel deposition and single molecule tracking experiments  

NASA Astrophysics Data System (ADS)

Single molecule trajectories of 1,1'-dioctadecyl-3,3,3'3'-tetramethylindocarbo - cyanine perchlorate (DiI) fluorophores diffusing on planar supported 1,2-dimyristoyl-snglycero- 3-phosphocholine (DMPC) lipid bilayers imaged through total internal reflection fluorescence (TIRF) microscopy at different temperatures are investigated. The spatial resolution limit for detecting molecular motion is evaluated by characterizing the apparent motion which arises from the limited signal-to-noise ratio (S/N) of imaged and simulated stationary DiI molecules. Statistical criteria for reliably distinguishing molecular motion from stationary molecules using F-test statistics, including the computation of local signal-to-noise ratios are then established and used for reliably detecting subdiffraction motion of DiI molecules on DMPC. The same single molecule tracking concept is used in investigating the temperature dependence of subdiffraction diffusional confinement of single Rhodamine 6G molecules in polymer brushes of poly (N-isopropylacrylamide), pNIPAAm, above and below its lower critical solution temperature (LCST) of 32°C. Reliably distinguishing subdiffraction molecular motion from stationary events is crucial in validating the application of single molecule tracking experiment in probing nanometersized hydrophobic environments of polymer structure. A versatile and rapid sol-gel technique for the fabrication of high quality one-dimensional photonic bandgap materials was developed. Silica/titania multilayer materials are fabricated by a sol-gel chemistry route combined with dip-coating onto planar or curved substrate. A shock-cooling step immediately following the thin film heat-treatment process is introduced. The versatility of this sol-gel method is demonstrated by the fabrication of various Bragg stack-type materials with fine-tuned optical properties. Measured optical properties show good agreement with theoretical simulations confirming the high quality of these sol-gel fabricated optical materials. Finally, magnetic functionalization studies of sol-gel derived Co-ion doped titania thin films using superconducting quantum interference device (SQUID) magnetometry and an attempt to measure their magneto-optical properties using a home-built Faraday rotation setup are discussed. The experimental limitations in reliably measuring magnetization responses of these thin films are introduced and discussed in detail. The summary and outlook chapters summarize the scientific significance of each research project and briefly introduce ongoing research based on the work and the results presented in this dissertation.

Barhoum, Moussa


Electron spin resonance and muon spin relaxation studies of single molecule magnets  

NASA Astrophysics Data System (ADS)

We use a combination of electron spin resonance, muon-spin relaxation and SQUID magnetometry to study polycrystalline and single crystal samples of various novel single molecule magnets (SMMs). We also describe a theoretical framework which can be used to analyse the results from each technique. Electron spin resonance measurements are performed using a millimetre vector network analyser and data are presented on several SMM systems using microwave frequencies from 40-300 GHz. Muon-spin relaxation measurements have been performed on several SMM systems in applied longitudinal magnetic field and in temperatures down to 20 mK. The results suggest that dynamic local magnetic field fluctuations are responsible for the relaxation of the muon spin ensemble. We discuss what can be learned from these experiments concerning SMMs and suggest experiments which can probe the quantum nature of SMMs. (Work in collaboration with S Sharmin, T Lancaster, A Ardavan, F L Pratt, E J L McInnes and R E P Winpenny) References: S. J. Blundell and F. L. Pratt, J. Phys.: Condens. Matter 16, R771 (2004); T. Lancaster et al., J. Phys.: Condens. Matter 16, S4563 (2004); S. Sharmin et al., Appl. Phys. Lett. in press.

Blundell, Stephen



Numerically exact, time-dependent treatment of vibrationally coupled electron transport in single-molecule junctions  

NASA Astrophysics Data System (ADS)

The multilayer multiconfiguration time-dependent Hartree (ML-MCTDH) theory within second quantization representation of the Fock space, a novel numerically exact methodology to treat many-body quantum dynamics for systems containing identical particles, is applied to study the effect of vibrational motion on electron transport in a generic model for single-molecule junctions. The results demonstrate the importance of electronic-vibrational coupling for the transport characteristics. For situations where the energy of the bridge state is located close to the Fermi energy, the simulations show the time-dependent formation of a polaron state that results in a pronounced suppression of the current corresponding to the phenomenon of phonon blockade. We show that this phenomenon cannot be explained solely by the polaron shift of the energy but requires methods that incorporate the dynamical effect of the vibrations on the transport. The accurate results obtained with the ML-MCTDH in this parameter regime are compared to results of nonequilibrium Green's function theory.

Wang, Haobin; Pshenichnyuk, Ivan; Härtle, Rainer; Thoss, Michael



Electronic states of pyrene single crystal and of its single molecule inserted in a molecular vessel of cyclodextrin  

NASA Astrophysics Data System (ADS)

Highly purified single crystals of pyrene were made by a gas phase crystal growth method from 180 times of zone-refined pyrene. The absorption spectra of the single crystal have been transformed from the reflection spectra between 2.5 and 6.5eV at 2, 77K and room temperature. The dry powder of ?-cyclodextrin including pyrene single molecule were prepared in vacuum to investigate the electronic states of the isolated molecule. The absorption spectra of the single molecule show similar spectra to those of the single crystal. The pyrene molecule keeps its electronic character even in the single crystal.

Takahashi, Nobuaki; Gombojav, Bold; Yoshinari, Takehisa; Nagasaka, Shin-Ichiro; Takahashi, Yoshio; Yamamoto, Aishi; Goto, Takenari; Kasuya, Atsuo



LeRoy Apker Award Talk: Electronics at the Nanoscale: Graphene, Carbon Nanotubes, and Single-Molecule Devices  

NASA Astrophysics Data System (ADS)

Low-dimensional nanostructures are emerging as model systems for fundamental studies of quantum transport, as well as promising candidates for novel post-silicon electronic devices incorporating quantum size effects. Key examples of these include few-layer graphene, carbon nanotubes, polymer nanofibers, and even single molecules. In this talk, I will summarize my work combining experimental and computational tools to study, control, and apply molecular nanomaterials of low dimensionality -- using scanning probe microscopy techniques to study electronic phenomena in few-layer graphene and carbon nanotubes, as well as to elucidate the structure of biochemically-functionalized carbon nanotubes; using computer simulations to investigate key electronic properties of single-molecule transistors; and demonstrating a straightforward chemical technique by which samples of few-layer graphene can be etched along their crystallographic directions, potentially enabling the creation of a variety of new graphene-based nanostructures.

Datta, Sujit



Electronic measurements of single-molecule processing by DNA polymerase I  

NASA Astrophysics Data System (ADS)

A single-molecule nanocircuit technique is applied to continuously monitor DNA replication activity by the enzyme DNA polymerase I (Pol I). Using single copies of Pol I bound to a single-walled carbon nanotube device, an electrical signal was generated to reveal enzymatic function and dynamic variability. Continuous, single-molecule-resolution recordings were obtained for Pol I processing homopolymeric DNA templates over 10 minutes and through 10,000 DNA replication events. Processivity of up to 40 nucleotide bases was directly observed, and statistical analysis of the recordings determined key kinetic parameters for the enzyme's open and closed conformations. We observe that the closed complex forms a phosphodiester bond in a highly efficient process 99.8% of the time, with a mean duration of only 0.3 ms for all four dNTPs. The rate-limiting step for replication occurs during the enzyme's open state, but with a duration that is nearly twice as long for dATP or dTTP incorporation than for dCTP or dGTP. Taken together, the results provide a wealth of new information complementing prior work on the mechanism and dynamics of DNA polymerase.

Choi, Yongki; Olsen, Tivoli; Gul, Tolga; Corso, Brad; Dong, Chengjun; Brown, William; Weiss, Gregory; Collins, Philip



Electron transport through Ni/1,4-benzenedithiol/Ni single-molecule junctions under magnetic field  

NASA Astrophysics Data System (ADS)

We have studied electron transport through Ni/1,4-benzenedithiol (BDT)/Ni single molecule junctions at cryogenic temperatures under magnetic field up to 250 mT. Instead of examining magnetoresistance (MR) of individual junctions, we measured the conductance of many junctions under a constant magnetic field and investigated how a single-molecule peak in a conductance histogram shifts with the field strength. We found that the single-molecule resistance at 77 K, deduced from the conductance peak position, shows a hysteresis against the field strength and takes a maximum around 50 mT when the magnetic field increases from 0 T to 150 mT. The observed resistance change yields a MR of ~(80-90)% for Ni/BDT/Ni single molecule junctions. This MR is higher than experimental MR reported for non-conjugating molecules but consistent with high theoretical MR predicted for ?-conjugated molecules such as BDT. We have also investigated the nonlinearity of the current-voltage (I-V) characteristics of Ni/BDT/Ni junctions under 0 T and 150 mT and found that the nonlinearity changes its sign from negative at 0 T to positive at 150 mT. This result suggests that the junction transmission spectrum at 0 T should have a low-lying peak within +/-0.1 eV from the Fermi level, but the peak moves out of the bias window when the magnetic field increases to 150 mT. The observed field-induced change in the I-V nonlinearity is qualitatively consistent with theoretical I-V curves of Ni/BDT/Ni calculated for magnetized and non-magnetized Ni electrodes.

Horiguchi, Kazunori; Sagisaka, Takami; Kurokawa, Shu; Sakai, Akira



Vibrationally Induced Decoherence in Single-Molecule Junctions: The Role of Electron-Hole Pair Creation Processes  

NASA Astrophysics Data System (ADS)

We investigate quantum interference effects and vibrationally induced decoherence in single-molecule junctions, employing nonequilibrium Green's function theory [1]. Molecular junctions often exhibit quasidegenerate electronic states that allow an electron to tunnel through the junction in different ways [2,3]. The respective outgoing wavefunctions interfere constructively or destructively, leading to an increase or decrease of the tunnel current, respectively. Interaction of the tunneling electrons with the vibrational degrees of freedom of the junction, however, gives 'which-path' information about the corresponding tunneling pathways because of the state-specific nature of electronic-vibrational coupling [2,3,4]. We demonstrate how this interplay between interference and vibrationally induced decoherence results in a strong temperature dependence of the current and highlight the role of electron-hole pair creation processes in this context [3,4]. To this end, we employ both generic models of single-molecule junctions as well as realistic models that are based on first-principles electronic structure calculations. [1] Phys. Rev. Lett. 102, 146801 (2009), [2] Phys. Rev. Lett. 107, 046802 (2011), [3] Phys. Rev. Lett. 109, 056801 (2012), [4] arXiv:1209.5619 (2012).

Hartle, Rainer; Butzin, Michael; Coto, Pedro B.; Ballmann, Stefan; Weber, Heiko B.; Thoss, Michael



Superior contact for single-molecule conductance: electronic coupling of thiolate and isothiocyanate on Pt, Pd, and Au.  


One of the critical issues for the realization of molecular electronics is the development of ideal molecule-electrode contacts that render efficient charge transportation and thus attenuate the unwanted voltage drop and power loss. The conductance at the single-molecule level has long been expected to be correlated strongly with the electrode materials. However, other than gold, systematic studies of a homologous series of molecules to extract the headgroup-metal contact conductance (G(n=0)) have not been reported. Carefully examined herein are the conductances of alkanedithiols anchored onto electrode materials of Au and Pt as well as the conductances of alkanediisothiocyanates on Au, Pd, and Pt by utilizing the method of STM-BJ (scanning tunneling microscopy break junction). In comparison with Au substrate, Pd and Pt are group 10 elements with stronger d-orbital characteristics, and larger local density of states near the Fermi level. The model compounds, SCN(CH(2))(n)NCS (n = 4, 6, and 8), are studied because the isothiocyanate (-NCS) headgroup is a versatile ligand for organometallics, an emerging class of molecular wires, and can bind to substrates of noble metals to complete a metal-molecule-metal configuration for external I-V measurements. Also studied include alkanedithiols, one of the most scrutinized systems in the field of single-molecule conductance. The results show that the conductance for single molecules bridged between a pair of Pt electrodes is about 3.5-fold superior to those between Au electrodes. On all electrode materials, observed are two sets of conductance values, with the smaller set being 1 order of magnitude less conductive. These findings are ascribed to the degree of electronic coupling between the headgroup and the electrode. PMID:20020686

Ko, Chih-Hung; Huang, Min-Jie; Fu, Ming-Dung; Chen, Chun-Hsien



Intramolecular Distances and Dynamics from the Combined Photon Statistics of Single-Molecule FRET and Photoinduced Electron Transfer.  


Single-molecule Förster resonance energy transfer (FRET) and photoinduced electron transfer (PET) have developed into versatile and complementary methods for probing distances and dynamics in biomolecules. Here we show that the two methods can be combined in one molecule to obtain both accurate distance information and the kinetics of intramolecular contact formation. In a first step, we show that the fluorescent dyes Alexa 488 and Alexa 594, which are frequently used as a donor and acceptor for single-molecule FRET, are also suitable as PET probes with tryptophan as a fluorescence quencher. We then performed combined FRET/PET experiments with FRET donor- and acceptor-labeled polyproline peptides. The placement of a tryptophan residue into the polyglycylserine tail incorporated in the peptides allowed us to measure both FRET efficiencies and the nanosecond dynamics of contact formation between one of the fluorescent dyes and the quencher. Variation of the linker length between the polyproline and the Alexa dyes and in the position of the tryptophan residue demonstrates the sensitivity of this approach. Modeling of the combined photon statistics underlying the combined FRET and PET process enables the accurate analysis of both the resulting transfer efficiency histograms and the nanosecond fluorescence correlation functions. This approach opens up new possibilities for investigating single biomolecules with high spatial and temporal resolution. PMID:23718771

Haenni, Dominik; Zosel, Franziska; Reymond, Luc; Nettels, Daniel; Schuler, Benjamin



Single-Molecule Electron Tunneling Spectroscopy of the Higher Plant Light-Harvesting Complex LHC II  

Microsoft Academic Search

Electronic spectroscopy of a single biological molecule is demonstrated with ?4 Å spatial resolution. The light-harvesting complex II (LHC II), in the ground and photo-excited states, was studied using scanning tunneling microscopy and spectroscopy of intact Photosystem II complexes. Analysis of the spectra indicates that the main mechanisms of tunneling between the STM tip and the surface involve delocalized electronic

Philip B. Lukins



Impact of edge shape on the functionalities of graphene-based single-molecule electronics devices  

NASA Astrophysics Data System (ADS)

We present an ab initio analysis of the impact of edge shape and graphene-molecule anchor coupling on the electronic and transport functionalities of graphene-based molecular electronics devices. We analyze how Fano-like resonances, spin filtering, and negative differential resistance effects may or may not arise by modifying suitably the edge shapes and the terminating groups of simple organic molecules. We show that the spin filtering effect is a consequence of the magnetic behavior of zigzag-terminated edges, which is enhanced by furnishing these with a wedge shape. The negative differential resistance effect is originated by the presence of two degenerate electronic states localized at each of the atoms coupling the molecule to graphene which are strongly affected by a bias voltage. The effect could thus be tailored by a suitable choice of the molecule and contact atoms if edge shape could be controlled with atomic precision.

Carrascal, D.; García-Suárez, V. M.; Ferrer, J.



Single molecule electron paramagnetic resonance spectroscopy: hyperfine splitting owing to a single nucleus.  


Individual pentacene-d(14) molecules doped into a p-terphenyl-d(14) host crystal have been studied by optically detected electron paramagnetic resonance spectroscopy. The magnetic resonance transitions between the triplet sublevels of the pentacene molecule and the splitting of the resonance lines for a molecule that contains a carbon-13 nucleus have been observed in an external magnetic field. This splitting is caused by the hyperfine interaction of the triplet electron spin with the single carbon-13 nuclear spin. PMID:17843664

Köhler, J; Brouwer, A C; Groenen, E J; Schmidt, J



Single Molecule Electron Paramagnetic Resonance Spectroscopy: Hyperfine Splitting Owing to a Single Nucleus  

Microsoft Academic Search

Individual pentacene-d14 molecules doped into a p-terphenyl-d14 host crystal have been studied by optically detected electron paramagnetic resonance spectroscopy. The magnetic resonance transitions between the triplet sublevels of the pentacene molecule and the splitting of the resonance lines for a molecule that contains a carbon-13 nucleus have been observed in an external magnetic field. This splitting is caused by the

J. Kohler; A. C. J. Brouwer; E. J. J. Groenen; J. Schmidt



Tailoring electronic states of a single molecule using adamantane-based molecular tripods.  


Adsorption structures and electronic states of molecular tripods, having a Br atom (BATT) and a ferrocene derivative (Ferrocene-ATT) at the head part of the adamantane-based trithiolate, adsorbed on Au(111) have been investigated using low temperature scanning tunneling microscopy (STM) and scanning tunneling spectroscopy (STS). We found that BATT and Ferrocene-ATT form self-assembled monolayers (SAMs), and their orderings are identical to one another, which suggests that the adsorption structure of adamantane-based molecular tripods is independent of the type of functional substituent attached to the head part. The electronic states originated from the ferrocene group were confirmed in the STS spectrum of Ferrocene-ATT whereas those are absent in the BATT spectrum. We note that the ferrocene part has few interactions with the Au substrate owing not only to the upright geometry of Ferrocene-ATT but also to the insulative properties of the adamantane base. The STS mapping revealed the spatial distribution of the electronic state of Ferrocene-ATT. PMID:23877197

Katano, Satoshi; Kim, Yousoo; Kitagawa, Toshikazu; Kawai, Maki



Single-molecule fluorescence spectroscopy studies of photo-induced electron transfer between CdSe\\/ZnS quantum dots and fullerene  

Microsoft Academic Search

Photo-induced electron transfer in CdSe\\/ZnS semiconductor quantum dot-fullerene conjugates was investigated by single molecule fluorescence spectroscopy. The average rate for photoinduced electron transfer is estimated around 108s-1. Quenching by electron transfer is observed in the \\

Zhihua Xu; Mircea Cotlet



Significant enhancement of energy barriers in dinuclear dysprosium single-molecule magnets through electron-withdrawing effects.  


The effect of electron-withdrawing ligands on the energy barriers of Single-Molecule Magnets (SMMs) is investigated. By introducing highly electron-withdrawing atoms on targeted ligands, the energy barrier was significantly enhanced. The structural and magnetic properties of five novel SMMs based on a dinuclear {Dy2} phenoxo-bridged motif are explored and compared with a previously studied {Dy2} SMM (1). All complexes share the formula [Dy2(valdien)2(L)2]·solvent, where H2valdien = N1,N3-bis(3-methoxysalicylidene) diethylenetriamine, the terminal ligand L = NO3(-) (1), CH3COO(-) (2), ClCH2COO(-) (3), Cl2CHCOO(-) (4), CH3COCHCOCH3(-) (5), CF3COCHCOCF3(-) (6), and solvent = 0.5 MeOH (4), 2 CH2Cl2 (5). Systematic increase of the barrier was observed for all complexes with the most drastic increase seen in 6 when the acac ligand of 5 was fluorinated resulting in a 7-fold enhancement of the anisotropic barrier. Ab initio calculations reveal more axial g tensors as well as higher energy first excited Kramers doublets in 4 and 6 leading to higher energy barriers for those complexes. PMID:23964606

Habib, Fatemah; Brunet, Gabriel; Vieru, Veacheslav; Korobkov, Ilia; Chibotaru, Liviu F; Murugesu, Muralee



Yeast cytochrome c integrated with electronic elements: a nanoscopic and spectroscopic study down to single-molecule level  

NASA Astrophysics Data System (ADS)

Various aspects of redox protein integration with nano-electronic elements are addressed by a multi-technique investigation of different yeast cytochrome c (YCC)-based hybrid systems. Three different immobilization strategies on gold via organic linkers are explored, involving either covalent bonding or electrostatic interaction. Specifically, Au surfaces are chemically modified by self-assembled monolayers (SAMs) exposing thiol-reactive groups, or by acid-oxidized single-wall carbon nanotubes (SWNTs). Atomic force microscopy and scanning tunnelling microscopy are employed to characterize the morphology and the electronic properties of single YCC molecules adsorbed on the modified gold surfaces. In each hybrid system, the protein molecules are stably assembled, in a native configuration. A standing-up arrangement of YCC on SAMs is suggested, together with an enhancement of the molecular conduction, as compared to YCC directly assembled on gold. The electrostatic interaction with functionalized SWNTs allows several YCC adsorption geometries, with a preferential high-spin haem configuration, as outlined by Raman spectroscopy. Moreover, the conduction properties of YCC, explored in different YCC nanojunctions by conductive atomic force microscopy, indicate the effectiveness of electrical conduction through the molecule and its dependence on the electrode material. The joint employment of several techniques confirms the key role of a well-designed immobilization strategy, for optimizing biorecognition capabilities and electrical coupling with conductive substrates at the single-molecule level, as a starting point for advanced applications in nano-biotechnology.

Delfino, I.; Bonanni, B.; Andolfi, L.; Baldacchini, C.; Bizzarri, A. R.; Cannistraro, S.



Electronic structure study by means of x-ray spectroscopy and theoretical calculations of the ``ferric star'' single molecule magnet  

NASA Astrophysics Data System (ADS)

The electronic structure of the single molecule magnet system {M[Fe(L1)2]3}.4CHCl3 [M=Fe,CrL1=CH3N(CH2CH2O)22-] has been studied using x-ray photoelectron spectroscopy, x-ray-absorption spectroscopy, soft-x-ray emission spectroscopy, as well as theoretical density-functional-based methods. There is a good agreement between theoretical calculations and experimental data. The valence band mainly consists of three bands between 2 and 30 eV. Both theory and experiments show that the top of the valence band is dominated by the hybridization between Fe 3d and O 2p bands. From the shape of the Fe 2p spectra it is argued that Fe in the molecule is most likely in the 2+ charge state. Its neighboring atoms (O,N) exhibit a magnetic polarization yielding effective spin S=5/2 per iron atom, giving a high-spin state molecule with a total S=5 effective spin for the case of M=Fe.

Takács, A. F.; Neumann, M.; Postnikov, A. V.; Kuepper, K.; Scheurer, A.; Sperner, S.; Saalfrank, R. W.; Prince, K. C.



Photoinduced electron transfer (PET)-probes for the study of enzyme activity at the ensemble and single molecule level  

NASA Astrophysics Data System (ADS)

Fluorescence based enzyme analysis is commonly done by FRET-probes, natural enzyme substrates flanked by two corresponding fluorophores, showing spectral changes upon distance variations between the fluorophores. However, the use of double labeled substrates displays several limitations such as reduction of sensitivity and high background signal accompanied by high costs for synthesis. Therefore, the development of new probes avoiding these factors is of general interest in enzyme research. A promising approach represents smart probes, i.e. singly labeled quenched enzyme substrates that increase fluorescence intensity upon enzymatic cleavage. Smart probes use the fact that certain rhodamine and oxazine dyes are selectively quenched upon contact formation with guanine or tryptophan residues via photoinduced electron transfer (PET). The rapid response time of the probes enables real-time monitoring of enzyme activity in ensemble as well as in single molecule measurements, which is an important prerequisite for the improved understanding of enzyme mechanisms. We present the design of smart probes for the detection of the two hydrolases, DNaseI and Carboxypeptidase A (CPA) with respect to stability and substrate specificity in ensemble measurements. Furthermore, we investigate the influence of the attached fluorophore on hydrolysis efficiency in case of CPA and demonstrate first applications of smart probes in single enzyme experiments.

Henkenjohann, Sigrun; van de Linde, Sebastian; Doose, Sören; Tinnefeld, Philip; Sauer, Markus



Movies of molecular motions and reactions: the single-molecule, real-time transmission electron microscope imaging technique.  


"The truth is, the Science of Nature has been already too long made only a work of the Brain and the Fancy: It is now high time that it should return to the plainness and soundness of Observations on material and obvious things," proudly declared Robert Hooke in his highly successful picture book of microscopic and telescopic images, "Micrographia" in 1665. Hooke's statement has remained true in chemistry, where a considerable work of the brain and the fancy is still necessary. Single-molecule, real-time transmission electron microscope (SMRT-TEM) imaging at an atomic resolution now allows us to learn about molecules simply by watching movies of them. Like any dream come true, the new analytical technique challenged the old common sense of the communities, and offers new research opportunities that are unavailable by conventional methods. With its capacity to visualize the motions and the reactions of individual molecules and molecular clusters, the SMRT-TEM technique will become an indispensable tool in molecular science and the engineering of natural and synthetic substances, as well as in science education. PMID:23280645

Nakamura, Eiichi



Single-molecule fluorescence spectroscopy studies of photo-induced electron transfer between CdSe/ZnS quantum dots and fullerene  

NASA Astrophysics Data System (ADS)

Photo-induced electron transfer in CdSe/ZnS semiconductor quantum dot-fullerene conjugates was investigated by single molecule fluorescence spectroscopy. The average rate for photoinduced electron transfer is estimated around 108s-1. Quenching by electron transfer is observed in the "on" state and it manifests both as reduced fluorescence intensity and as shortening in fluorescence lifetime. As a result, the electron transfer changes the on/off dynamics of the fluorescence intensity of individual quantum dots.

Xu, Zhihua; Cotlet, Mircea



Single Molecule Spectroscopy  

Microsoft Academic Search

We report unambiguous optical detection of single molecules of pentacene in p-terphenyl crystal. The relative shift of the molecular resonance frequencies with temperature changes demonstrates the phenomenon of activated spectral diffusion. These results show the feasibility of the optical study of a single molecule and its local environment.

M. Orrit; J. Bernard



Single Molecule Spectroscopy  

NASA Astrophysics Data System (ADS)

We report unambiguous optical detection of single molecules of pentacene in p-terphenyl crystal. The relative shift of the molecular resonance frequencies with temperature changes demonstrates the phenomenon of activated spectral diffusion. These results show the feasibility of the optical study of a single molecule and its local environment.

Orrit, M.; Bernard, J.


Single-molecule optoelectronics.  


With discrete states, several-atom Ag(n) nanoclusters exhibit molecule-like behavior with strong visible fluorescence and robust optical properties. This new class of single-molecule fluorophores has been created and electrically contacted in thin films to produce the first electroluminescent single molecules. A direct reporter of nanoscale charge injection and transport through discrete energy levels, bright Ag(n) electroluminescence has been harnessed to create single-molecule light-emitting diodes (LEDs) and optoelectronic logic gates and even to demonstrate full addition operations. These experiments utilizing the small size and quantum behavior of individual Ag nanoclusters usher in the new field of single-molecule optoelectronics. PMID:16028887

Lee, Tae-Hee; Gonzalez, Jose I; Zheng, Jie; Dickson, Robert M



Electronic states of pyrene single crystal and of its single molecule inserted in a molecular vessel of cyclodextrin  

Microsoft Academic Search

Highly purified single crystals of pyrene were made by a gas phase crystal growth method from 180 times of zone-refined pyrene. The absorption spectra of the single crystal have been transformed from the reflection spectra between 2.5 and 6.5eV at 2, 77K and room temperature. The dry powder of ?-cyclodextrin including pyrene single molecule were prepared in vacuum to investigate

Nobuaki Takahashi; Bold Gombojav; Takehisa Yoshinari; Shin-Ichiro Nagasaka; Yoshio Takahashi; Aishi Yamamoto; Takenari Goto; Atsuo Kasuya



Probing single molecule dynamics  

SciTech Connect

The room temperature dynamics of single sulforhodamine 101 molecules dispersed on a glass surface are investigated on two different time scales with near-field optics. On the 10[sup [minus]2] - to 10[sup 2]-second time scale, intensity fluctuations in the emission from single molecules are examined with polarization measurements, providing insight into their spectroscopic properties. On the nanosecond time scale, the fluorescence lifetimes of single molecules are measured, and their excited-state energy transfer to the aluminum coating of the near-field probe is characterized. A movie of the time-resolved emission demonstrates the feasibility of fluorescence lifetime imaging with single molecule sensitivity, picosecond temporal resolution, and a spatial resolving power beyond the diffraction limit.

Xie, X.S.; Dunn, R.C. (Pacific Northwest Lab., Richland, WA (United States))



Clay Nanoparticle-Supported Single-Molecule Fluorescence Spectroelectrochemistry  

SciTech Connect

We report single-molecule fluorescence spectroelectrochemistry on a clay-modified ITO electrode using cresyl violet as a redox fluorescent probe. Ensemble averaged experiments show that cresyl violet displays well-defined cyclic voltammograms when adsorbed on the clay-modified electrode. By probing the fluorescence intensity of a single cresyl violet molecule absorbed on clay surface, we can trace the redox reaction of individual molecules induced by the cyclic voltammetric potential scanning. Inhomogeneous interfacial electron transfer dynamics of the immobilized single cresyl violet molecules on the clay-modified surface were observed.

Lei, Chenghong; Hu, Dehong; Ackerman, Eric J.



Single-Molecule Cellular Biophysics  

NASA Astrophysics Data System (ADS)

1. Once upon a (length and) time (scale) -; 2. The molecules of life; 3. Making the invisible visible: part 1; 4. Making the invisible visible: part 2; 5. Measuring forces and manipulating single molecules; 6. Single molecule biophysics; 7. Molecules from beyond; 8. Into the membrane; 9. Inside cells; 10. Single molecule biophysics beyond the single cell and beyond the single molecule; Index.

Leake, Mark C.



Image charge effects in single-molecule junctions: Breaking of symmetries and negative-differential resistance in a benzene single-electron transistor  

NASA Astrophysics Data System (ADS)

Both experiments and theoretical studies have demonstrated that the interaction between the current-carrying electrons and the induced polarization charge in single-molecule junctions leads to a strong renormalization of molecular charging energies. However, the effect on electronic excitations and molecular symmetries remain unclear. Using a theoretical framework developed for semiconductor-nanostructure-based single-electron transistors (SETs), we demonstrate that the image charge interaction breaks the molecular symmetries in a benzene-based single-molecule transistor operating in the Coulomb blockade regime. This results in the appearance of a so-called blocking state, which gives rise to negative-differential resistance (NDR). We show that the appearance of NDR and its magnitude in the symmetry-broken benzene SET depends in a complicated way on the interplay between the many-body matrix elements, the lead tunnel coupling asymmetry, and the bias polarity. In particular, the current reducing property of the blocking state causing the NDR is shown to vanish under strongly asymmetric tunnel couplings, when the molecule is coupled stronger to the drain electrode. The calculated I-V characteristic may serve as an indicator for image charge broken molecular symmetries in experimental situations.

Kaasbjerg, K.; Flensberg, K.



Single Molecule Transcription Elongation  

PubMed Central

Single molecule optical trapping assays have now been applied to a great number of macromolecular systems including DNA, RNA, cargo motors, restriction enzymes, DNA helicases, chromosome remodelers, DNA polymerases and both viral and bacterial RNA polymerases. The advantages of the technique are the ability to observe dynamic, unsynchronized molecular processes, to determine the distributions of experimental quantities and to apply force to the system while monitoring the response over time. Here, we describe the application of these powerful techniques to study the dynamics of transcription elongation by RNA polymerase II from Saccharomyces cerevisiae.

Galburt, Eric A.; Grill, Stephan W.; Bustamante, Carlos



Single Molecule Manipulation  

NASA Astrophysics Data System (ADS)

Single-molecule manipulation studies open a door for a close-up investigation of complex biological interactions at the molecular level. In these studies, single biomolecules are pulled while their force response is being monitored. The process is often nonequilibrium, and interpretation of the results has been challenging. We used the atomic force microscope to pull proteins and DNA, and determined the equilibrium properties of the molecules using the recently derived nonequilibrium work theorem. I will present applications of the technique in areas ranging from fundamental biological problems such as DNA mechanics, to complex medical processes such as the mechanical activation of von Willebrand Factor, a key protein in blood coagulation.

Kiang, Ching-Hwa



Single molecule sensing with carbon nanotube devices  

NASA Astrophysics Data System (ADS)

Nanoscale electronic devices like field-effect transistors have long promised to provide sensitive, label-free detection of biomolecules. In particular, single-walled carbon nanotubes have the requisite sensitivity to detect single molecule events and sufficient bandwidth to directly monitor single molecule dynamics in real time. Recent measurements have demonstrated this premise by monitoring the dynamic, single-molecule processivity of three different enzymes: lysozyme, protein Kinase A, and the Klenow fragment of DNA polymerase I. In each case, recordings resolved detailed trajectories of tens of thousands of individual chemical events and provided excellent statistics for single-molecule events. This electronic technique has a temporal resolution approaching 1 microsecond, which provides a new window for observing brief, intermediate transition states. In addition, the devices are indefinitely stable, so that the same molecule can be observed for minutes and hours. The extended recordings provide new insights into rare events like transitions to chemically-inactive conformations.

Choi, Yongki; Sims, Patrick C.; Olsen, Tivoli J.; Iftikhar, Mariam; Corso, Brad L.; Gul, O. Tolga; Weiss, Gregory A.; Collins, Philip G.



Explosion on a Single Molecule Level: A Conceptual Model Based on Ionization and Fragmentation of TNT under Electron Impact.  

National Technical Information Service (NTIS)

Several studies of the mass spectra of nitroaromatic compounds have appeared in recent literature with emphasis on either analytical applications or on the modes of electron impact fragmentation. The chemistry of the nitroaromatic compounds is of consider...

S. Bulusu



Yeast cytochrome c integrated with electronic elements: a nanoscopic and spectroscopic study down to single-molecule level  

Microsoft Academic Search

Various aspects of redox protein integration with nano-electronic elements are addressed by a multi-technique investigation of different yeast cytochrome c (YCC)-based hybrid systems. Three different immobilization strategies on gold via organic linkers are explored, involving either covalent bonding or electrostatic interaction. Specifically, Au surfaces are chemically modified by self-assembled monolayers (SAMs) exposing thiol-reactive groups, or by acid-oxidized single-wall carbon nanotubes

I. Delfino; B. Bonanni; L. Andolfi; C. Baldacchini; A. R. Bizzarri; S. Cannistraro



Statistics and kinetics of single-molecule electron transfer dynamics in complex environments: A simulation model study  

SciTech Connect

Dynamics of the environments of complex systems such as biomolecules, polar solvents, and glass plays an important role in controlling electron transfer reactions. The kinetics is determined by the nature of a complex multidimensional landscape. By quantifying the mean and high-order statistics of the first-passage time and the associated ratios, the dynamics in electron transfer reactions controlled by the environments can be revealed. We consider real experimental conditions with finite observation time windows. At high temperatures, exponential kinetics is observed and there are multiple kinetic paths leading to the product state. At and below an intermediate temperature, nonexponential kinetics starts to appear, revealing the nature of the distribution of local traps on the landscape. Discrete kinetic paths emerge. At very low temperatures, nonexponential kinetics continues to be observed. We point out that the size of the observational time window is crucial in revealing the intrinsic nature of the real kinetics. The mean first-passage time is defined as a characteristic time. Only when the observational time window is significantly larger than this characteristic time does one have the opportunity to collect enough statistics to capture rare statistical fluctuations and characterize the kinetics accurately.

Paula, Luciana C. [Departamento de Fisica, Instituto de Biociencias Letras e Ciencias Exatas, Universidade Estadual Paulista, Sao Jose do Rio Preto, Sao Paulo 15054-000 (Brazil); Departamento de Estudos Basicos e Instrumentais, Universidade Estadual do Sudoeste da Bahia, Itapetinga, Bahia 45700-000 (Brazil); Wang Jin [Department of Chemistry, Physics and Applied Mathematics, State University of New York at Stony Brook, Stony Brook, New York 11794-3800 (United States); State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry of Chinese Academy of Sciences, Changchun 130022 (China); Leite, Vitor B. P. [Departamento de Fisica, Instituto de Biociencias Letras e Ciencias Exatas, Universidade Estadual Paulista, Sao Jose do Rio Preto, Sao Paulo 15054-000 (Brazil)



Electronic transport in biphenyl single-molecule junctions with carbon nanotubes electrodes: The role of molecular conformation and chirality  

SciTech Connect

We investigate, by means of ab initio calculations, electronic transport in molecular junctions composed of a biphenyl molecule attached to metallic carbon nanotubes. We find that the conductance is proportional to cos{sup 2} {theta}, with {theta} the angle between phenyl rings, when the Fermi level of the contacts lies within the frontier molecular orbitals energy gap. This result, which agrees with experiments in biphenyl junctions with nonorganic contacts, suggests that the cos{sup 2} {theta} law has a more general applicability, irrespective of the nature of the electrodes. We calculate the geometrical degree of chirality of the junction, which only depends on the atomic positions, and demonstrate that it is not only proportional to cos{sup 2} {theta} but also is strongly correlated with the current through the system. These results indicate that molecular conformation plays the preponderant role in determining transport properties of biphenyl-carbon nanotubes molecular junctions.

Brito Silva, C. A. Jr.; Granhen, E. R. [Pos-Graduacao em Engenharia Eletrica, Universidade Federal do Para, 66075-900 Belem, PA (Brazil); Silva, S. J. S. da; Leal, J. F. P. [Pos-Graduacao em Fisica, Universidade Federal do Para, 66075-110 Belem, PA (Brazil); Del Nero, J. [Departamento de Fisica, Universidade Federal do Para, 66075-110 Belem, PA (Brazil); Divisao de Metrologia de Materiais, Instituto Nacional de Metrologia, Normalizacao e Qualidade Industrial, 25250-020 Duque de Caxias, RJ (Brazil); Instituto de Fisica, Universidade Federal do Rio de Janeiro, 21941-972 Rio de Janeiro, RJ (Brazil); Pinheiro, F. A. [Instituto de Fisica, Universidade Federal do Rio de Janeiro, 21941-972 Rio de Janeiro, RJ (Brazil)



The single-molecule conductance and electrochemical electron-transfer rate are related by a power law.  


This study examines quantitative correlations between molecular conductances and standard electrochemical rate constants for alkanes and peptide nucleic acid (PNA) oligomers as a function of the length, structure, and charge transport mechanism. The experimental data show a power-law relationship between conductances and charge transfer rates within a given class of molecules with the same bridge chemistry, and a lack of correlation when a more diverse group of molecules is compared, in contrast with some theoretical predictions. Surprisingly, the PNA duplexes exhibit the lowest charge-transfer rates and the highest molecular conductances. The nonlinear rate-conductance relationships for structures with the same bridging chemistries are attributed to differences in the charge-mediation characteristics of the molecular bridge, energy barrier shifts and electronic dephasing, in the two different experimental settings. PMID:23692478

Wierzbinski, Emil; Venkatramani, Ravindra; Davis, Kathryn L; Bezer, Silvia; Kong, Jing; Xing, Yangjun; Borguet, Eric; Achim, Catalina; Beratan, David N; Waldeck, David H



Molecular spintronics using single-molecule magnets  

Microsoft Academic Search

A revolution in electronics is in view, with the contemporary evolution of the two novel disciplines of spintronics and molecular electronics. A fundamental link between these two fields can be established using molecular magnetic materials and, in particular, single-molecule magnets. Here, we review the first progress in the resulting field, molecular spintronics, which will enable the manipulation of spin and

Lapo Bogani; Wolfgang Wernsdorfer



Optically detected spin coherence of single molecules  

NASA Astrophysics Data System (ADS)

Optically detected electron paramagnetic resonance of single molecules of pentacene in a p-terphenyl crystal at 1.8 K is presented. Transient nutation of a single electronic spin is demonstrated, showing a coherence damping within several microseconds. The fluorescence photons of a single molecule can be used as an internal timebase to trigger the application of microwave pulses. Because of this it is possible to enhance or switch off the optically detected magnetic resonance effect, depending on the delay between the triggering photon and the microwave pulse.

Wrachtrup, J.; von Borczyskowski, C.; Bernard, J.; Orrit, M.; Brown, R.



Nanochannel Based Single Molecule Recycling  

PubMed Central

We present a method for measuring the fluorescence from a single molecule hundreds of times without surface immobilization. The approach is based on the use of electroosmosis to repeatedly drive a single target molecule in a fused silica nanochannel through a stationary laser focus. Single molecule fluorescence detected during the transit time through the laser focus is used to repeatedly reverse the electrical potential controlling the flow direction. Our method does not rely on continuous observation and therefore is less susceptible to fluorescence blinking than existing fluorescence-based trapping schemes. The variation in the turnaround times can be used to measure the diffusion coefficient on a single molecule level. We demonstrate the ability to recycle both proteins and DNA in nanochannels and show that the procedure can be combined with single-pair Förster energy transfer. Nanochannel-based single molecule recycling holds promise for studying conformational dynamics on the same single molecule in solution and without surface tethering.

Lesoine, John F.; Venkataraman, Prahnesh A.; Maloney, Peter C.; Dumont, Mark



Introduction to Single-Molecule Transistor  

NASA Astrophysics Data System (ADS)

Basic concepts, within the so-called Coulomb-blockade model, of a single-molecule transistor are reviewed. Beginning with a short summary of the experimental methods, the basic model of the electron transport theory, its single-electron-transistor limit, transport through a multi-level quantum dot (molecule), and the transport through a molecule with excited (vibrational) levels are discussed. Representative experimental results are given. Theories on the interaction between tunneling electrons and the molecular vibration are briefly described.

Nishijima, Mitsuaki


Fluorescence Excitation of Single Molecules.  

National Technical Information Service (NTIS)

A few years ago, many people would have deemed the optical observation of single molecules nearly impossible. Yet, new experiments at room and at liquid helium temperatures have started to remove this psychological barrier. Several applications to trace d...

M. Orrit J. Bernard



IPET and FETR: Experimental Approach for Studying Molecular Structure Dynamics by Cryo-Electron Tomography of a Single-Molecule Structure  

PubMed Central

The dynamic personalities and structural heterogeneity of proteins are essential for proper functioning. Structural determination of dynamic/heterogeneous proteins is limited by conventional approaches of X-ray and electron microscopy (EM) of single-particle reconstruction that require an average from thousands to millions different molecules. Cryo-electron tomography (cryoET) is an approach to determine three-dimensional (3D) reconstruction of a single and unique biological object such as bacteria and cells, by imaging the object from a series of tilting angles. However, cconventional reconstruction methods use large-size whole-micrographs that are limited by reconstruction resolution (lower than 20 Å), especially for small and low-symmetric molecule (<400 kDa). In this study, we demonstrated the adverse effects from image distortion and the measuring tilt-errors (including tilt-axis and tilt-angle errors) both play a major role in limiting the reconstruction resolution. Therefore, we developed a “focused electron tomography reconstruction” (FETR) algorithm to improve the resolution by decreasing the reconstructing image size so that it contains only a single-instance protein. FETR can tolerate certain levels of image-distortion and measuring tilt-errors, and can also precisely determine the translational parameters via an iterative refinement process that contains a series of automatically generated dynamic filters and masks. To describe this method, a set of simulated cryoET images was employed; to validate this approach, the real experimental images from negative-staining and cryoET were used. Since this approach can obtain the structure of a single-instance molecule/particle, we named it individual-particle electron tomography (IPET) as a new robust strategy/approach that does not require a pre-given initial model, class averaging of multiple molecules or an extended ordered lattice, but can tolerate small tilt-errors for high-resolution single “snapshot” molecule structure determination. Thus, FETR/IPET provides a completely new opportunity for a single-molecule structure determination, and could be used to study the dynamic character and equilibrium fluctuation of macromolecules.

Zhang, Lei; Ren, Gang



Photon and electron stimulated surface dynamics of single molecules. Final report on D.O.E. No. DE-FG0295ER14563  

SciTech Connect

The initial goal of this work was to build up an entirely new low-temperature scanning tunneling microscopy (STM) and ultrahigh vacuum system to examine the electron- and photon-induced chemistry of single molecules at low surface temperatures where thermal diffusion would be quenched. The photochemistry of methyl bromide on Pt(111) was first examined at 90 K using liquid nitrogen cooling. Br atoms were quite mobile even at 90 K, and were only visible by STM when coalesced along Pt step edges or in Br islands structures. The 193 nm photofragmentation of methyl bromide efficiently created monovacancies in the substrate at 90 K. It was found that at elevated temperatures there is considerable restructuring and reactive attack of the Pt surface by halogens, but for traditional, lower temperature studies of alkyl radicals prepared by thermal dissociative adsorption of alkyl iodides there is probably no problem with adsorbing I generating monovacancies on the surface. The dynamics of the ho t Br atoms formed by dissociative adsorption of Br{sub 2} was also examined. It was discovered that hot Br atoms from Br{sub 2} dissociative adsorption travel farther than hot O atoms from O{sub 2} dissociative adsorption; hot atom motion from different dissociative adsorption systems had not previously been compared for the same metal substrate. The experimental results strengthened the theoretical case that corrugation of the adsorbate/substrate potential is the key issue in determining hot atom travel. In addition, the data provided strong evidence for the transient existence of a weakly adsorbed and mobile Br{sub 2} precursor to dissociative adsorption. Some experiments imaging individual molecules at 15 K were also conducted.

Harrison, Ian



Imaging Photoinduced Charge Transport in Single Molecules  

NASA Astrophysics Data System (ADS)

The ability to perform sub-Angstrom imaging of photoinduced charge transport in single molecules in ultrahigh vacuum with a low temperature scanning tunneling microscope (STM) indicates the defeat of diffraction limited resolution and the opportunity to understand in new directions nanostructures and their functions. The experiments are enabled by the enhanced field due to coupling of the light from cw and femtosecond lasers to the plasmons in the STM nano-junction and the enhanced field to the molecule that can be monitored by tunneling electrons. By imaging the tunneling electrons as a function of energy and time, it is then possible to record the spatial and temporal evolution of the topography, and the electronic, vibrational, and magnetic states of single molecules. The observation of photoinduced phenomena in nanostructures, including molecules, is relevant to a number of technologies, such as photocatalysis, solar energy harvesting, and optical communication.

Ho, Wilson



Probing Inhomogeneous Vibrational Reorganization Energy Barriers of Interfacial Electron Transfer  

SciTech Connect

We report an atomic force microscopy (AFM) and confocal Raman microscopy study on the interfacial electron transfer of a dye-sensitization system, alizarin adsorbed upon TiO2 nanoparticles. Resonance Raman and absorption spectral analyses revealed the distribution of the mode-specific vibrational reorganization energies encompassing different local sites (~250 nm spatial resolution), suggesting spatially inhomogeneous vibrational reorganization energy and different Franck-Condon coupling factors of the interfacial electron transfer. We found that the total vibrational reorganization energy was inhomogeneous from site to site, and specifically, the mode-specific analyses indicated that the energy distributions were inhomogeneous for bridging normal modes and homogeneous for nonbridging normal modes, especially for modes far away from the alizarin- TiO2 coupling hydroxyl modes. Our results demonstrate a significant step forward in characterizing site-specific inhomogeneous interfacial charge transfer dynamics.

Pan, Duohai; Hu, Dehong; Lu, H. Peter



Single-Molecule DNA Analysis  

NASA Astrophysics Data System (ADS)

The ability to detect single molecules of DNA or RNA has led to an extremely rich area of exploration of the single most important biomolecule in nature. In cases in which the nucleic acid molecules are tethered to a solid support, confined to a channel, or simply allowed to diffuse into a detection volume, novel techniques have been developed to manipulate the DNA and to examine properties such as structural dynamics and protein-DNA interactions. Beyond the analysis of the properties of nucleic acids themselves, single-molecule detection has enabled dramatic improvements in the throughput of DNA sequencing and holds promise for continuing progress. Both optical and nonoptical detection methods that use surfaces, nanopores, and zero-mode waveguides have been attempted, and one optically based instrument is already commercially available. The breadth of literature related to single-molecule DNA analysis is vast; this review focuses on a survey of efforts in molecular dynamics and nucleic acid sequencing.

Efcavitch, J. William; Thompson, John F.



Introduction to Single-Molecule Transistor  

NASA Astrophysics Data System (ADS)

Effects, beyond the Coulomb-blockade model, of a single-molecule transistor are reviewed. The image-charge effect, elastic cotunneling effect (conduction via the Kondo effect), and the inelastic cotunneling effect [non-resonance (vibrational) inelastic electron tunneling spectroscopy] are discussed. Theories on the interaction between the Kondo effect and the molecular vibration are briefly reviewed. Inelastic Kondo effect, switching phenomena and spin-blockade effect are described. Representative experimental results are given.

Nishijima, Mitsuaki


Single-Molecule DNA Analysis  

Microsoft Academic Search

The ability to detect single molecules of DNA or RNA has led to an extremely rich area of exploration of the single most important biomolecule in nature. In cases in which the nucleic acid molecules are tethered to a solid support, confined to a channel, or simply allowed to diffuse into a detection volume, novel techniques have been developed to

J. William Efcavitch; John F. Thompson



Single molecule fluorescence spectroscopy: Toward observation of single molecule reaction  

NASA Astrophysics Data System (ADS)

Advances in room temperature single molecule spectroscopy by laser induced fluorescence provide new tools for the study of individual biological macromolecules under physiological conditions. Two properties of a single fluorescent probe attached to a macromolecule can be exploited to provide local, dynamic structural information. The first, is the very high sensitivity of the fluorophore to its immediate local environment, including the sensitivity to the presence of other fluorophores and quenchers near-by. The second is its unique absorption and emission transition dipoles, which can be interrogated by polarized excitation light and/or by analyzing the emission polarization. Conformational changes can be detected by measuring distance and orientation changes on the macromolecule. Distance changes between two sites on the macromolecule can be measured via single-pair fluorescence resonance energy transfer (spFRET). Orientation changes can be detected by measuring the changes in the dipole orientation of a rigidly attached probe or changes in the rotational freedom of motion of a tethered probe via single molecule fluorescence polarization anisotropy (smFPA). We report spFRET and smFPA measurements done on individual biological macromolecules (DNA, proteins) under physiological condition. Strong inhomogeneity among molecules and time dependent behavior, which would otherwise be hidden in conventional ensemble studies, are clearly revealed. spFRET and smFPA are ideal tools for observing single molecule reactions such as folding/unfolding and enzymatic activities of proteins. Recent results toward establishing structure-function relationship using these tools will be discussed.

Ha, Taekjip



Single-molecule identification via electric current noise  

PubMed Central

Label-free and real-time single-molecule detection may aid the development of high-throughput biosensing platforms. Molecular fluctuations are a source of noise that often hinders single-molecule identification by obscuring the fine details of molecular identity. In this study, we report molecular identification through direct observation of quantum-fluctuation-induced inelastic noise in single organic molecules. We investigated current fluctuations flowing through a single molecule that is chemically connected to two electrodes. We found increased current oscillations synchronous to electric field excitations of characteristic molecular vibrational modes that contribute to inelastic electron tunnelling. This finding demonstrates a large contribution of charge interaction with nuclear dynamics on noise properties of single-molecule bridges and suggests a potential use of inelastic noise as a valuable molecular signature for single-molecule identification.

Tsutsui, Makusu; Taniguchi, Masateru; Kawai, Tomoji



Recording Single Molecule Dynamics and Function using Carbon Nanotube Circuits  

NASA Astrophysics Data System (ADS)

Nanoscale electronic devices like field-effect transistors (FETs) have long promised to provide sensitive, label-free detection of biomolecules. In particular, single-walled carbon nanotubes (SWNTs) have the requisite sensitivity to detect single molecule events, and have sufficient bandwidth to directly monitor single molecule dynamics in real time. Recent measurements have demonstrated this premise by monitoring the dynamic, single-molecule processivity of three different enzymes: lysozyme, protein Kinase A, and the Klenow fragment of polymerase I. Initial successes in each case indicate the generality and attractiveness of SWNT FETs as a new tool to complement other single molecule techniques. Furthermore, our focused research on transduction mechanisms provides the design rules necessary to further generalize this SWNT FET technique. This presentation will summarize these rules, and demonstrate how the purposeful incorporation of just one amino acid is sufficient to fabricate effective, single molecule nanocircuits from a wide range of enzymes or proteins.

Choi, Yongki; Sims, Patrick; Moody, Issa; Olsen, Tivoli; Corso, Brad L.; Tolga Gul, O.; Weiss, Gregory A.; Collins, Philip G.



Single-Molecule Optomechanical Cycle  

NASA Astrophysics Data System (ADS)

Light-powered molecular machines are conjectured to be essential constituents of future nanoscale devices. As a model for such systems, we have synthesized a polymer of bistable photosensitive azobenzenes. Individual polymers were investigated by single-molecule force spectroscopy in combination with optical excitation in total internal reflection. We were able to optically lengthen and contract individual polymers by switching the azo groups between their trans and cis configurations. The polymer was found to contract against an external force acting along the polymer backbone, thus delivering mechanical work. As a proof of principle, the polymer was operated in a periodic mode, demonstrating for the first time optomechanical energy conversion in a single-molecule device.

Hugel, Thorsten; Holland, Nolan B.; Cattani, Anna; Moroder, Luis; Seitz, Markus; Gaub, Hermann E.



Nanodevices for Single Molecule Studies  

Microsoft Academic Search

During the last two decades, biotechnology research has resulted in progress in fields as diverse as the life sciences, agriculture\\u000a and healthcare. While existing technology enables the analysis of a variety of biological systems, new tools are needed for\\u000a increasing the efficiency of current methods, and for developing new ones altogether. Interest has grown in single molecule\\u000a analysis for these

H. G. Craighead; S. M. Stavis; K. T. Samiee


Introduction to Single-Molecule Transistor  

NASA Astrophysics Data System (ADS)

Experimental and theoretical studies on the 2-terminal systems, which are related with a (3-terminal) single-molecule transistor, are reviewed. The measured current- bias-voltage (I-V) characteristics for the representative molecules are compared with the ab initio calculations. Molecular-vibration-induced loss of coherence and the change of the electron-transport mechanism are discussed. Mechanisms for rectification, negative differential resistance and switching of the 2-terminal systems are described. Shot-noise and thermopower studies are briefly mentioned.

Nishijima, Mitsuaki


Nuclear transport of single molecules  

PubMed Central

The mechanism by which macromolecules are selectively translocated through the nuclear pore complex (NPC) is still essentially unresolved. Single molecule methods can provide unique information on topographic properties and kinetic processes of asynchronous supramolecular assemblies with excellent spatial and time resolution. Here, single-molecule far-field fluorescence microscopy was applied to the NPC of permeabilized cells. The nucleoporin Nup358 could be localized at a distance of 70 nm from POM121-GFP along the NPC axis. Binding sites of NTF2, the transport receptor of RanGDP, were observed in cytoplasmic filaments and central framework, but not nucleoplasmic filaments of the NPC. The dwell times of NTF2 and transportin 1 at their NPC binding sites were 5.8 ± 0.2 and 7.1 ± 0.2 ms, respectively. Notably, the dwell times of these receptors were reduced upon binding to a specific transport substrate, suggesting that translocation is accelerated for loaded receptor molecules. Together with the known transport rates, our data suggest that nucleocytoplasmic transport occurs via multiple parallel pathways within single NPCs.

Kubitscheck, Ulrich; Grunwald, David; Hoekstra, Andreas; Rohleder, Daniel; Kues, Thorsten; Siebrasse, Jan Peter; Peters, Reiner



Single Molecule Studies of Chromatin  

SciTech Connect

In eukaryotic cells, DNA is packaged as chromatin, a highly ordered structure formed through the wrapping of the DNA around histone proteins, and further packed through interactions with a number of other proteins. In order for processes such as DNA replication, DNA repair, and transcription to occur, the structure of chromatin must be remodeled such that the necessary enzymes can access the DNA. A number of remodeling enzymes have been described, but our understanding of the remodeling process is hindered by a lack of knowledge of the fine structure of chromatin, and how this structure is modulated in the living cell. We have carried out single molecule experiments using atomic force microscopy (AFM) to study the packaging arrangements in chromatin from a variety of cell types. Comparison of the structures observed reveals differences which can be explained in terms of the cell type and its transcriptional activity. During the course of this project, sample preparation and AFM techniques were developed and optimized. Several opportunities for follow-up work are outlined which could provide further insight into the dynamic structural rearrangements of chromatin.

Jeans, C; Thelen, M P; Noy, A



Signatures of molecular magnetism in single-molecule transport spectroscopy.  


We report single-molecule-transistor measurements on devices incorporating magnetic molecules. By studying the electron-tunneling spectrum as a function of magnetic field, we are able to identify signatures of magnetic states and their associated magnetic anisotropy. A comparison of the data to simulations also suggests that sequential electron tunneling may enhance the magnetic relaxation of the magnetic molecule. PMID:16968018

Jo, Moon-Ho; Grose, Jacob E; Baheti, Kanhayalal; Deshmukh, Mandar M; Sokol, Jennifer J; Rumberger, Evan M; Hendrickson, David N; Long, Jeffrey R; Park, Hongkun; Ralph, D C



Extracting Models in Single Molecule Experiments  

NASA Astrophysics Data System (ADS)

Single molecule experiments can now monitor the journey of a protein from its assembly near a ribosome to its proteolytic demise. Ideally all single molecule data should be self-explanatory. However data originating from single molecule experiments is particularly challenging to interpret on account of fluctuations and noise at such small scales. Realistically, basic understanding comes from models carefully extracted from the noisy data. Statistical mechanics, and maximum entropy in particular, provide a powerful framework for accomplishing this task in a principled fashion. Here I will discuss our work in extracting conformational memory from single molecule force spectroscopy experiments on large biomolecules. One clear advantage of this method is that we let the data tend towards the correct model, we do not fit the data. I will show that the dynamical model of the single molecule dynamics which emerges from this analysis is often more textured and complex than could otherwise come from fitting the data to a pre-conceived model.

Presse, Steve



Electronic structures of interfacial states formed at polymeric semiconductor heterojunctions  

NASA Astrophysics Data System (ADS)

Heterojunctions between organic semiconductors are central to the operation of light-emitting and photovoltaic diodes, providing respectively for electron-hole capture and separation. However, relatively little is known about the character of electronic excitations stable at the heterojunction. We have developed molecular models to study such interfacial excited electronic excitations that form at the heterojunction between model polymer donor and polymer acceptor systems: poly(9,9-dioctylfluorene-co-bis-N,N-(4-butylphenyl)-bis-N,N-phenyl-1,4-phenylenediamine) (PFB) with poly(9,9-dioctylfluorene-co-benzothiadiazole) (F8BT), and poly(9,9-dioctylfluorene-co-N-(4-butylphenyl)diphenylamine) (TFB) with F8BT. We find that for stable ground-state geometries the excited state has a strong charge-transfer character. Furthermore, when partly covalent, modelled radiative lifetimes (~10-7s) and off-chain axis polarization (30?) match observed `exciplex' emission. Additionally for the PFB:F8BT blend, geometries with fully ionic character are also found, thus accounting for the low electroluminescence efficiency of this system.

Huang, Ya-Shih; Westenhoff, Sebastian; Avilov, Igor; Sreearunothai, Paiboon; Hodgkiss, Justin M.; Deleener, Caroline; Friend, Richard H.; Beljonne, David



Time-Resolved, Single Molecule Spectroelectrochemistry of Conjugated Polymers in Contact with ITO  

NASA Astrophysics Data System (ADS)

Time-resolved, single molecule spectroelectrochemistry was used to study excited-state interfacial electron transfer between single conjugated polymer (MEH-PPV) molecules (possessing about 200 redox sites) and an indium tin oxide (ITO) electrode. Decay kinetics and emission yields were obtained while cycling the electrode potential in the range of -.5V to +.5V (Silver wire), which lies between the reduction (-1.5 eV) and oxidation potentials (0.8 eV) of the ground state. At +0.5 V, the emission intensities and average lifetimes were observed to increase about 20% whereas at -0.5 V both values decrease by the same amount. Several possible origins of the potential-induced intensity modulation are proposed.

Grey, John; Palacios, Rodrigo; Chang, Wei-Shun; Miller, William; Bard, Allen; Barbara, Paul



Optical detection of magnetic resonance in a single molecule  

Microsoft Academic Search

MAGNETIC resonance spectroscopy1 is a powerful tool for molecular characterization and structure determination. The sensitivity of conventional approaches is limited to about 1010 electron spins or 1016 nuclear spins; this sensitivity can be improved to about 105 spins by polarizing the spins via optical pumping and detecting optical rather than microwave photons2. Recently, fluorescence from single molecules was detected by

J. Wrachtrup; C. von Borczyskowski; J. Bernard; M. Orritt; R. Brown



Single-molecule fluorescence of nucleic acids.  


Single-molecule fluorescence studies of nucleic acids are revolutionizing our understanding of fundamental cellular processes related to DNA and RNA processing mechanisms. Detailed molecular insights into DNA repair, replication, transcription, and RNA folding and function are continuously being uncovered by using the full repertoire of single-molecule fluorescence techniques. The fundamental reason behind the stunning growth in the application of single-molecule techniques to study nucleic acid structure and dynamics is the unmatched ability of single-molecule fluorescence, and mostly single-molecule FRET, to resolve heterogeneous static and dynamic populations and identify transient and low-populated states without the need for sample synchronization. New advances in DNA and RNA synthesis, post-synthetic dye-labeling methods, immobilization and passivation strategies, improved dye photophysics, and standardized analysis methods have enabled the implementation of single-molecule techniques beyond specialized laboratories. In this chapter, we introduce the practical aspects of applying single-molecule techniques to investigate nucleic acid structure, dynamics, and function. PMID:24108654

McCluskey, Kaley; Shaw, Euan; Lafontaine, Daniel A; Penedo, J Carlos



Single molecule studies of homologous recombination†  

PubMed Central

Single molecule methods offer an unprecedented opportunity to examine complex macromolecular reactions that are obfuscated by ensemble averaging. The application of single molecule techniques to study DNA processing enzymes has revealed new mechanistic details that are unobtainable from bulk biochemical studies. Homologous DNA recombination is a multi-step pathway that is facilitated by numerous enzymes that must precisely and rapidly manipulate diverse DNA substrates to repair potentially lethal breaks in the DNA duplex. In this review, we present an overview of single molecule assays that have been developed to study key aspects of homologous recombination and discuss the unique information gleaned from these experiments.

Finkelstein, Ilya J.; Greene, Eric C.



Single molecule junction conductance and binding geometry  

NASA Astrophysics Data System (ADS)

This Thesis addresses the fundamental problem of controlling transport through a metal-organic interface by studying electronic and mechanical properties of single organic molecule-metal junctions. Using a Scanning Tunneling Microscope (STM) we image, probe energy-level alignment and perform STM-based break junction (BJ) measurements on molecules bound to a gold surface. Using Scanning Tunneling Microscope-based break-junction (STM-BJ) techniques, we explore the effect of binding geometry on single-molecule conductance by varying the structure of the molecules, metal-molecule binding chemistry and by applying sub-nanometer manipulation control to the junction. These experiments are performed both in ambient conditions and in ultra high vacuum (UHV) at cryogenic temperatures. First, using STM imaging and scanning tunneling spectroscopy (STS) measurements we explore binding configurations and electronic properties of an amine-terminated benzene derivative on gold. We find that details of metal-molecule binding affect energy-level alignment at the interface. Next, using the STM-BJ technique, we form and rupture metal-molecule-metal junctions ˜104 times to obtain conductance-vs-extension curves and extract most likely conductance values for each molecule. With these measurements, we demonstrated that the control of junction conductance is possible through a choice of metal-molecule binding chemistry and sub-nanometer positioning. First, we show that molecules terminated with amines, sulfides and phosphines bind selectively on gold and therefore demonstrate constant conductance levels even as the junction is elongated and the metal-molecule attachment point is modified. Such well-defined conductance is also obtained with paracyclophane molecules which bind to gold directly through the pi system. Next, we are able to create metal-molecule-metal junctions with more than one reproducible conductance signatures that can be accessed by changing junction geometry. In the case of pyridine-linked molecules, conductance can be reliably switched between two distinct conductance states using sub-nanometer mechanical manipulation. Using a methyl sulfide linker attached to an oligoene backbone, we are able to create a 3-nm-long molecular potentiometer, whose resistance can be tuned exponentially with Angstom-scale modulations in metal-molecule configuration. These experiments points to a new paradigm for attaining reproducible electrical characteristics of metal-organic devices which involves controlling linker-metal chemistry rather than fabricating identically structured metal-molecule interfaces. By choosing a linker group which is either insensitive to or responds reproducibly to changes in metal-molecule configuration, one can design single molecule devices with functionality more complex than a simple resistor. These ambient temperature experiments were combined with UHV conductance measurements performed in a commercial STM on amine-terminated benzene derivatives which conduct through a non-resonant tunneling mechanism, at temperatures varying from 5 to 300 Kelvin. Our results indicate that while amine-gold binding remains selective irrespective of environment, conductance is not temperature independent, in contrast to what is expected for a tunneling mechanism. Furthermore, using temperature-dependent measurements in ambient conditions we find that HOMO-conducting amines and LUMO-conducting pyridines show opposite dependence of conductance on temperature. These results indicate that energy-level alignment between the molecule and the electrodes changes as a result of varying electrode structure at different temperatures. We find that temperature can serve as a knob with which to tune transport properties of single molecule-metal junctions.

Kamenetska, Maria


Pair Tunneling through Single Molecules  

NASA Astrophysics Data System (ADS)

Coupling to molecular vibrations induces a polaronic shift, and can lead to a negative charging energy, U. For negative U, the occupation of the ground state of the molecule is even. In this situation, virtual pair transitions between the molecule and the leads can dominate electron transport. At low temperature, T, these transitions give rise to the charge-Kondo effect [1]. We developed the electron transport theory through the negative-U molecule [2] at relatively high T, when the Kondo correlations are suppressed. Two physical ingredients distinguish our theory from the transport through a superconducting grain coupled to the normal leads [3]: (i) in parallel with sequential pair-tunneling processes, single-particle cotunneling processes take place; (ii) the electron pair on the molecule can be created (or annihilated) by two electrons tunneling in from (or out to) opposite leads. We found that, even within the rate-equation description, the behavior of differential conductance through the negative-U molecule as function of the gate voltage is quite peculiar: the height of the peak near the degeneracy point is independent of temperature, while its width is proportional to T. This is in contrast to the ordinary Coulomb-blockade conductance peak, whose integral strength is T-independent. At finite source-drain bias, V>>T, the width of the conductance peak is ˜V, whereas the conventional Coulomb-blockade peak at finite V splits into two sharp peaks at detunings V/2, and -V/2. Possible applications to the gate-controlled current rectification and switching will be discussed. [1] A. Taraphder and P. Coleman, Phys. Rev. Lett. 66, 2814 (1991). [2] J. Koch, M. E. Raikh, and F. von Oppen, Phys. Rev. Lett. 96, 056803 (2006). [3] F. W. J. Hekking, L. I. Glazman, K. A. Matveev, and R. I. Shekhter, Phys. Rev. Lett. 70, 4138 (1993).

Raikh, Mikhail



Ceramic Translations. Volume 41. Grain Boundaries and Interfacial Phenomena in Electronic Ceramics.  

National Technical Information Service (NTIS)

This symposium and therefore this proceedings was organized to bring together worldwide expertise in grain boundary and interfacial phenomena in electronic ceramics. Thes issues are key to understanding technological advances in fields as diverse as ferro...

L. M. Levinson S. Hirano



Fluorescent probes and delivery methods for single-molecule experiments.  


The recent explosion in papers utilising single-molecule experiments pushes the envelope further for increased spatial and temporal resolution. In order to achieve this, a combination of novel fluorescent probes and spectroscopy techniques are required. Herein, we provide an overview on our contribution to developments in the field of fluorescent probes along with a palette of alternative delivery methods for introducing the probes into living cells. We discuss probe requirements arising from the use of single-molecule spectroscopy methods and the customisation of probes that depends on the target molecule, the chemical state of the molecule as well as the distance and the type of interaction between sensor and ligand. We explain how Förster resonance energy transfer (FRET) and photon-induced electron transfer (PET) can increase the probe customisation. We also discuss additional requirements that arise when performing experiments in living cells like toxicity and cell permeability. Regarding the latter, we devote a special paragraph on the different ways to introduce the desired probe into the cell and how the different properties of each probe and cell type may require different delivery methods. We offer insights based on our experience working with a variety of single-molecule methods, fluorescent probes and delivery systems. Overall, we encompass the latest developments on probe design and delivery and illustrate that the wealth of information provided by single-molecule studies goes along with increased complexity. PMID:19960557

Lymperopoulos, Konstantinos; Kiel, Alexander; Seefeld, Anne; Stöhr, Katharina; Herten, Dirk-Peter



Sample preparation for single molecule localization microscopy.  


Single molecule localization-based optical nanoscopy was introduced in 2006, surpassing traditional diffraction-limited resolutions by an order of magnitude. Seven years later, this superresolution technique is continuing to follow a trend of increasing popularity and pervasiveness, with the proof-of-concept work long finished and commercial implementations now available. However one important aspect that tends to become lost in translation is the importance of proper sample preparation, with very few resources addressing the considerations that must be made when preparing samples for imaging with single molecule level sensitivity. Presented here is a an in-depth analysis of all aspects of sample preparation for single molecule superresolution, including both live and fixed cell preparation, choice of fluorophore, fixation and staining techniques, and imaging buffer considerations. PMID:24084850

Allen, John R; Ross, Stephen T; Davidson, Michael W



A Practical Guide to Single Molecule FRET  

PubMed Central

Despite the explosive growth in the biological applications of single molecule methods over the last decade, these techniques have thus far been practiced mostly by researchers who are biophysically oriented. This is partly because of the lack of commercial instruments in many cases and also because of the perceived steep learning curve and need for expensive equipments. We wish to provide a practical guide to using Förster (or Fluorescence) Resonance Energy Transfer (FRET) at the single molecule level, focusing on the study of immobilized molecules that allow measurements of single molecule reaction trajectories from about 1 millisecond to many minutes. An instrument can be built at a reasonable cost using various off-the-shelf components and operated reliably using current well-established protocols and freely available software.

Roy, Rahul; Hohng, Sungchul



Protein folding at single-molecule resolution  

PubMed Central

The protein folding reaction carries great significance for cellular function and hence continues to be the research focus of a large interdisciplinary protein science community. Single-molecule methods are providing new and powerful tools for dissecting the mechanisms of this complex process by virtue of their ability to provide views of protein structure and dynamics without associated ensemble averaging. This review briefly introduces common FRET and force methods, and then explores several areas of protein folding where single-molecule experiments have yielded insights. These include exciting new information about folding landscapes, dynamics, intermediates, unfolded ensembles, intrinsically disordered proteins, assisted folding and biomechanical unfolding. Emerging and future work is expected to include advances in single-molecule techniques aimed at such investigations, and increasing work on more complex systems from both the physics and biology standpoints, including folding and dynamics of systems of interacting proteins and of proteins in cells and organisms.

Ferreon, Allan Chris M.; Deniz, Ashok A.



Life at the Single Molecule Level  


In a living cell, gene expression—the transcription of DNA to messenger RNA followed by translation to protein—occurs stochastically, as a consequence of the low copy number of DNA and mRNA molecules involved. Can one monitor these processes in a living cell in real time? How do cells with identical genes exhibit different phenotypes? Recent advances in single-molecule imaging in living bacterial cells allow these questions to be answered at the molecular level in a quantitative manner. It was found that rare events of single molecules can have important biological consequences.


Studies of Interfacial Electronic Processes in Nanoporous TiO2 Thin-Films  

NASA Astrophysics Data System (ADS)

Metal-oxide nanoparticles sensitized to visible light by covalent attachment of molecular adsorbates have attracted considerable attention in recent years due their central role in technologies for solar energy conversion, including dye-sensitized solar cells (DSSCs) and solar photocatalysis. However, the mechanisms of interfacial electron transfer and subsequent electron transport induced by photoexcitation of the molecular adsorbates remain only partially understood. We report recent progress in studies of nanoporous TiO2 thin-films functionalized with molecular adsorbates, with emphasis on interfacial electron injection, molecular rectification and the mechanism of electron transport through sintered TiO2 nanoparticles in thin-films relevant to DSSCs.

Batista, Victor



Density Functional Theory with Dissipation: Transport through Single Molecules  

SciTech Connect

A huge amount of fundamental research was performed on this grant. Most of it focussed on fundamental issues of electronic structure calculations of transport through single molecules, using density functional theory. Achievements were: (1) First density functional theory with dissipation; (2) Pseudopotential plane wave calculations with master equation; (3) Weak bias limit; (4) Long-chain conductance; and (5) Self-interaction effects in tunneling.

Kieron Burke



Interfacial misfit dislocation scattering effect in two-dimensional electron gas channel of GaN heterojunction  

NASA Astrophysics Data System (ADS)

Interfacial misfit dislocation in AlxGa1 - xN/GaN heterojunction was studied as a function of barrier layer thickness and lattice mismatch degree. The sheet charge density induced by interfacial dislocation was obtained. Based on the interfacial dislocation model, the mobility was calculated in two-dimensional electron gas channel. It was found that the mobility was dominated by the interfacial dislocation scattering at low temperature, and depended on the barrier layer thickness and lattice mismatch degree.

Liu, Bing; Lu, Yanwu; Huang, Yan; Liu, Guipeng; Zhu, Qinsheng; Wang, Zhanguo



Single molecule measurements and biological motors  

Microsoft Academic Search

Recent technological advances in lasers and optical detectors have enabled a variety of new, single molecule technologies to be developed. Using intense and highly collimated laser light sources in addition to super-sensitive cameras, the fluorescence of single fluorophores can now be imaged in aqueous solution. Also, laser optical tweezers have enabled the piconewton forces produced by pair of interacting biomolecules

Alex E. Knight; Gregory Mashanov; Justin E. Molloy



Towards single molecule detection using photoacoustic microscopy  

NASA Astrophysics Data System (ADS)

Recently, a number of optical imaging modalities have achieved single molecule sensitivity, including photothermal imaging, stimulated emission microscopy, ground state depletion microscopy, and transmission microscopy. These optical techniques are based on optical absorption contrast, extending single-molecule detection to non-fluorescent chromophores. Photoacoustics is a hybrid technique that utilizes optical excitation and ultrasonic detection, allowing it to scale both the optical and acoustic regimes with 100% sensitivity to optical absorption. However, the sensitivity of photoacoustics is limited by thermal noise, inherent in the medium itself in the form of acoustic black body radiation. In this paper, we investigate the molecular sensitivity of photoacoustics in the context of the thermal noise limit. We show that single molecule sensitivity is achievable theoretically at room temperature for molecules with sufficiently fast relaxation times. Hurdles to achieve single molecule sensitivity in practice include development of detection schemes that work at short working distance, <100 microns, high frequency, <100 MHz, and low loss, <10 dB.

Winkler, Amy M.; Maslov, Konstantin; Wang, Lihong V.



Single-molecule analysis using DNA origami.  


During the last two decades, scientists have developed various methods that allow the detection and manipulation of single molecules, which have also been called "in singulo" approaches. Fundamental understanding of biochemical reactions, folding of biomolecules, and the screening of drugs were achieved by using these methods. Single-molecule analysis was also performed in the field of DNA nanotechnology, mainly by using atomic force microscopy. However, until recently, the approaches used commonly in nanotechnology adopted structures with a dimension of 10-20 nm, which is not suitable for many applications. The recent development of scaffolded DNA origami by Rothemund made it possible for the construction of larger defined assemblies. One of the most salient features of the origami method is the precise addressability of the structures formed: Each staple can serve as an attachment point for different kinds of nanoobjects. Thus, the method is suitable for the precise positioning of various functionalities and for the single-molecule analysis of many chemical and biochemical processes. Here we summarize recent progress in the area of single-molecule analysis using DNA origami and discuss the future directions of this research. PMID:22121063

Rajendran, Arivazhagan; Endo, Masayuki; Sugiyama, Hiroshi



Single-molecule magnets: Iron lines up  

NASA Astrophysics Data System (ADS)

For more than a decade, single-molecule magnets have relied on multinuclear transition metal clusters and lanthanide compounds. Now, a mononuclear, two-coordinate iron(I) complex has shown that single transition metals can compete with the lanthanides when certain design principles from magnetochemistry are borne in mind.

Bill, Eckhard



Hafnium metallocene compounds used as cathode interfacial layers for enhanced electron transfer in organic solar cells  

PubMed Central

We have used hafnium metallocene compounds as cathode interfacial layers for organic solar cells [OSCs]. A metallocene compound consists of a transition metal and two cyclopentadienyl ligands coordinated in a sandwich structure. For the fabrication of the OSCs, poly[3,4-ethylenedioxythiophene]:poly(styrene sulfonate), poly(3-hexylthiophene-2,5-diyl) + [6,6]-phenyl C61 butyric acid methyl ester, bis-(ethylcyclopentadienyl)hafnium(IV) dichloride, and aluminum were deposited as a hole transport layer, an active layer, a cathode interfacial layer, and a cathode, respectively. The hafnium metallocene compound cathode interfacial layer improved the performance of OSCs compared to that of OSCs without the interfacial layer. The current density-voltage characteristics of OSCs with an interfacial layer thickness of 0.7 nm and of those without an interfacial layer showed power conversion efficiency [PCE] values of 2.96% and 2.34%, respectively, under an illumination condition of 100 mW/cm2 (AM 1.5). It is thought that a cathode interfacial layer of an appropriate thickness enhances the electron transfer between the active layer and the cathode, and thus increases the PCE of the OSCs.



Hafnium metallocene compounds used as cathode interfacial layers for enhanced electron transfer in organic solar cells.  


We have used hafnium metallocene compounds as cathode interfacial layers for organic solar cells [OSCs]. A metallocene compound consists of a transition metal and two cyclopentadienyl ligands coordinated in a sandwich structure. For the fabrication of the OSCs, poly[3,4-ethylenedioxythiophene]:poly(styrene sulfonate), poly(3-hexylthiophene-2,5-diyl) + 66-phenyl C61 butyric acid methyl ester, bis-(ethylcyclopentadienyl)hafnium(IV) dichloride, and aluminum were deposited as a hole transport layer, an active layer, a cathode interfacial layer, and a cathode, respectively. The hafnium metallocene compound cathode interfacial layer improved the performance of OSCs compared to that of OSCs without the interfacial layer. The current density-voltage characteristics of OSCs with an interfacial layer thickness of 0.7 nm and of those without an interfacial layer showed power conversion efficiency [PCE] values of 2.96% and 2.34%, respectively, under an illumination condition of 100 mW/cm2 (AM 1.5). It is thought that a cathode interfacial layer of an appropriate thickness enhances the electron transfer between the active layer and the cathode, and thus increases the PCE of the OSCs. PMID:22230259

Park, Keunhee; Oh, Seungsik; Jung, Donggeun; Chae, Heeyeop; Kim, Hyoungsub; Boo, Jin-Hyo



Diamond-based single-molecule magnetic resonance spectroscopy  

NASA Astrophysics Data System (ADS)

The detection of a nuclear spin in an individual molecule represents a key challenge in physics and biology whose solution has been pursued for many years. The small magnetic moment of a single nucleus and the unavoidable environmental noise present the key obstacles for its realization. In this paper, we demonstrate theoretically that a single nitrogen-vacancy center in diamond can be used to construct a nano-scale single-molecule spectrometer that is capable of detecting the position and spin state of a single nucleus and can determine the distance and alignment of a nuclear or electron spin pair. The proposed device would find applications in single-molecule spectroscopy in chemistry and biology, for example in determining the protein structure or in monitoring macromolecular motions, and can thus provide a tool to help unravel the microscopic mechanisms underlying bio-molecular function.

Cai, Jianming; Jelezko, Fedor; Plenio, Martin B.; Retzker, Alex



Quantum dynamics simulations of interfacial electron transfer in sensitized TiO2 semiconductors  

Microsoft Academic Search

A mixed quantum-classical method combining ab initio-DFT molecular dynamics simulations with electronic relaxation calculations is used to investigate interfacial electron transfer in catechol\\/TiO2-anatase nanostructures under vacuum conditions at finite temperature. The calculations demonstrate that the injection mechanism is accelerated by thermal nuclear motion. In particular, electron-phonon scattering leads to ultrafast adsorbate monolayer electron transfer and the disappearance of the anisotropic

Sabas G. Abuabara; Luis G. C. Rego; Victor S. Batista



Enzymatic single-molecule kinetic isotope effects.  


Ensemble-based measurements of kinetic isotope effects (KIEs) have advanced physical understanding of enzyme-catalyzed reactions, but controversies remain. KIEs are used as reporters of rate-limiting H-transfer steps, quantum mechanical tunnelling, dynamics and multiple reactive states. Single molecule (SM) enzymatic KIEs could provide new information on the physical basis of enzyme catalysis. Here, single pair fluorescence energy transfer (spFRET) was used to measure SM enzymatic KIEs on the H-transfer catalyzed by the enzyme pentaerythritol tetranitrate reductase. We evaluated a range of methods for extracting the SM KIE from single molecule spFRET time traces. The SM KIE enabled separation of contributions from nonenzymatic protein and fluorophore processes and H-transfer reactions. Our work demonstrates SM KIE analysis as a new method for deconvolving reaction chemistry from intrinsic dynamics. PMID:23402437

Pudney, Christopher R; Lane, Richard S K; Fielding, Alistair J; Magennis, Steven W; Hay, Sam; Scrutton, Nigel S



Superresolution Imaging using Single-Molecule Localization  

PubMed Central

Superresolution imaging is a rapidly emerging new field of microscopy that dramatically improves the spatial resolution of light microscopy by over an order of magnitude (?10–20-nm resolution), allowing biological processes to be described at the molecular scale. Here, we discuss a form of superresolution microscopy based on the controlled activation and sampling of sparse subsets of photoconvertible fluorescent molecules. In this single-molecule based imaging approach, a wide variety of probes have proved valuable, ranging from genetically encodable photoactivatable fluorescent proteins to photoswitchable cyanine dyes. These have been used in diverse applications of superresolution imaging: from three-dimensional, multicolor molecule localization to tracking of nanometric structures and molecules in living cells. Single-molecule-based superresolution imaging thus offers exciting possibilities for obtaining molecular-scale information on biological events occurring at variable timescales.

Patterson, George; Davidson, Michael; Manley, Suliana; Lippincott-Schwartz, Jennifer



Single-Molecule Analysis of Biomembranes  

NASA Astrophysics Data System (ADS)

Biomembranes are more than just a cell's envelope - as the interface to the surrounding of a cell they carry key signalling functions. Consequentially, membranes are highly complex organelles: they host about thousand different types of lipids and about half of the proteome, whose interaction has to be orchestrated appropriately for the various signalling purposes. In particular, knowledge on the nanoscopic organization of the plasma membrane appears critical for understanding the regulation of interactions between membrane proteins. The high localization precision of ˜20 nm combined with a high time resolution of ˜1 ms made single molecule tracking an excellent technology to obtain insights into membrane nanostructures, even in a live cell context. In this chapter, we will highlight concepts to achieve superresolution by single molecule imaging, summarize tools for data analysis, and review applications on artificial and live cell membranes.

Schmidt, Thomas; Schütz, Gerhard J.


Single molecule transcription profiling with AFM  

NASA Astrophysics Data System (ADS)

Established techniques for global gene expression profiling, such as microarrays, face fundamental sensitivity constraints. Due to greatly increasing interest in examining minute samples from micro-dissected tissues, including single cells, unorthodox approaches, including molecular nanotechnologies, are being explored in this application. Here, we examine the use of single molecule, ordered restriction mapping, combined with AFM, to measure gene transcription levels from very low abundance samples. We frame the problem mathematically, using coding theory, and present an analysis of the critical error sources that may serve as a guide to designing future studies. We follow with experiments detailing the construction of high density, single molecule, ordered restriction maps from plasmids and from cDNA molecules, using two different enzymes, a result not previously reported. We discuss these results in the context of our calculations. Based on invited talk at the International Conference on Nanoscience and Technology 2006.

Reed, Jason; Mishra, Bud; Pittenger, Bede; Magonov, Sergei; Troke, Joshua; Teitell, Michael A.; Gimzewski, James K.



Single molecule transcription profiling with AFM*  

PubMed Central

Established techniques for global gene expression profiling, such as microarrays, face fundamental sensitivity constraints. Due to greatly increasing interest in examining minute samples from micro-dissected tissues, including single cells, unorthodox approaches, including molecular nanotechnologies, are being explored in this application. Here, we examine the use of single molecule, ordered restriction mapping, combined with AFM, to measure gene transcription levels from very low abundance samples. We frame the problem mathematically, using coding theory, and present an analysis of the critical error sources that may serve as a guide to designing future studies. We follow with experiments detailing the construction of high density, single molecule, ordered restriction maps from plasmids and from cDNA molecules, using two different enzymes, a result not previously reported. We discuss these results in the context of our calculations.

Reed, Jason; Mishra, Bud; Pittenger, Bede; Magonov, Sergei; Troke, Joshua; Teitell, Michael A; Gimzewski, James K



Interfacial electron dynamics and hot-electron-driven surface photochemistry of carbon tetrachloride on Ag(111)  

SciTech Connect

We used time-resolved two-photon photoemission (2PPE) spectroscopy to investigate the photochemical behavior, the interfacial electronic structure, and the fate of photogenerated hot electron for carbon tetrachloride adsorbed on Ag(111). The photodissociation cross section was determined over a wide range of photon energy from 1.62 to 5.69 eV, which suggested a low-lying electron affinity level of adsorbed CCl{sub 4}. A CCl{sub 4}-derived unoccupied state located at 3.41 eV above the Fermi level was attributed to an image potential (IP) state based on its binding energy and effective mass. Polarization dependence of the 2PPE signal revealed that the IP state was populated by an indirect excitation process involving scattering of photoexcited hot electrons rather than direct electronic transition from a bulk band. The lifetime of the IP state was much shorter on the CCl{sub 4}-covered Ag(111) surface than on the clean one, implying that the electron in the IP state is scavenged effectively by CCl{sub 4}, probably through dissociative attachment to it. These results are significant in the sense that they provide dynamical evidence for a new relaxation pathway of the IP state in addition to the more common pathway involving back transfer of electron to the substrate.

Ryu, Sunmin; Chang, Jinyoung; Kim, Seong Keun [School of Chemistry, Seoul National University, Seoul 151-747 (Korea, Republic of)



Detection of single molecules in microspheres  

SciTech Connect

We have investigated the use of micron-sized liquid droplets as sample medium to detect single fluorescent molecules in solution. The use of microdroplets (5--15 {mu}m diameter) offers several powerful advantages over single-molecule detection schemes involving measurements on bulk liquids where the probe volume is defined by the laser beam. In addition, cavity-quantum electrodynamical (QED) effects have been observed which influence both spontaneous emission rates and fluorescence yields of dye molecules in these microspheres.

Barnes, M.D.; Whitten, W.B.; Ramsey, J.M. [Oak Ridge National Lab., TN (United States); Ng, K.C. [California State Univ., Fresno, CA (United States). Dept. of Chemistry; Arnold, S. [Polytechnic Inst. of Brooklyn, NY (United States). Microparticle Photophysics Lab.



Single-molecule mechanical identification and sequencing  

Microsoft Academic Search

High-throughput, low-cost DNA sequencing has emerged as one of the challenges of the postgenomic era. Here we present the proof of concept for a single-molecule platform that allows DNA identification and sequencing. In contrast to most present methods, our scheme is not based on the detection of the fluorescent nucleotides but on DNA hairpin length. By pulling on magnetic beads

Fangyuan Ding; Maria Manosas; Michelle M Spiering; Stephen J Benkovic; David Bensimon; Jean-François Allemand; Vincent Croquette



Modeling of N@C60 single-molecule transistors  

NASA Astrophysics Data System (ADS)

We report on recent experimental and theoretical results for single-molecule transistors involving endohedral N@C60 fullerene molecules. In this talk, we will focus on the theoretical modeling. The observed differential conductance shows strong evidence for the exchange interaction between electrons in the fullerene LUMO and the nitrogen p-electrons, favoring an antiferromagnetic interaction. In addition, soft vibrational modes are seen, which are attributed to oscillations of the molecule as a whole. We discuss a model Hamiltonian that reproduces the main features of the experimental conductance.

Timm, Carsten; Grose, Jacob E.; Harneit, Wolfgang; Ralph, Daniel C.



Kondo effects in a C60 single-molecule transistor  

NASA Astrophysics Data System (ADS)

We have used the electromigration technique to fabricate a $\\rm{C_{{60}}}$ single-molecule transistor (SMT). We present a full experimental study as a function of temperature, down to 35 mK, and as a function of magnetic field up to 8 T in a SMT with odd number of electrons, where the usual spin-1/2 Kondo effect occurs, with good agreement with theory. In the case of even number of electrons, a low temperature magneto-transport study is provided, which demonstrates a Zeeman splitting of the zero-bias anomaly at energies well below the Kondo scale.

Roch, N.; Winkelmann, C. B.; Florens, S.; Bouchiat, V.; Wernsdorfer, W.; Balestro, F.



Single-Molecule Imaging of Cellular Signaling  

NASA Astrophysics Data System (ADS)

Single-molecule microscopy is an emerging technique to understand the function of a protein in the context of its natural environment. In our laboratory this technique has been used to study the dynamics of signal transduction in vivo. A multitude of signal transduction cascades are initiated by interactions between proteins in the plasma membrane. These cascades start by binding a ligand to its receptor, thereby activating downstream signaling pathways which finally result in complex cellular responses. To fully understand these processes it is important to study the initial steps of the signaling cascades. Standard biological assays mostly call for overexpression of the proteins and high concentrations of ligand. This sets severe limits to the interpretation of, for instance, the time-course of the observations, given the large temporal spread caused by the diffusion-limited binding processes. Methods and limitations of single-molecule microscopy for the study of cell signaling are discussed on the example of the chemotactic signaling of the slime-mold Dictyostelium discoideum. Single-molecule studies, as reviewed in this chapter, appear to be one of the essential methodologies for the full spatiotemporal clarification of cellular signaling, one of the ultimate goals in cell biology.

De Keijzer, Sandra; Snaar-Jagalska, B. Ewa; Spaink, Herman P.; Schmidt, Thomas


Model systems for single molecule polymer dynamics  

PubMed Central

Double stranded DNA (dsDNA) has long served as a model system for single molecule polymer dynamics. However, dsDNA is a semiflexible polymer, and the structural rigidity of the DNA double helix gives rise to local molecular properties and chain dynamics that differ from flexible chains, including synthetic organic polymers. Recently, we developed single stranded DNA (ssDNA) as a new model system for single molecule studies of flexible polymer chains. In this work, we discuss model polymer systems in the context of “ideal” and “real” chain behavior considering thermal blobs, tension blobs, hydrodynamic drag and force–extension relations. In addition, we present monomer aspect ratio as a key parameter describing chain conformation and dynamics, and we derive dynamical scaling relations in terms of this molecular-level parameter. We show that asymmetric Kuhn segments can suppress monomer–monomer interactions, thereby altering global chain dynamics. Finally, we discuss ssDNA in the context of a new model system for single molecule polymer dynamics. Overall, we anticipate that future single polymer studies of flexible chains will reveal new insight into the dynamic behavior of “real” polymers, which will highlight the importance of molecular individualism and the prevalence of non-linear phenomena.

Latinwo, Folarin



Single molecule observations of DNA hybridization kinetics  

NASA Astrophysics Data System (ADS)

Two 25 base-pair complementary DNA strands are encapsulated within an optically trapped nano-droplet, and we observe the kinetics of their hybridization in dynamic equilibrium via single molecule fluorescence resonance energy transfer (FRET) as a function of temperature and of the solution's NaCl concentration. We have observed binding and unbinding events between the two freely diffusing DNA strands, and our measurements reveal that the duplex can exist in multiple conformational states at elevated temperatures and low concentrations of NaCl.

Jofre, Ana; Case, Jason; Hicks, Sean



Probing Single-Molecule Protein Conformational Dynamics  

SciTech Connect

Protein conformational fluctuations and dynamics, often associated with static and dynamic inhomogeneities, play a crucial role in biomolecular functions. It is extremely difficult to characterize such spatially and temporally inhomogeneous dynamics in an ensemble-averaged measurement, especially when the proteins involve in a multiple-step and multiple-conformation complex chemical interactions and transformations, such as in enzymatic reactions, protein-protein interactions, protein-DNA interactions, and ion-channel membrane protein activities. Single-molecule spectroscopy is a powerful approach to analyze protein conformational dynamics under physiological conditions, providing dynamic perspectives on a molecular-level understanding of protein structure-function mechanisms.




Periodic acceptor excitation spectroscopy of single molecules  

PubMed Central

Alternating-laser excitation (ALEX) spectroscopy has recently been added to the single-molecule spectroscopy toolkit. ALEX monitors interaction and stoichiometry of biomolecules, reports on biomolecular structure by measuring accurate Förster resonance energy transfer (FRET) efficiencies, and allows sorting of subpopulations on the basis of stoichiometry and FRET. Here, we demonstrate that a simple combination of one continuous-wave donor-excitation laser and one directly modulated acceptor-excitation laser (Periodic Acceptor eXcitation) is sufficient to recapitulate the capabilities of ALEX while minimizing the cost and complexity associated with use of modulation techniques.

Doose, Soren; Heilemann, Mike; Michalet, Xavier; Weiss, Shimon



Periodic acceptor excitation spectroscopy of single molecules.  


Alternating-laser excitation (ALEX) spectroscopy has recently been added to the single-molecule spectroscopy toolkit. ALEX monitors interaction and stoichiometry of biomolecules, reports on biomolecular structure by measuring accurate Förster resonance energy transfer (FRET) efficiencies, and allows sorting of subpopulations on the basis of stoichiometry and FRET. Here, we demonstrate that a simple combination of one continuous-wave donor-excitation laser and one directly modulated acceptor-excitation laser (Periodic Acceptor eXcitation) is sufficient to recapitulate the capabilities of ALEX while minimizing the cost and complexity associated with use of modulation techniques. PMID:17279362

Doose, Sören; Heilemann, Mike; Michalet, Xavier; Weiss, Shimon; Kapanidis, Achillefs N



Single-molecule Analysis of Translational Dynamics  

PubMed Central

Decades of extensive biochemical and biophysical research have outlined the mechanism of translation. Rich structural studies have provided detailed snapshots of the translational machinery at all stages of the translation cycle. However the relationship between structural dynamics, composition, and function remains unknown. The heterogeneous and asynchronous nature of translation hinders elucidation of its underlying molecular mechanisms by conventional methods. Single-molecule approaches unsusceptible to these complications have led to the first glances at both compositional and conformational dynamics on the ribosome. These experiments provide the necessary link between static structure and mechanism, often providing new perspectives. Here we review recent advances in the field and their relationship to structural and biochemical data.

Petrov, Alexey; Chen, Jin; O'Leary, Sean; Tsai, Albert; Puglisi, Joseph D.



Single-molecule electrophoresis. Final report  

SciTech Connect

A novel method for the detection and identification of single molecules in solution has been devised, computer-simulated, and experimentally achieved. The technique involves the determination of electrophoretic velocities by measuring the time required by individual molecules to travel a fixed distance between two laser beams. Computer simulations of the process were performed beforehand in order to estimate the experimental feasibility of the method, and to determine the optimum values for the various experimental parameters. Examples of the use of the technique for the ultrasensitive detection and identification of rhodamine-6G, a mixture of DNA restriction fragments, and a mixture of proteins in aqueous solution are presented.

Castro, A.; Shera, E.B.



Hybrid photodetector for single-molecule spectroscopy and microscopy  

PubMed Central

We report benchmark tests of a new single-photon counting detector based on a GaAsP photocathode and an electron-bombarded avalanche photodiode developed by Hamamatsu Photonics. We compare its performance with those of standard Geiger-mode avalanche photodiodes. We show its advantages for FCS due to the absence of after-pulsing and for fluorescence lifetime measurements due to its excellent time resolution. Its large sensitive area also greatly simplifies setup alignment. Its spectral sensitivity being similar to that of recently introduced CMOS SPADs, this new detector could become a valuable tool for single-molecule fluorescence measurements, as well as for many other applications.

Michalet, X.; Cheng, Adrian; Antelman, Joshua; Suyama, Motohiro; Arisaka, Katsushi; Weiss, Shimon



Single Molecule Studies of Nucleocytoplasmic Transport  

PubMed Central

Molecular traffic between the cytoplasm and the nucleoplasm of eukaryotic cells is mediated by nuclear pore complexes (NPCs). Hundreds, if not thousands, of molecules interact with and transit through each NPC every second. The pore is blocked by a permeability barrier, which consists of a network of intrinsically unfolded polypeptides containing thousands of phenylalanine-glycine (FG) repeat motifs. This FG-network rejects larger molecules and admits smaller molecules or cargos bound to nuclear transport receptors (NTRs). For a cargo transport complex, minimally consisting of a cargo molecule plus an NTR, access to the permeability barrier is provided by interactions between the NTR and the FG repeat motifs. Numerous models have been postulated to explain the controlled accessibility and the transport characteristics of the FG-network, but the amorphous, flexible nature of this structure has hindered characterization. A relatively recent development is the ability to monitor the real-time movement of single molecules through individual NPCs via single molecule fluorescence (SMF) microscopy. A major advantage of this approach is that it can be used to continuously monitor a series of specific molecular interactions in an active pore with millisecond time resolution, which therefore allows one to distinguish between kinetic and thermodynamic control. Novel insights and prospects for the future are outlined in this review.

Tu, Li-Chun; Musser, Siegfried M.



Single-molecule chemical denaturation of riboswitches  

PubMed Central

To date, single-molecule RNA science has been developed almost exclusively around the effect of metal ions as folding promoters and stabilizers of the RNA structure. Here, we introduce a novel strategy that combines single-molecule Förster resonance energy transfer (FRET) and chemical denaturation to observe and manipulate RNA dynamics. We demonstrate that the competing interplay between metal ions and denaturant agents provides a platform to extract information that otherwise will remain hidden with current methods. Using the adenine-sensing riboswitch aptamer as a model, we provide strong evidence for a rate-limiting folding step of the aptamer domain being modulated through ligand binding, a feature that is important for regulation of the controlled gene. In the absence of ligand, the rate-determining step is dominated by the formation of long-range key tertiary contacts between peripheral stem-loop elements. In contrast, when the adenine ligand interacts with partially folded messenger RNAs, the aptamer requires specifically bound Mg2+ ions, as those observed in the crystal structure, to progress further towards the native form. Moreover, despite that the ligand-free and ligand-bound states are indistinguishable by FRET, their different stability against urea-induced denaturation allowed us to discriminate them, even when they coexist within a single FRET trajectory; a feature not accessible by existing methods.

Dalgarno, Paul A.; Bordello, Jorge; Morris, Rhodri; St-Pierre, Patrick; Dube, Audrey; Samuel, Ifor D. W.; Lafontaine, Daniel A.; Penedo, J. Carlos



Mechanoenzymatics and Nanoassembly of Single Molecules  

NASA Astrophysics Data System (ADS)

We investigated the muscle enzyme, titin kinase, by means of single-molecule force spectroscopy. Our results show that the binding of ATP, which is the first step of its signaling cascade controlling the muscle gene expression and protein turnover, is mechanically induced. The detailed determination of barrier positions in the mechanical activation pathway and the corresponding functional states allow structural insight, by comparing the experiment with molecular dynamics simulations. From our results, we conclude that titin kinase acts as a natural force sensor controlling the muscle build-up. To study the interplay of functional units, we developed the single-molecule cut-and-paste technique, which combines the precision of AFM with the selectivity of DNA hybridization. Functional units can be assembled one-by-one in an arbitrarily predefined pattern, with an accuracy that is better than 11 nm. The cyclic assembly process is optically monitored and mechanically recorded by force-extension traces. Using biotin as a functional unit attached to the transported DNA, patterns of binding sites may be created, to which streptavidin-modified nanoobjects like fluorescent nanoparticles can specifically self-assemble in a second step.

Puchner, Elias M.; Gaub, Hermann E.


Single-molecule chemical denaturation of riboswitches.  


To date, single-molecule RNA science has been developed almost exclusively around the effect of metal ions as folding promoters and stabilizers of the RNA structure. Here, we introduce a novel strategy that combines single-molecule Förster resonance energy transfer (FRET) and chemical denaturation to observe and manipulate RNA dynamics. We demonstrate that the competing interplay between metal ions and denaturant agents provides a platform to extract information that otherwise will remain hidden with current methods. Using the adenine-sensing riboswitch aptamer as a model, we provide strong evidence for a rate-limiting folding step of the aptamer domain being modulated through ligand binding, a feature that is important for regulation of the controlled gene. In the absence of ligand, the rate-determining step is dominated by the formation of long-range key tertiary contacts between peripheral stem-loop elements. In contrast, when the adenine ligand interacts with partially folded messenger RNAs, the aptamer requires specifically bound Mg(2+) ions, as those observed in the crystal structure, to progress further towards the native form. Moreover, despite that the ligand-free and ligand-bound states are indistinguishable by FRET, their different stability against urea-induced denaturation allowed us to discriminate them, even when they coexist within a single FRET trajectory; a feature not accessible by existing methods. PMID:23446276

Dalgarno, Paul A; Bordello, Jorge; Morris, Rhodri; St-Pierre, Patrick; Dubé, Audrey; Samuel, Ifor D W; Lafontaine, Daniel A; Penedo, J Carlos



Single molecule dynamics in lipid membranes  

NASA Astrophysics Data System (ADS)

Lipid membranes are self-assembled molecular materials that form the membranes of cells. Because of their biological function, lipid membranes are important from a biomedical and biotechnological standpoint. Because of their complex fluid properties, they also provide a rich testbed for studying the structure and dynamics in self-assembled materials and for developing other bio-mimetic structures. In this work, we studied the dynamics of single lipid molecules using experimental and computational techniques. Using single molecule fluorescence microscopy, we tracked the diffusive motion of lipids in phase separated lipid membranes. With the additional techniques of atomic force microscopy and Monte Carlo simulation, we were able to, for the first time experimentally, directly correlate the observed obstructed diffusion with lipid membrane organization. The single molecule tracking tracking experiments required the addition of impurity fluorescent molecules and the assumption that the impurities do not alter the dynamics of the system. To test this assumption, we performed atomistic molecular dynamics simulations of a fluorescently labeled lipid in a lipid membrane. We showed that the fluorescent impurity could have a significant impact on some membrane properties, such as phase behavior, but that relative changes in diffusive behavior are unaffected.

Skaug, Michael James


Single-molecule detection with active transport  

NASA Astrophysics Data System (ADS)

A glass capillary is used near the focal region of a custom-built confocal microscope to investigate the use of active transport for single-molecule detection in solution, with both one and two-photon laser excitation. The capillary tip has a diameter of several microns and is carefully aligned nearby to the sub-micron laser beam waist, collinear to the optical axis, so that a negative pressure-difference causes molecules to be drawn into the capillary, along the laser beam axis. The flow of solution, which is characterized by fluorescence correlation spectroscopy (FCS), can increase the single-molecule detection rate for slowly diffusing proteins by over a factor of 100, while the mean rate of photons during each burst is similar to that for random diffusional transport. Also, the flow is along the longest axis of the ellipsoidally-shaped confocal volume, which results in more collected photons per molecule than that for transverse flow at the same speed. When transport is dominated by flow, FCS can no longer distinguish molecules with differing translational diffusion, and hence a fluorescence fluctuation spectroscopy method based on differences in fluorescence brightness is investigated as a means for assaying different solution components, for applications in pharmaceutical drug discovery. Multi-channel fluctuation spectroscopy techniques can also be used for assays with the flow system and hence this dissertation also reports the characterization of a prototype 4-channel single-photon detector with a two-wavelength polarization-resolved optical set-up.

Ball, David Allan


Single Molecule Dynamics of Adenylate Kinase  

NASA Astrophysics Data System (ADS)

Enzymes are complex molecules evolving on a very convoluted and restricted free energy surface. It is expected that, upon closer examination, their dynamics and kinetics will exhibit complicated behavior. We use single molecule fluorescence spectroscopy to study these spatial and temporal heterogeneities. Adenylate Kinase (AK) is an important enzyme in the regulation of the cellular energy balance. It catalyzes the interconversion of ATP, ADP, and AMP. Because of its critical roles in cellular function, any spatial or temporal heterogeneities in the activity of AK may have important consequences on the cellular level. We have labeled a mutant of E. Coli AK with a pair of fluorescent substituents to monitor its dynamics using single-molecule Förster Resonance Energy Transfer. To accurately measure the distance between these two residues in real time, we have developed a new non-parametric method of analysis that delivers time and distance resolution approaching the theoretical limit. Our maximum information algorithm analyzes the trajectory on a photon-by-photon basis, utilizing the information carried by each photon to achieve sufficient time resolution to observe the reactive complex in detail. These measurements allow us to calculate the static conformational distributions of individual molecules. The time dependent observations also allow us to measure temporal heterogeneity in the reactivity of AK. The measured reactive trajectories indicate a correlated structural flexibility and reaction dynamics. This newly developed methodology may allow us to begin to study the ways in which reactive heterogeneity affects its cellular function.

Watkins, Lucas; Yang, Haw



Single-molecule Imaging of Chaperonin Functions  

NASA Astrophysics Data System (ADS)

Single-molecule imaging is a powerful technique to study functions of biological molecules. As an application of this method, dynamics of the chaperonin (GroEL)-cochaperonin (GroES) interaction and chaperonin-asisited protein folding were visualized by using total internal reflection fluorescence microscopy. Single molecule analysis revealed for the first time that the release of GroES from GroEL was governed by the two successive timers; a lag period (-3s) preceded the net release process (-5s). The protein folding in the immobilized cis complex, monitored using green fluorescent protein and triggered by photolysis of caged-ATP, also started after a lag period (-3s). Thus, the first timer defines the 3s lifetime of a novel intermediate state in which both non-native protein and GroES attach to GroEL. Although increasing number of reports on the observation and analysis of a single protein molecule have been published recently, the study presented here is the first “live-scene” visualization of functional association-dissociation between proteins and of protein folding. The power of this approach will not be restricted in the studies on chaperonin but be extended to the studies on other protein-protein interaction.

Ueno, Taro; Tadakuma, Hisashi; Funatsu, Takashi


Optical Studies of Single Molecules at Room Temperature  

Microsoft Academic Search

Recent developments in optical studies of single molecules at room temperature are reviewed, with an emphasis on the underlying principles and the potential of single-molecule experiments. Examples of single-molecule studies are given, including photophysics and photochemistry pertinent to single-molecule measurements, spectral fluctuations, Raman spectroscopy, diffusional motions, conformational dynamics, fluorescence resonant energy transfer, exciton dynamics, and enzymatic turnovers. These studies illustrate

Xiaoliang Xie; Jay K. Trautman



Coherent Control of Single Molecules at Room Temp  

NASA Astrophysics Data System (ADS)

Electronic coherence plays a key role in natural processes like ultrafast energy transfer and charge separation. Coherent control has proven powerful, however in complex biosystems with different conformations and environments, the intrinsic inhomogeneity of the synchronized subset severely limits the achievable degree of control. The ultimate solution to overcome intrinsic inhomogeneities is the investigation of the behavior of one molecule at a time. Here we report the observation and manipulation of vibrational wave-packet interference and electronic coherence in individual molecules at ambient conditions. Adapting time and phase distribution of the optical excitation field to the dynamics of each molecule we achieve a superior degree of control. The time-phase maps show distinct diversity between different, yet chemically identical, molecules. We induce Rabi-oscillations and control the coherent superposition state in a single molecule. Broadly distributed coherence decay times are found for different individual molecules giving direct insight into the structural heterogeneity of the local surroundings. Our approach allows single-molecule coherent control in a variety of complex inhomogeneous systems and thus to study the role of coherence in energy transfer of single biocomplexes under natural conditions. D.Brinks et al. Nature 465, 905 (2010); R.Hildner et al. Nat.Physics doi:10.1038/nphys1858 (2010).

van Hulst, Niek; Brinks, Daan; Hildner, Richard



Single-Molecule STM Studies on Atomically-Flat Nanoparticles  

NASA Astrophysics Data System (ADS)

The scanning tunneling microscope (STM) has been broadly applied to measure electronic characteristics of individual molecules supported in an inert monolayer matrix, which is typically grown on gold thin films on mica or bulk single crystal substrates. Although these substrates are excellent for electronic measurements, they have serious disadvantages for optical measurements because they are not optically transparent and the metal surface can quench the molecular excited state. We demonstrate that single molecule electronic measurements can also be performed using atomically-flat gold nanoparticles (FGNPs) supported on indium tin oxide coated glass as a replacement for the typical gold substrate. These substrates are optically transparent and each of the FGNP ``nanosubstrates'' is an optically resonant photonic antenna, thus they have the added advantage that optical measurements can be performed.

Dahayanaka, D. H.; Kelle, D. W.; Wasielewski, D. J.; Day, E. S.; White, D. R.; Bumm, L. A.; Waite, C. M.; Moore, J. L.; Halterman, R. L.



Vibrationally induced decoherence in single-molecule junctions  

NASA Astrophysics Data System (ADS)

We investigate the interplay of quantum interference effects and electronic-vibrational coupling in electron transport through single-molecule junctions, employing a nonequilibrium Green's function approach. Our findings show that inelastic processes lead, in general, to a quenching of quantum interference effects. This quenching is more pronounced for increasing bias voltages and levels of vibrational excitation. As a result of this vibrationally induced decoherence, vibrational signatures in the transport characteristics of a molecular contact may strongly deviate from a simple Franck-Condon picture. This includes signatures in both the resonant and the nonresonant transport regimes. Moreover, it is shown that local cooling by electron-hole pair creation processes can influence the transport characteristics profoundly, giving rise to a significant temperature dependence of the electrical current.

Härtle, R.; Butzin, M.; Thoss, M.



Protein folding studied by single molecule FRET  

PubMed Central

A complete understanding of a protein folding mechanism requires description of the distribution of microscopic pathways that connect the folded and unfolded states. This distribution can, in principle, be described by computer simulations and theoretical models of protein folding, but is hidden in conventional experiments on large ensembles of molecules because only average properties are measured. A long-term goal of single molecule fluorescence studies is to time-resolve the structural events as individual molecules make transitions between folded and unfolded states. Although such studies are still in their infancy, the work up to now shows great promise and has already produced novel and important information on current issues in protein folding that has been impossible or difficult to obtain from ensemble measurements.

Schuler, Benjamin; Eaton, William A.



Single molecule detection for in vitro diagnostics  

NASA Astrophysics Data System (ADS)

In this paper we present a novel highly sensitive detection system for diagnostic applications. The system is designed to meet the needs of medical diagnostics for reliable measurements of pathogens and biomarkers in the low concentration regime. It consists of a confocal detection unit, micro-structured sampling cells, and a "Virtual lab" analysis software. The detection unit works with laser induced fluorescence and is designed to provide accurate and highly sensitive measurement at the single molecule level. Various sampling cells are micro-structured in glass, silicon or polymers to enable measurements under flow and nonflow conditions. Sampling volume is below one microliter. The "Virtual lab" software analyzes the light intensity online according to the patent pending "Accurate Stochastic Fluorescence Spectroscopy" (ASFS) developed by FluIT Biosystems GmbH. Tools for simulation and experiment optimization are included as well. Experimental results for various applications with relevance for in vitro diagnostics will be presented.

Kirner, Thomas; Ackermann, Jörg; Mathis, Harald P.; Greiner, Benjamin; Tonn, Thomas; Tschachojan, David; Kukoc-Zivojnov, Natasa; Giehring, Sebastian



High throughput single molecule detection for monitoring biochemical reactions  

PubMed Central

The design, performance and application of a novel optical system for high throughput single molecule detection (SMD) configured in a continuous flow format using microfluidics is reported. The system consisted of a microfabricated polymer-based multi-channel fluidic network situated within the optical path of a laser source (?ex = 660 nm) with photon transduction accomplished using an electron-multiplying charge coupled device (EMCCD) operated in a frame transfer mode that allowed tracking single molecules as they passed through a large field-of-view (FoV) illumination zone. The microfluidic device consisted of 30 microchannels possessing dimensions of 30 ?m (width) × 20 ?m (depth) with a 25 mm pitch. Individual molecules were electrokinetically driven through the fluidic network and excited within the wide-field illumination area with the resulting fluorescence collected via an objective and imaged onto the EMCCD camera. The detection system demonstrated sufficient sensitivity to detect single DNA molecules labeled with a fluorescent tag (AlexaFluor 660) identified through their characteristic emission wavelength and the burst of photons produced during their transit through the excitation volume. In its present configuration and fluidic architecture, the sample processing throughput was ?4.02 × 105 molecules s?1, but could be increased dramatically through the use of narrower channels and a smaller pitch. The system was further evaluated using a single molecule-based fluorescence quenching assay for measuring the population differences between duplexed and single-stranded DNA molecules as a function of temperature for determining the duplex melting temperature, Tm.

Okagbare, Paul I.; Soper, Steven A.



Bringing single-molecule spectroscopy to macromolecular protein complexes  

PubMed Central

Single-molecule fluorescence spectroscopy offers real-time, nanometer-resolution information. Over the past two decades, this emerging single-molecule technique has been rapidly adopted to investigate the structural dynamics and biological functions of proteins. Despite this remarkable achievement, single-molecule fluorescence techniques must be extended to macromolecular protein complexes that are physiologically more relevant for functional studies. In this review, we present recent major breakthroughs for investigating protein complexes within cell extracts using single-molecule fluorescence. We outline the challenges, future prospects and potential applications of these new single-molecule fluorescence techniques in biological and clinical research.

Joo, Chirlmin; Fareh, Mohamed; Kim, V. Narry



Probing interfacial electronic structures in atomic layer LaMnO{sub3} and SrTiO{sub 3} superlattices.  

SciTech Connect

The interfacial electronic structure characterization of a m x (LaMnO{sub 3})/n x (SrTiO{sub 3}) superlattice based on scanning transmission electron microscopy and electron energy loss spectroscopy. Evidence of interfacial band alignment and electron transfer are presented based on the observation of O K edge of individual transition metal and oxygen atomic columns. Electron probe aberration correction was essential for the high spatial resolution mapping of interfacial electronic states.

Shah, A. B.; Ramasse, Q. M.; Zhai, X.; Wen, J. G.; May, S. J.; Petrov, I.; Bhattacharya, A.; Abbamonte, P.; Eckstein, J. N.; Zuo, J.-M.; Univ. of Illinois; LBNL



Optical detection of magnetic resonance in a single molecule  

NASA Astrophysics Data System (ADS)

MAGNETIC resonance spectroscopy1 is a powerful tool for molecular characterization and structure determination. The sensitivity of conventional approaches is limited to about 1010 electron spins or 1016 nuclear spins; this sensitivity can be improved to about 105 spins by polarizing the spins via optical pumping and detecting optical rather than microwave photons2. Recently, fluorescence from single molecules was detected by tuning a single-frequency laser in the inhomogeneously broadened fluorescence excitation band of a dilute dispersion of pentacene in a host crystal of p-terphenyl3,4. Here we report that, by combining single-molecule fluorescence spectroscopy with optically detected magnetic resonance for the pentacene-doped p-terphenyl system, we can detect magnetic resonance in a single pentacene molecule. We observe two of the three possible transitions between sublevels of the metastable triplet state. The spectral lineshapes indicate that the proton nuclear spin states change during the measurement, leading to spectral diffusion within the magnetic resonance line.

Wrachtrup, J.; von Borczyskowski, C.; Bernard, J.; Orritt, M.; Brown, R.



Conduction mechanisms in biphenyl dithiol single-molecule junctions  

NASA Astrophysics Data System (ADS)

Based on density-functional theory calculations, we report a detailed study of the single-molecule charge-transport properties for a series of recently synthesized biphenyl-dithiol molecules [D. Vonlanthen , Angew. Chem., Int. Ed.1433-785110.1002/anie.200903946 48, 8886 (2009); A. Mishchenko , Nano Lett.NALEFD1530-698410.1021/nl903084b 10, 156 (2010)]. The torsion angle ? between the two phenyl rings, and hence the degree of ? conjugation, is controlled by alkyl chains and methyl side groups. We consider three different coordination geometries, namely, top-top, bridge-bridge, and hollow-hollow, with the terminal sulfur atoms bound to one, two, and three gold surface atoms, respectively. Our calculations show that different coordination geometries give rise to conductances that vary by one order of magnitude for the same molecule. Irrespective of the coordination geometries, the charge transport calculations predict a cos2? dependence of the conductance, which is confirmed by our experimental measurements. We demonstrate that the calculated transmission through biphenyl dithiols is typically dominated by a single transmission eigenchannel formed from ? electrons. For perpendicular orientation of the rings a residual conductance arises from ?-? couplings. But only for a single molecule with a completely broken conjugation we find a nearly perfect degeneracy of the ?-? eigenchannels for the hollow-hollow-type contact in our theory.

Bürkle, M.; Viljas, J. K.; Vonlanthen, D.; Mishchenko, A.; Schön, G.; Mayor, M.; Wandlowski, T.; Pauly, F.



Single-molecule fluorescence quenching near small nanoparticles  

NASA Astrophysics Data System (ADS)

We study theoretically radiative and nonradiative decay of a single molecule near small gold nanoparticle. The local field enhancement leads to an increased radiative decay rate while the energy transfer from molecule to optically-inactive electronic states in nanoparticle results in a decrease in fluorescence quantum efficiency for small molecule-nanoparticle distances. We performed a DFT-TDLDA calculation of both the enhancement and the quenching for small nanometer-sized gold nanoparticles. We found that in a close proximity to the surface, the non-radiative decay rate is dominated by generation of electron-hole pairs out of the Fermi sea resulting in a significantly lower quantum efficiency as compared to that obtained from electromagnetic calculations. For large distances, the efficiency is maximal for molecule polarized normal to the surface, whereas for small distances it is maximal for parallel orientation.

Pustovit, V. N.; Shahbazyan, T. V.



Single molecule force spectroscopy of asphaltene aggregates.  


Asphaltene aggregation and deposition cause severe problems in nearly all phases of petroleum processing. To resolve those problems, understanding the aggregation mechanisms is a prerequisite and has attracted the interest of a great number of investigators. However, to date, the nature and extent of asphaltene aggregation remain widely debated. In the present study, we attempt to investigate asphaltene aggregation from a completely new perspective. The technique of single molecule force spectroscopy (SMFS) was used to investigate the response of single asphaltene aggregates under an external pulling force. Force curves representing the stretching of single asphaltene aggregates were obtained in simple electrolyte solutions (KCl and calcium) and organic solvents (toluene and heptane). These force curves were well-fitted by the modified worm-like chain model, indicating that those asphaltene aggregates acted like long-chain polymers under pulling by an external force. It was found that lower solution pH values and the presence of divalent cations resulted in a lower bending rigidity of the formed aggregates. The information retrieved from the force curves suggests that asphaltene molecules with a structure featuring small aromatic clusters connected by aliphatic chains do exist and that asphaltene aggregation could occur through a linear polymerization mechanism. The current study extends the application scope of SMFS. PMID:17441741

Long, Jun; Xu, Zhenghe; Masliyah, Jacob H



Single-Molecule Fluorescent Particle Tracking  

NASA Astrophysics Data System (ADS)

One of the most fascinating processes in biology is the directed movement of organisms, subcellular compartments, and single proteins. Tracking the cellular motion is of great interest to single-molecule biophysicists to understand the mechanism of wide variety of biological processes, from basic mechanism of molecular machines to protein--protein interactions. In the last two decades, random diffusion of proteins and lipids has been tracked under the fluorescence microscope to understand how they associate with their targeted molecules. However, cellular motility is not limited to diffusion of small particles. Many fundamental processes occur by discrete physical movements upon each enzymatic cycle. For example, motor proteins of cytoskeleton can transport intracellular cargoes by taking nanometer-sized steps along the linear tracks within the cell. Several high precision techniques have been developed to understand the working principles and kinetics of motors in a detailed manner. This chapter summarizes the recent advances in fluorescence microscopy techniques that allow high precision tracking of biological molecules.

Yildiz, Ahmet


Single-Molecule Manipulation Using Optical Traps  

NASA Astrophysics Data System (ADS)

One of the most sensitive tools for manipulating single molecules and measuring their properties is the optical trap, also known as optical tweezers. Consisting essentially of a strongly focused light beam, optical traps were first developed and demonstrated in the 1970s and 1980s by Arthur Ashkin and colleagues (Ashkin et al. 1986). These early pioneers showed that micron-sized dielectric particles could be held and manipulated in solution by using optical forces to create a stable, three-dimensional potential well. Since then, optical trapping instrumentation has been refined and developed such that piconewton forces are now routinely applied, while at the same time measuring the resultant displacements to nanometre or even angström resolution. As a result of these advances, optical traps have been applied widely, from cytometry to the study of mesoscopic colloids and polymers and of course the properties of single biological macromolecules. This chapter begins with a description of the theory and design of optical traps, followed by an illustrative discussion of applications to the study of structure formation and molecular motors, a description of typical "tricks of the trade" for using optical traps, and a brief look at techniques for extending the capabilities of traps.

Woodside, Michael T.; Valentine, Megan T.


Single-molecule, single-particle fluorescence imaging of TiO2-based photocatalytic reactions.  


Nanostructured metal oxide semiconductors, such as TiO(2) and ZnO, have attracted great attention as the promising material for photovoltaic devices, photocatalysts for water splitting and environmental purification, sensors, batteries, etc. In this critical review, we have focused on the on-site observation of interfacial chemical reactions involving charge carriers and reactive oxygen species (ROS), such as singlet oxygen and the hydroxyl radical, generated by the photoexcitation of TiO(2) nanoparticles using single-molecule, single-particle fluorescence spectroscopy. Advanced fluorescence imaging techniques enable us to determine the location of the photocatalytically active sites that are closely related to the defects heterogeneously distributed on the surface. Consequently, this review provides a great opportunity to understand the temporal and spatial heterogeneities within an individual catalyst particle, allowing for the potential use of single-molecule, single-particle approaches in the analysis of photocatalytic reactions (189 references). PMID:20824247

Tachikawa, Takashi; Majima, Tetsuro



Dissecting contact mechanics from quantum interference in single-molecule junctions of stilbene derivatives.  


Electronic factors in molecules such as quantum interference and cross-conjugation can lead to dramatic modulation and suppression of conductance in single-molecule junctions. Probing such effects at the single-molecule level requires simultaneous measurements of independent junction properties, as conductance alone cannot provide conclusive evidence of junction formation for molecules with low conductivity. Here, we compare the mechanics of the conducting para-terminated 4,4'-di(methylthio)stilbene and moderately conducting 1,2-bis(4-(methylthio)phenyl)ethane to that of insulating meta-terminated 3,3'-di(methylthio)stilbene single-molecule junctions. We simultaneously measure force and conductance across single-molecule junctions and use force signatures to obtain independent evidence of junction formation and rupture in the meta-linked cross-conjugated molecule even when no clear low-bias conductance is measured. By separately quantifying conductance and mechanics, we identify the formation of atypical 3,3'-di(methylthio)stilbene molecular junctions that are mechanically stable but electronically decoupled. While theoretical studies have envisaged many plausible systems where quantum interference might be observed, our experiments provide the first direct quantitative study of the interplay between contact mechanics and the distinctively quantum mechanical nature of electronic transport in single-molecule junctions. PMID:22352939

Aradhya, Sriharsha V; Meisner, Jeffrey S; Krikorian, Markrete; Ahn, Seokhoon; Parameswaran, Radha; Steigerwald, Michael L; Nuckolls, Colin; Venkataraman, Latha



From single molecule to single tubules  

NASA Astrophysics Data System (ADS)

Biological systems often make decisions upon conformational changes and assembly of single molecules. In vivo, epithelial cells (such as the mammary gland cells) can respond to extracellular matrix (ECM) molecules, type I collagen (COL), and switch their morphology from a lobular lumen (100-200 micron) to a tubular lumen (1mm-1cm). However, how cells make such a morphogenetic decision through interactions with each other and with COL is unclear. Using a temporal control of cell-ECM interaction, we find that epithelial cells, in response to a fine-tuned percentage of type I collagen (COL) in ECM, develop various linear patterns. Remarkably, these patterns allow cells to self-assemble into a tubule of length ˜ 1cm and diameter ˜ 400 micron in the liquid phase (i.e., scaffold-free conditions). In contrast with conventional thought, the linear patterns arise through bi-directional transmission of traction force, but not through diffusible biochemical factors secreted by cells. In turn, the transmission of force evokes a long-range (˜ 600 micron) intercellular mechanical interaction. A feedback effect is encountered when the mechanical interaction modifies cell positioning and COL alignment. Micro-patterning experiments further reveal that such a feedback is a novel cell-number-dependent, rich-get-richer process, which allows cells to integrate mechanical interactions into long-range (> 1mm) linear coordination. Our results suggest a mechanism cells can use to form and coordinate long-range tubular patterns, independent of those controlled by diffusible biochemical factors, and provide a new strategy to engineer/regenerate epithelial organs using scaffold-free self-assembly methods.

Guo, Chin-Lin



Microarray analysis at single molecule resolution  

PubMed Central

Bioanalytical chip-based assays have been enormously improved in sensitivity in the recent years; detection of trace amounts of substances down to the level of individual fluorescent molecules has become state of the art technology. The impact of such detection methods, however, has yet not fully been exploited, mainly due to a lack in appropriate mathematical tools for robust data analysis. One particular example relates to the analysis of microarray data. While classical microarray analysis works at resolutions of two to 20 micrometers and quantifies the abundance of target molecules by determining average pixel intensities, a novel high resolution approach [1] directly visualizes individual bound molecules as diffraction limited peaks. The now possible quantification via counting is less susceptible to labeling artifacts and background noise. We have developed an approach for the analysis of high-resolution microarray images. It consists first of a single molecule detection step, based on undecimated wavelet transforms, and second, of a spot identification step via spatial statistics approach (corresponding to the segmentation step in the classical microarray analysis). The detection method was tested on simulated images with a concentration range of 0.001 to 0.5 molecules per square micron and signal-to-noise ratio (SNR) between 0.9 and 31.6. For SNR above 15 the false negatives relative error was below 15%. Separation of foreground/background proved reliable, in case foreground density exceeds background by a factor of 2. The method has also been applied to real data from high-resolution microarray measurements.

Muresan, Leila; Jacak, Jaroslaw; Klement, Erich Peter; Hesse, Jan; Schutz, Gerhard J.



Single-molecule refrigerators: Substitution and gate effects  

NASA Astrophysics Data System (ADS)

Using a first-principles approach, we investigate the quantum cooling effects in single-molecule junctions. In comparison with the unsubstituted butanethiol single-molecule junction as a refrigerator, the amino-substituted butanethiol single-molecule junction shows significant enhancement in the coefficient of performance (COP). The enhancement is attributed to the appearance of new states in the neighborhood of chemical potentials due to amino substitution. The COP of butanethiol refrigerator can be improved further by the gate voltages.

Liu, Yu-Shen; Chen, Yu-Chang



Single-Molecule Fluorescence Studies of RNA: A Decade's Progress  

PubMed Central

Over the past decade, single-molecule fluorescence studies have elucidated the structure-function relationship of RNA molecules. The real-time observation of individual RNAs by single-molecule fluorescence has unveiled the dynamic behavior of complex RNA systems in unprecedented detail, revealing the presence of transient intermediate states and their kinetic pathways. This review provides an overview of how single-molecule fluorescence has been used to explore the dynamics of RNA folding and catalysis.

Karunatilaka, Krishanthi S.; Rueda, David



Single Molecule Manipulation of Co-Salophene-Br2  

NASA Astrophysics Data System (ADS)

Using Low Temperature Scanning Tunneling Microscopy (LT-STM) manipulation we studied the system of individual Co-Salophene-Br2 molecules adsorbed on a Au(111) surface. Co-Salophene-Br2 has a metallic ion caged on three sides, and two of three ?-rings have bromine termination. This molecule adsorbs onto Au(111) in a planar orientation and forms ordered molecular clusters. With the STM tip single molecules were pulled out from molecular clusters and then the bromine atoms were individually dissociated to form Co-Salophene molecules having two, one, or zero attached bromine atoms. The bond dissociation was selectively done by locally injecting tunneling electrons. In conjunction with our manipulation studies Kondo resonances of the intact and de-brominated molecules were probed by means of local tunneling spectroscopy and spectroscopic mapping. This work is a step towards the engineering of molecular systems on a surface from basic molecular units.

Dilullo, Andrew; Chang, Shih-Hsin; Hoffman, Germar; Wiesendanger, Roland; Hla, Saw-Wai



Single-molecule chemical reactions on DNA origami  

NASA Astrophysics Data System (ADS)

DNA nanotechnology and particularly DNA origami, in which long, single-stranded DNA molecules are folded into predetermined shapes, can be used to form complex self-assembled nanostructures. Although DNA itself has limited chemical, optical or electronic functionality, DNA nanostructures can serve as templates for building materials with new functional properties. Relatively large nanocomponents such as nanoparticles and biomolecules can also be integrated into DNA nanostructures and imaged. Here, we show that chemical reactions with single molecules can be performed and imaged at a local position on a DNA origami scaffold by atomic force microscopy. The high yields and chemoselectivities of successive cleavage and bond-forming reactions observed in these experiments demonstrate the feasibility of post-assembly chemical modification of DNA nanostructures and their potential use as locally addressable solid supports.

Voigt, Niels V.; Tørring, Thomas; Rotaru, Alexandru; Jacobsen, Mikkel F.; Ravnsbæk, Jens B.; Subramani, Ramesh; Mamdouh, Wael; Kjems, Jørgen; Mokhir, Andriy; Besenbacher, Flemming; Gothelf, Kurt Vesterager



A molecular platinum cluster junction: a single-molecule switch.  


We present a theoretical study of electron transport through single-molecule junctions incorporating a Pt(6) metal cluster bound within an organic framework. The insertion of this molecule between a pair of electrodes leads to a fully atomically engineered nanometallic device with high conductance at the Fermi level and two sequential high on/off switching states. The origin of this property can be traced back to the existence of a degenerate HOMO consisting of two asymmetric orbitals with energies close to the Fermi level of the metal leads. The degeneracy is broken when the molecule is contacted to the leads, giving rise to two resonances that become pinned to the Fermi level and display destructive interference. PMID:23330549

Zotti, Linda A; Leary, Edmund; Soriano, Maria; Cuevas, Juan Carlos; Palacios, Juan Jose



Quantum phase transition in a single-molecule quantum dot.  


Quantum criticality is the intriguing possibility offered by the laws of quantum mechanics when the wave function of a many-particle physical system is forced to evolve continuously between two distinct, competing ground states. This phenomenon, often related to a zero-temperature magnetic phase transition, is believed to govern many of the fascinating properties of strongly correlated systems such as heavy-fermion compounds or high-temperature superconductors. In contrast to bulk materials with very complex electronic structures, artificial nanoscale devices could offer a new and simpler means of understanding quantum phase transitions. Here we demonstrate this possibility in a single-molecule quantum dot, where a gate voltage induces a crossing of two different types of electron spin state (singlet and triplet) at zero magnetic field. The quantum dot is operated in the Kondo regime, where the electron spin on the quantum dot is partially screened by metallic electrodes. This strong electronic coupling between the quantum dot and the metallic contacts provides the strong electron correlations necessary to observe quantum critical behaviour. The quantum magnetic phase transition between two different Kondo regimes is achieved by tuning gate voltages and is fundamentally different from previously observed Kondo transitions in semiconductor and nanotube quantum dots. Our work may offer new directions in terms of control and tunability for molecular spintronics. PMID:18509439

Roch, Nicolas; Florens, Serge; Bouchiat, Vincent; Wernsdorfer, Wolfgang; Balestro, Franck



Quantum dynamics simulations of interfacial electron transfer in sensitized TiO2 semiconductors  

NASA Astrophysics Data System (ADS)

A mixed quantum-classical method combining ab initio-DFT molecular dynamics simulations with electronic relaxation calculations is used to investigate interfacial electron transfer in catechol/TiO2-anatase nanostructures under vacuum conditions at finite temperature. The calculations demonstrate that the injection mechanism is accelerated by thermal nuclear motion. In particular, electron-phonon scattering leads to ultrafast adsorbate monolayer electron transfer and the disappearance of the anisotropic charge delocalization (i.e., carrier diffusion) identified in frozen lattice studies, due to increased coupling between quasistationary molecular orbitals localized on the adsorbates and those orbitals delocalized throughout the semiconductor bulk. The results are particularly relevant to the understanding of surface charge separation in efficient mechanisms of molecular-based photovoltaic devices.

Abuabara, Sabas G.; Rego, Luis G. C.; Batista, Victor S.



Structure and mechanics of proteins from single molecules to cells  

NASA Astrophysics Data System (ADS)

Physical factors drive evolution and play important roles in motility and attachment as well as in differentiation. As animal cells adhere to survive, they generate force and "feel" various mechanical features of their surroundings and respond to externally applied forces. This mechanosensitivity requires a substrate for cells to adhere to and a mechanism for cells to apply force, followed by a cellular response to the mechanical properties of the substrate. We have taken an outside-in approach to characterize several aspects of cellular mechanosensitivity. First, we used single molecule force spectroscopy to measure how fibrinogen, an extracellular matrix protein that forms the scaffold of blood clots, responds to applied force and found that it rapidly unfolds in 23 nm steps at forces around 100 pN. Second, we used tensile testing to measure the force-extension behavior of fibrin gels and found that they behave almost linearly to strains of over 100%, have extensibilities of 170 +/- 15%, and undergo a large volume decrease that corresponds to a large and negative peak in compressibility at low strain, which indicates a structural transition. Using electron microscopy and X-ray scattering we concluded that these properties are likely due to coiled-coil unfolding, as observed at the single molecule level in fibrinogen. Moving inside cells, we used total internal reflection fluorescence and atomic force microscopy to image self-assembled myosin filaments. These filaments of motor proteins that are responsible for cell and muscle contractility were found to be asymmetric, with an average of 32% more force generating heads on one half than the other. This could imply a force imbalance, so that rather than being simply contractile, myosin filaments may also be motile in cells.

Brown, Andre E.


Sorting Single Molecules: Application to Diagnostics and Evolutionary Biotechnology  

Microsoft Academic Search

A method is described that provides for detection and identification of single molecules in solution. The method is based on fluoresence correlation spectroscopy, which recoords spatio-temporaal correlation among fluctuating light singles, coupled with devices for trapping single molecules in an electric field. This technique is applied to studies of molecular evolution, where it allows fast screening of large mutant spectra

Manfred Eigen; Rudolf Rigler



An electrostatic gate for mechanically controlled single-molecule junctions  

NASA Astrophysics Data System (ADS)

We present a fabrication scheme for a tunable single-molecule transistor that allows for controlling the electrode separation and provides an electrostatic gate. The experimental approach is based on the mechanically controlled break junction technique but integrates an additional bottom gate electrode and an uninterrupted high-? gate dielectric. The device performance is demonstrated for a single-molecule junction showing Coulomb blockade characteristics.

Ballmann, Stefan; Weber, Heiko B.



iBioSeminar: Single Molecule Manipulation in Biochemistry  

NSDL National Science Digital Library

Dr. Bustamante begins his talk by explaining why one would wish to study biochemical reactions at the level of single molecules. He explains that many processes within the cell are carried out by very few molecules. By studying single molecules, it is possible to obtain details about the mechanism of a reaction that cannot be ascertained by studying a population of molecules.

Carlos Bustamante (University of California, Berkeley and Howard Hughes Medical Institute;)



Single-molecule electrochemistry: present status and outlook.  


The development of methods for detecting and manipulating matter at the level of individual macromolecules represents one of the key scientific advancements of recent decades. These techniques allow us to get information that is largely unobtainable otherwise, such as the magnitudes of microscopic forces, mechanistic details of catalytic processes, macromolecular population heterogeneities, and time-resolved, step-by-step observation of complex kinetics. Methods based on optical, mechanical, and ionic-conductance signal transduction are particularly developed. However, there is scope for new approaches that can broaden the range of molecular systems that we can study at this ultimate level of sensitivity and for developing new analytical methods relying on single-molecule detection. Approaches based on purely electrical detection are particularly appealing in the latter context, since they can be easily combined with microelectronics or fluidic devices on a single microchip to create large parallel assays at relatively low cost. A form of electrical signal transduction that has so far remained relatively underdeveloped at the single-molecule level is the direct detection of the charge transferred in electrochemical processes. The reason for this is simple: only a few electrons are transferred per molecule in a typical faradaic reaction, a heterogeneous charge-transfer reaction that occurs at the electrode's surface. Detecting this tiny amount of charge is impossible using conventional electrochemical instrumentation. A workaround is to use redox cycling, in which the charge transferred is amplified by repeatedly reducing and oxidizing analyte molecules as they randomly diffuse between a pair of electrodes. For this process to be sufficiently efficient, the electrodes must be positioned within less than 100 nm of each other, and the analyte must remain between the electrodes long enough for the measurement to take place. Early efforts focused on tip-based nanoelectrodes, descended from scanning electrochemical microscopy, to create suitable geometries. However, it has been challenging to apply these technologies broadly. In this Account, we describe our alternative approach based on electrodes embedded in microfabricated nanochannels, so-called nanogap transducers. Microfabrication techniques grant a high level of reproducibility and control over the geometry of the devices, permitting systematic development and characterization. We have employed these devices to demonstrate single-molecule sensitivity. This method shows good agreement with theoretical analysis based on the Brownian motion of discrete molecules, but only once the finite time resolution of the experimental apparatus is taken into account. These results highlight both the random nature of single-molecule signals and the complications that it can introduce in data interpretation. We conclude this Account with a discussion on how scientists can overcome this limitation in the future to create a new experimental platform that can be generally useful for both fundamental studies and analytical applications. PMID:23270398

Lemay, Serge G; Kang, Shuo; Mathwig, Klaus; Singh, Pradyumna S



Manipulating NiFe/AlOx interfacial chemistry for the spin-polarized electrons transport  

NASA Astrophysics Data System (ADS)

Through vacuum annealing, interfacial chemical composition of sputter-deposited AlOx/NiFe/AlOx can be controlled for electron transport manipulation. Chemical status change at the NiFe/AlOx interface was quantified by X-ray photoelectron spectroscopy and correlated to the structure and electron transport properties of the heterostructure. It is found that elemental Al existed in the insulting AlOx after annealing at intermediate temperature can improve the AlOx/NiFe interface and thus favor the electronic transport. Annealing at higher temperature will result in native AlOx formation and degrade transport properties due to the NiFe/AlOx interfaces deterioration caused by significant difference in thermal expansion coefficients of the two materials.

Zhao, Chong-Jun; Sun, Li; Ding, Lei; Li, Jian-Wei; Zhang, Jing-Yan; Cao, Yi; Yu, Guang-Hua



Evidence on single-molecule transport in electrostatically-gated molecular transistors  

NASA Astrophysics Data System (ADS)

We show that, if adequately formulated for molecular electronics, the barrier picture can quantitatively reproduce the currents and describe the orbital gating in the molecular transistors fabricated by Song et al. [H. Song, Y. Kim, Y.H. Jang, H. Jeong, M.A. Reed, T. Lee, Nature 462 (2009) 1039]. Based on our results, we demonstrate (i) that the measured current represents the contribution of a single molecule, and (ii) the linear dependence of the molecular orbital energy offset ?g on the voltage Vt at the Fowler-Nordheim minimum, validating thereby the transition voltage spectroscopy for the gated single molecule devices of Song et al.

Bâldea, Ioan; Köppel, Horst



Viewing the interior of a single molecule: vibronically resolved photon imaging at submolecular resolution.  


We report the spatial imaging of the photon transition probability of a single molecule at submolecular resolution. Photon imaging of a ringlike pattern is further resolved as two orthogonal vibronic transitions after incorporating spectral selectivity. A theoretical model and the calculated intensity images reveal that the transition probability is dominated by the symmetry of the positions of the tip and the transition dipole moment. This imaging technique enables the probing of the electronic and optical properties in the interior of a single molecule. PMID:21231352

Chen, Chi; Chu, Ping; Bobisch, C A; Mills, D L; Ho, W



Nucleosome-remodelling machines and other molecular motors observed at the single-molecule level.  


Through its capability to transiently pack and unpack our genome, chromatin is a key player in the regulation of gene expression. Single-molecule approaches have recently complemented conventional biochemical and biophysical techniques to decipher the complex mechanisms ruling chromatin dynamics. Micromanipulations with tweezers (magnetic or optical) and imaging with molecular microscopy (electron or atomic force) have indeed provided opportunities to handle and visualize single molecules, and to measure the forces and torques produced by molecular motors, along with their effects on DNA or nucleosomal templates. By giving access to dynamic events that tend to be blurred in traditional biochemical bulk experiments, these techniques provide critical information regarding the mechanisms underlying the regulation of gene activation and deactivation by nucleosome and chromatin structural changes. This minireview describes some single-molecule approaches to the study of ATP-consuming molecular motors acting on DNA, with applications to the case of nucleosome-remodelling machines. PMID:21810177

Lavelle, Christophe; Praly, Elise; Bensimon, David; Le Cam, Eric; Croquette, Vincent



Conductance measurement of pyridyl-based single molecule junctions with Cu and Au contacts.  


We studied the conductance of pyridyl-based single molecule junctions with Cu contacts by using an electrochemical jump-to-contact scanning tunneling microscopy break junction (ECSTM-BJ) approach. The single molecule junctions of 4,4'-bipyridine (BPY), 1,2-di(pyridin-4-yl)ethene (BPY-EE) and 1,2-di(pyridin-4-yl)ethane (BPY-EA) bridged with Cu clusters show three sets of conductance values. These values are smaller than the conductance values of single molecule junctions with Au electrodes measured by the traditional scanning tunneling microscopy break junction in acidic or neutral solutions, which can be attributed to the different electronic coupling efficiencies between molecules and electrodes. The consistent conductance of pyridyl-based molecules in acidic and neutral solutions may show that the protonated pyridyl group contacts to the electrode through the deprotonated form. PMID:24164714

Zhou, Xiao-Yi; Peng, Zheng-Lian; Sun, Yan-Yan; Wang, Li-Na; Niu, Zhen-Jiang; Zhou, Xiao-Shun



Orbital Gating of Single Molecule Transistors  

NASA Astrophysics Data System (ADS)

Electron devices containing molecules as the active region have been an active area of research over the last few years. In molecular-scale devices, a longstanding challenge has been to create a true three-terminal device; e.g., one that operates by modifying the internal energy structure of the molecule, analogous to conventional FETs. Here we report the observation of such a solid-state molecular device, in which transport current is directly modulated by an external gate voltage. We have realized a molecular transistor made from the prototype molecular junction, benzene dithiol, and have used a combination of spectroscopies to determine the internal energetic structure of the molecular junction. Resonance-enhanced coupling to the nearest molecular orbital is revealed by electron tunneling spectroscopy, demonstrating for the first time direct molecular orbital gating in a molecular electronic device.

Reed, Mark



Microscopy beyond the diffraction limit using actively controlled single molecules.  


In this short review, the general principles are described for obtaining microscopic images with resolution beyond the optical diffraction limit with single molecules. Although it has been known for several decades that single-molecule emitters can blink or turn on and off, in recent work the addition of on/off control of molecular emission to maintain concentrations at very low levels in each imaging frame combined with sequential imaging of sparse subsets has enabled the reconstruction of images with resolution far below the optical diffraction limit. Single-molecule active control microscopy provides a powerful window into information about nanoscale structures that was previously unavailable. PMID:22582796

Moerner, W E



Microscopy beyond the diffraction limit using actively controlled single molecules  

PubMed Central

Summary In this short review, the general principles are described for obtaining microscopic images with resolution beyond the optical diffraction limit with single molecules. Although it has been known for several decades that single-molecule emitters can blink or turn on and off, in recent work the addition of on/off control of molecular emission to maintain concentrations at very low levels in each imaging frame combined with sequential imaging of sparse subsets has enabled the reconstruction of images with resolution far below the optical diffraction limit. Single-molecule active control microscopy provides a powerful window into information about nanoscale structures that was previously unavailable.




Single-molecule detection: applications to ultrasensitive biochemical analysis  

NASA Astrophysics Data System (ADS)

Recent developments in laser-based detection of fluorescent molecules have made possible the implementation of very sensitive techniques for biochemical analysis. We present and discuss our experiments on the applications of our recently developed technique of single-molecule detection to the analysis of molecules of biological interest. These newly developed methods are capable of detecting and identifying biomolecules at the single-molecule level of sensitivity. In one case, identification is based on measuring fluorescence brightness from single molecules. In another, molecules are classified by determining their electrophoretic velocities.

Castro, Alonso; Shera, E. Brooks



Single-molecule imaging at high hydrostatic pressure  

NASA Astrophysics Data System (ADS)

Direct microscopic fluorescence imaging of single molecules can provide a wealth of mechanistic information, but up to now, it has not been possible under high pressure conditions, due to limitations in microscope pressure cell design. We describe a pressure cell window design that makes it possible to image directly single molecules at high hydrostatic pressure. We demonstrate our design by imaging single molecules of Alexa Fluor 647 dye bound to DNA, at 120 and 210 bar, and following their fluorescence photodynamics. We further show that the failure pressure of this type of pressure cell window can be in excess of 1 kbar.

Vass, Hugh; Lucas Black, S.; Flors, Cristina; Lloyd, Diarmuid; Bruce Ward, F.; Allen, Rosalind J.



Kinetic equations for transport through single-molecule transistors  

NASA Astrophysics Data System (ADS)

We present explicit kinetic equations for quantum transport through a general molecular quantum dot, accounting for all contributions up to fourth order perturbation theory in the tunneling Hamiltonian and the complete molecular density matrix. Such a full treatment describes not only sequential, cotunneling, and pair tunneling, but also contains terms contributing to renormalization of the molecular resonances as well as their broadening. Due to the latter all terms in the perturbation expansion are automatically well defined for any set of system parameters: no divergences occur and no by-hand regularization is required. Additionally we show that, in contrast to second order perturbation theory, in fourth order it is essential to account for quantum coherence between nondegenerate states, entering the theory through the nondiagonal elements of the density matrix. As a first application, we study a single-molecule transistor coupled to a localized vibrational mode (Anderson-Holstein model). We find that cotunneling-assisted sequential tunneling processes involving the vibration give rise to current peaks, i.e., negative differential conductance in the Coulomb-blockade regime. Such peaks occur in the crossover to strong electron-vibration coupling, where inelastic cotunneling competes with Franck-Condon suppressed sequential tunneling, and thereby indicate the strength of the electron-vibration coupling. The peaks depend sensitively on the coupling to a dissipative bath, thus providing also an experimental probe of the Q factor of the vibrational motion.

Leijnse, M.; Wegewijs, M. R.



Fluorescence Image of a Single Molecule in a Microsphere: Model.  

National Technical Information Service (NTIS)

We model fluorescence images of single molecules in spherical dielectric microcavities. Molecules are treated as time-harmonic dipoles. Images are integrated over emission frequencies. Because of the strong refractive properties of the enclosing sphere, t...

S. C. Hill P. Nachman S. Arnold J. M. Ramsey M. D. Barnes



Label-free, single-molecule detection with optical microcavities.  


Current single-molecule detection techniques require labeling the target molecule. We report a highly specific and sensitive optical sensor based on an ultrahigh quality (Q) factor (Q > 10(8)) whispering-gallery microcavity. The silica surface is functionalized to bind the target molecule; binding is detected by a resonant wavelength shift. Single-molecule detection is confirmed by observation of single-molecule binding events that shift the resonant frequency, as well as by the statistics for these shifts over many binding events. These shifts result from a thermo-optic mechanism. Additionally, label-free, single-molecule detection of interleukin-2 was demonstrated in serum. These experiments demonstrate a dynamic range of 10(12) in concentration, establishing the microcavity as a sensitive and versatile detector. PMID:17615303

Armani, Andrea M; Kulkarni, Rajan P; Fraser, Scott E; Flagan, Richard C; Vahala, Kerry J



Understanding Enzyme Activity Using Single Molecule Tracking (Poster)  

SciTech Connect

This poster describes single-molecule tracking and total internal reflection fluorescence microscopy. It discusses whether the carbohydrate-binding module (CBM) moves on cellulose, how the CBM binds to cellulose, and the mechanism of cellulosome assembly.

Liu, Y.-S.; Zeng, Y.; Luo, Y.; Xu, Q.; Himmel, M.; Smith S.; Wei, H.; Ding, S.-Y.



Multiparameter single-molecule fluorescence measurements of DNA intercalating fluorophores  

NASA Astrophysics Data System (ADS)

Experiments using single-molecules of TOTO-1 intercalated into dsDNA were performed to investigate the DNA sequence dependence on the fluorescence detectable with single-molecule fluorescence spectroscopy. Previous work has shown that there is a difference in the fluorescence lifetime when TOTO-1 is intercalated in poly-AT DNA or in poly-GC DNA. The fluorescence detected from single-molecules in this work for poly-GC and poly-AT DNA showed fluorescence lifetimes of 2.1 and 1.8 nsec, respectively. Analysis of the fluorescence intensity detected from single-molecules of TOTO-1 was performed by fluorescence cross-correlation spectroscopy. TOTO-1 is shown to spend large amounts of time in dark states. These dark states reduce the detectable fluorescence intensity to approximately 10 photons per millisecond on average.

Bowen, Benjamin P.; Enderlein, Jörg; Woodbury, Neal W. T.



Nano\\/micro technologies for single molecule manipulation and detection  

Microsoft Academic Search

A sensitive, rapid, and efficient single-molecule detection method was developed by combining fluorescence correlation spectroscopy (FCS) and on-chip molecular manipulation techniques. The highly restricted measurement volume in the FCS system significantly reduces the intrinsic background noise and facilitates single-molecule sensitivity. A microchannel integrated with multiple 3-D electrodes was fabricated and used for molecular sensing and manipulation by which individual DNA

Tza-huei Wang; Chih-ming Ho



Muscle contraction mechanism based on single molecule measurements.  


Single molecule measurements have shown that a muscle myosin step is driven by biased Brownian movement. Furthermore, they have also demonstrated that in response to strain in the backward direction a detached myosin head preferentially attaches to the forward direction due to an accelerated transition from a weak binding to strong binding state. Because they are consistent with the original Huxley model for muscle contraction, we have built a model that describes macroscopic muscle characteristics based on these single molecule results. PMID:23203295

Yanagida, Toshio; Ishii, Yoshiharu



Single-Molecule FRET: Methods and Biological Applications  

NASA Astrophysics Data System (ADS)

Since the first single-molecule fluorescence resonance energy transfer (FRET) measurement in 1996, the technique has contributed substantially to our understanding of biological molecules and processes by probing the structure and dynamics of nucleic acids, protein molecules, and their complexes with other molecules. This review discusses basic concepts and current developments in single-molecule FRET methodology, as well as examples of applications to systems such as nucleic acid machines and molecular motors.

Hwang, Ling Chin; Hohlbein, Johannes; Holden, Seamus J.; Kapanidis, Achillefs N.


A practical guide to single-molecule FRET  

Microsoft Academic Search

Single-molecule fluorescence resonance energy transfer (smFRET) is one of the most general and adaptable single-molecule techniques. Despite the explosive growth in the application of smFRET to answer biological questions in the last decade, the technique has been practiced mostly by biophysicists. We provide a practical guide to using smFRET, focusing on the study of immobilized molecules that allow measurements of

Rahul Roy; Sungchul Hohng; Taekjip Ha




Microsoft Academic Search

The kinetics and mechanisms of transcription are now being investi- gated by a repertoire of single-molecule techniques, including opti- cal and magnetic tweezers, high-sensitivity fluorescence techniques, and atomic force microscopy. Single-molecule techniques comple- ment traditional biochemical and crystallographic approaches, are capable of detecting the motions and dynamics of individual RNAP molecules and transcription complexes in real time, and make it

Lu Bai; Thomas J. Santangelo; Michelle D. Wang



Nanometer-localized multiple single-molecule fluorescence microscopy  

PubMed Central

Fitting the image of a single molecule to the point spread function of an optical system greatly improves the precision with which single molecules can be located. Centroid localization with nanometer precision has been achieved when a sufficient number of photons are collected. However, if multiple single molecules reside within a diffraction-limited spot, this localization approach does not work. This paper demonstrates nanometer-localized multiple single-molecule (NALMS) fluorescence microscopy by using both centroid localization and photobleaching of the single fluorophores. Short duplex DNA strands are used as nanoscale “rulers” to validate the NALMS microscopy approach. Nanometer accuracy is demonstrated for two to five single molecules within a diffraction-limited area. NALMS microscopy will greatly facilitate single-molecule study of biological systems because it covers the gap between fluorescence resonance energy transfer-based (<10 nm) and diffraction-limited microscopy (>100 nm) measurements of the distance between two fluorophores. Application of NALMS microscopy to DNA mapping with <10-nm (i.e., 30-base) resolution is demonstrated.

Qu, Xiaohui; Wu, David; Mets, Laurens; Scherer, Norbert F.



Magnetic anisotropy and high-spin effects in single-molecule transistors  

NASA Astrophysics Data System (ADS)

Fabrication of single-molecule transistors where electron transport occurs through an individual molecule has become possible due to the recent progress in molecular electronics. Three-terminal configuration allows charging molecules and performing transport spectroscopy in multiple redox states. Single-molecule magnets combining large spin with uniaxial anisotropy are of special interest as appealing candidates for high density memory applications and quantum information processing. We study single-molecule magnets Fe4. Three-terminal junctions are fabricated using electromigration of gold nanowires followed by a self-breaking. High-spin Kondo effect and inelastic cotunneling excitations show up in transport measurements. Several excitations feature the energy close to the energy of zero-field splitting (ZFS) of a ground spin multiplet in bulk. This splitting is caused by the anisotropy and is a hallmark of single-molecule magnets. We observe nonlinear Zeeman effect due to a misalignment of an anisotropy axis and a magnetic field direction. The ZFS energy is increased in oxidized and reduced states of the molecule indicating enhancement of the anisotropy in these states.

Zyazin, Alexander; van den Berg, Johan; Osorio, Edgar; Konstantinidis, Nikos; Leijnse, Martin; May, Falk; Hofstetter, Walter; Danieli, Chiara; Cornia, Andrea; Wegewijs, Maarten; van der Zant, Herre



Charge Transport in Azobenzene-Based Single-Molecule Junctions  

NASA Astrophysics Data System (ADS)

The azobenzene class of molecules has become an archetype of molecular photoswitch research, due to their simple structure and the significant difference of the electronic system between their cis and trans isomers. However, a detailed understanding of the charge transport for the two isomers, when embedded in a junction with electrodes is still lacking. In order to clarify this issue, we investigate charge transport properties through single Azobenzene-ThioMethyl (AzoTM) molecules in a mechanically controlled break junction (MCBJ) system at 4.2 K. Single-molecule conductance, I-V characteristics, and IETS spectra of molecular junctions are measured and compared with first-principles transport calculations. Our studies elucidate the origin of a slightly higher conductance of junctions with cis isomer and demonstrate that IETS spectra of cis and trans forms show distinct vibrational fingerprints that can be used for identifying the isomer.[1] 1. Y. Kim, A. Garcia-Lekue, D. Sysoiev, T. Frederiksen, U. Groth, E. Scheer, Phys. Rev. Lett. (accepted).

Garcia-Lekue, Aran; Kim, Youngsang; Sysoiev, Dmytro; Frederiksen, Thomas; Groth, Ulrich; Scheer, Elke



Molecular wires in single-molecule junctions: charge transport and vibrational excitations.  


We investigate the effect of vibrations on the electronic transport through single-molecule junctions, using the mechanically controlled break junction technique. The molecules under investigation are oligoyne chains with appropriate end groups, which represent both an ideally linear electrical wire and an ideal molecular vibrating string. Vibronic features can be detected as satellites to the electronic transitions, which are assigned to longitudinal modes of the string by comparison with density functional theory data. PMID:20521299

Ballmann, Stefan; Hieringer, Wolfgang; Secker, Daniel; Zheng, Qinglin; Gladysz, John A; Görling, Andreas; Weber, Heiko B



Asymmetric Coulomb blockade and Kondo temperature of single-molecule transistors  

NASA Astrophysics Data System (ADS)

Recent experiments on single-molecule transistors made of cobalt complexes exhibited anomalously weak gate voltage dependence of the Kondo temperature accompanied by a strong asymmetry in the Coulomb blockade peaks. We show that these observations can both be explained by strong electron vibron interactions when including anharmonicities of the molecular potential surfaces. The strong electron vibron interactions may originate from a tendency of the cobalt complexes toward Jahn Teller distortion.

Elste, Florian; von Oppen, Felix



Physical Mechanism of Interfacial Thermal Resistance in Electronic Packaging Based on a Mixed MD\\/FE Model  

Microsoft Academic Search

A multiscale model is constructed to study interfacial thermal resistance in electronic packaging. The model combines a molecular dynamics simulation for the critical regions within the system with a finite element (FE) method for a continuum description of the remainder of the system. For nonequilibrium simulations, the establishment of the proper boundary condition is very difficult. In this mixed model,

Ping Yang; Ningbo Liao



Massively Parallel Single-Molecule Manipulation Using Centrifugal Force  

NASA Astrophysics Data System (ADS)

Precise manipulation of single molecules has led to remarkable insights in physics, chemistry, biology, and medicine. However, two issues that have impeded the widespread adoption of these techniques are equipment cost and the laborious nature of making measurements one molecule at a time. To meet these challenges, we have developed an approach that enables massively parallel single- molecule force measurements using centrifugal force [1]. This approach is realized in the centrifuge force microscope, an instrument in which objects in an orbiting sample are subjected to a calibration-free, macroscopically uniform force- field while their micro-to-nanoscopic motions are observed. We demonstrate high- throughput single-molecule force spectroscopy with this technique by performing thousands of rupture experiments in parallel, characterizing force-dependent unbinding kinetics of an antibody-antigen pair in minutes rather than days. Currently, we are taking steps to integrate high-resolution detection, fluorescence, temperature control and a greater dynamic range in force. With significant benefits in efficiency, cost, simplicity, and versatility, single-molecule centrifugation has the potential to expand single-molecule experimentation to a wider range of researchers and experimental systems.[4pt] [1] K. Halvorsen, W.P. Wong, Biophysical Journal - Letters 98 (11), (2010).

Wong, Wesley; Halvorsen, Ken



Towards physiological complexity with in vitro single-molecule biophysics.  


Single-molecule biology has matured in recent years, driven to greater sophistication by the development of increasingly advanced experimental techniques. A progressive appreciation for its unique strengths is attracting research that spans an exceptionally broad swath of physiological phenomena--from the function of nucleosomes to protein diffusion in the cell membrane. Newfound enthusiasm notwithstanding, the single-molecule approach is limited to an intrinsically defined set of biological questions; such limitation applies to all experimental approaches, and an explicit statement of the boundaries delineating each set offers a guide to most fruitfully orienting in vitro single-molecule research in the future. Here, we briefly describe a simple conceptual framework to categorize how submolecular, molecular and intracellular processes are studied. We highlight the domain of single-molecule biology in this scheme, with an emphasis on its ability to probe various forms of heterogeneity inherent to populations of discrete biological macromolecules. We then give a general overview of our high-throughput DNA curtain methodology for studying protein-nucleic acid interactions, and by contextualizing it within this framework, we explore what might be the most enticing avenues of future research. We anticipate that a focus on single-molecule biology's unique strengths will suggest a new generation of experiments with greater complexity and more immediately translatable physiological relevance. PMID:23267187

Duzdevich, Daniel; Greene, Eric C



Observing single molecule chemical reactions on metal nanoparticles.  

SciTech Connect

We report the study of the photodecomposition of single Rhodamine 6G (R6G) dye molecules adsorbed on silver nanoparticles. The nanoparticles were immobilized and spatially isolated on polylysine-derivatized glass coverslips, and confocal laser microspectroscopy was used to obtain surface-enhanced Raman scattering (SERS) spectra from individual R6G molecules. The photodecomposition of these molecules was observed with 150-ms temporal resolution. The photoproduct was identified as graphitic carbon based on the appearance of broad SERS vibrational bands at 1592 cm{sup -1} and 1340 cm{sup -1} observed in both bulk and averaged single-molecule photoproduct spectra. In contrast, when observed at the single-molecule level, the photoproduct yielded sharp SERS spectra. The inhomogeneous broadening of the bulk SERS spectra is due to a variety of photoproducts in different surface orientations and is a characteristic of ensemble-averaged measurements of disordered systems. These single-molecule studies indicate a photodecomposition pathway by which the R6G molecule desorbs from the metal surface, an excited-state photoreaction occurs, and the R6G photoproduct(s) readsorbs to the surface. A SERS spectrum is obtained when either the intact R6G or the R6G photoproduct(s) are adsorbed on a SERS-active site. This work further illustrates the power of single-molecule spectroscopy (SMS) to reveal unique behaviors of single molecules that are not discernable with bulk measurements.

Emory, S. R. (Steven R.); Ambrose, W. Patrick; Goodwin, P. M. (Peter M); Keller, Richard A.



Presence and spatial distribution of interfacial electronic states in LaMnO{sub 3}-SrMnO{sub 3} superlattices.  

SciTech Connect

We report direct evidence of interfacial states at the onset of O K edge confined to a spatial distance of 1 unit-cell full-width at half maximum at the sharp interfaces between epitaxial films of LaMnO{sub 3} and SrMnO{sub 3} from electron energy-loss spectroscopy (EELS) measurements. The interfacial states are sensitive to interface sharpness; at rough interfaces with interfacial steps of 1-2 unit cells in height, experimental data shows a reduction, or suppression, of the interfacial states. The EELS measurements were performed using a fine electron probe obtained by electron lens aberration correction. By scanning the electron probe across the interface, we are able to map the spatial distribution of the interfacial states across interfaces at high resolution.

Shah, A. B.; Ramasse, Q. M.; May, S. J.; Kavich, J.; Wen, J. G.; Zhai, X.; Eckstein, J. N.; Freeland, J.; Bhattacharya, A.; Zuo, J. M.; Univ. of Illinois; LBNL



Interfacial orientation of Thermomyces lanuginosa lipase on phospholipid vesicles investigated by electron spin resonance relaxation spectroscopy.  


The binding orientation of the interfacially activated Thermomyces lanuginosa lipase (TLL, EC on phospholipid vesicles was investigated using site-directed spin labeling and electron spin resonance (ESR) relaxation spectroscopy. Eleven TLL single-cysteine mutants, each with the mutation positioned at the surface of the enzyme, were selectively spin labeled with the nitroxide reagent (1-oxyl-2,2,5,5-tetramethyl-Delta(3)-pyrroline-3-methyl) methanethiosulfonate. These were studied together with small unilamellar vesicles (SUV) consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG), to which TLL has previously been shown to bind in a catalytically active form [Cajal, Y., et al. (2000) Biochemistry 39, 413-423]. The orientation of TLL with respect to the lipid membrane was investigated using a water-soluble spin relaxation agent, chromium(III) oxalate (Crox), and a recently developed ESR relaxation technique [Lin, Y., et al. (1998) Science 279, 1925-1929], here modified to low microwave amplitude (<0.36 G). The exposure to Crox for the spin label at the different positions on the surface of TLL was determined in the absence and presence of vesicles. The spin label at positions Gly61-Cys and Thr267-Cys, closest to the active site nucleophile Ser146 of the positions analyzed, displayed the lowest exposure factors to the membrane-impermeable spin relaxant, indicating the proximity to the vesicle surface. As an independent technique, fluorescence spectroscopy was employed to measure fluorescence quenching of dansyl-labeled POPG vesicles as exerted by the protein-bound spin labels. The resulting Stern-Volmer quenching constants showed excellent agreement with the ESR exposure factors. An interfacial orientation of TLL is proposed on the basis of the obtained results. PMID:12450382

Hedin, Eva M K; Høyrup, Pernille; Patkar, Shamkant A; Vind, Jesper; Svendsen, Allan; Fransson, Linda; Hult, Karl



Simultaneous time and frequency resolved fluorescence microscopy of single molecules.  

SciTech Connect

Single molecule fluorophores were studied for the first time with a new confocal fluorescence microscope that allows the wavelength and emission time to be simultaneously measured with single molecule sensitivity. In this apparatus, the photons collected from the sample are imaged through a dispersive optical system onto a time and position sensitive detector. This detector records the wavelength and emission time of each detected photon relative to an excitation laser pulse. A histogram of many events for any selected spatial region or time interval can generate a full fluorescence spectrum and correlated decay plot for the given selection. At the single molecule level, this approach makes entirely new types of temporal and spectral correlation spectroscopy of possible. This report presents the results of simultaneous time- and frequency-resolved fluorescence measurements of single rhodamine 6G (R6G), tetramethylrhodamine (TMR), and Cy3 embedded in thin films of polymethylmethacrylate (PMMA).

Hayden, Carl C.; Gradinaru, Claudiu C.; Chandler, David W.; Luong, A. Khai



Detectors for single-molecule fluorescence imaging and spectroscopy  

PubMed Central

Single-molecule observation, characterization and manipulation techniques have recently come to the forefront of several research domains spanning chemistry, biology and physics. Due to the exquisite sensitivity, specificity, and unmasking of ensemble averaging, single-molecule fluorescence imaging and spectroscopy have become, in a short period of time, important tools in cell biology, biochemistry and biophysics. These methods led to new ways of thinking about biological processes such as viral infection, receptor diffusion and oligomerization, cellular signaling, protein-protein or protein-nucleic acid interactions, and molecular machines. Such achievements require a combination of several factors to be met, among which detector sensitivity and bandwidth are crucial. We examine here the needed performance of photodetectors used in these types of experiments, the current state of the art for different categories of detectors, and actual and future developments of single-photon counting detectors for single-molecule imaging and spectroscopy.




Probing Cellular Protein Complexes via Single Molecule Pull-down  

PubMed Central

Proteins perform most cellular functions in macromolecular complexes. The same protein often participates in different complexes to exhibit diverse functionality. Current ensemble approaches of identifying cellular protein interactions cannot reveal physiological permutations of these interactions. Here, we describe a single molecule pull-down (SiMPull) assay that combines the principles of conventional pull-down assay with single molecule fluorescence microscopy and enables direct visualization of individual cellular protein complexes. SiMPull can reveal how many proteins and of which kinds are present in the in vivo complex, as we show using protein kinase A. We then demonstrate a wide applicability to various signaling proteins found in cytosol, membrane, and cellular organelles, and to endogenous protein complexes from animal tissue extracts. The pulled down proteins are functional and are used, without further processing, for single molecule biochemical studies. SiMPull should provide a rapid, sensitive and robust platform for analyzing protein assemblies in biological pathways.

Jain, Ankur; Liu, Ruijie; Ramani, Biswarathan; Arauz, Edwin; Ishitsuka, Yuji; Ragunathan, Kaushik; Park, Jeehae; Chen, Jie; Xiang, Yang K.; Ha, Taekjip



Transchip: Single Molecule Detection of Transcriptional Elongation Complexes  

PubMed Central

A new single molecule system – Transchip – was developed for analysis of transcription products at their genomic origins. The bacteriophage T7 RNA polymerase and its promoters were used in a model system, and resultant RNAs were imaged and detected at their positions along single template DNA molecules. This system, Transchip, has drawn from critical aspects of Optical Mapping, a single molecule system that enables the construction of high resolution, ordered restriction maps of whole genomes from single DNA molecules. Through statistical analysis of hundreds of single molecule template/transcript complexes, Transchip enables analysis of the locations and strength of promoters, the direction and processivity of transcription reactions and termination of transcription. These novel results suggest that the new system may serve as a high-throughput platform to investigate transcriptional events on a large, genome-wide scale.

Wu, Tian; Schwartz, David C.



Single-molecule dynamics and mechanisms of metalloregulators and metallochaperones.  


Understanding how cells regulate and transport metal ions is an important goal in the field of bioinorganic chemistry, a frontier research area that resides at the interface of chemistry and biology. This Current Topic reviews recent advances from the authors' group in using single-molecule fluorescence imaging techniques to identify the mechanisms of metal homeostatic proteins, including metalloregulators and metallochaperones. It emphasizes the novel mechanistic insights into how dynamic protein-DNA and protein-protein interactions offer efficient pathways via which MerR-family metalloregulators and copper chaperones can fulfill their functions. This work also summarizes other related single-molecule studies of bioinorganic systems and provides an outlook toward single-molecule imaging of metalloprotein functions in living cells. PMID:24053279

Chen, Peng; Keller, Aaron M; Joshi, Chandra P; Martell, Danya J; Andoy, Nesha May; Benítez, Jaime J; Chen, Tai-Yen; Santiago, Ace George; Yang, Feng



Making connections--strategies for single molecule fluorescence biophysics.  


Fluorescence spectroscopy and fluorescence microscopy carried out on the single molecule level are elegant methods to decipher complex biological systems; it can provide a wealth of information that frequently is obscured in the averaging of ensemble measurements. Fluorescence can be used to localise a molecule, study its binding with interaction partners and ligands, or to follow conformational changes in large multicomponent systems. Efficient labelling of proteins and nucleic acids is very important for any fluorescence method, and equally the development of novel fluorophores has been crucial in making biomolecules amenable to single molecule fluorescence methods. In this paper we review novel coupling strategies that permit site-specific and efficient labelling of proteins. Furthermore, we will discuss progressive single molecule approaches that allow the detection of individual molecules and biomolecular complexes even directly isolated from cellular extracts at much higher and much lower concentrations than has been possible so far. PMID:23769868

Grohmann, Dina; Werner, Finn; Tinnefeld, Philip



Single molecule FRET to resolve conformational fluctuations in proteins  

NASA Astrophysics Data System (ADS)

Proteins fold into complex shapes that are intimately linked to their function. High-resolution techniques are capable of determining static images of these structures with atomic detail, but biological function and regulation is achieved through dynamic changes in protein conformation. Single molecule fluorescence techniques have a unique capability to detect transient molecular conformations. The power of the single molecule approach arises because it avoids the averaging over molecules and over time that are inherent in ensemble measurements. We report application of single molecule fluorescence resonance energy transfer (smFRET) to tSNARE to directly observe a conformational transition that is postulated to have an auto-regulatory function. We present a series of measurements using mutants of the proteins as well as homologues of different species in order to gain a molecular level understanding of these transitions. The techniques demonstrated here are directly applicable to investigations of conformational dynamics in other protein based macro-molecular machines.

Weninger, Keith



Single molecule methods with applications in living cells.  


Our knowledge about dynamic processes in biological cells systems has been obtained roughly on two levels of detail; molecular level experiments with purified components in test tubes and system wide experiments with indirect readouts in living cells. However, with the development of single molecule methods for application in living cells, this partition has started to dissolve. It is now possible to perform detailed biophysical experiments at high temporal resolution and to directly observe processes at the level of molecules in living cells. In this review we present single molecule methods that can easily be implemented by readers interested to venture into this exciting and expanding field. We also review some recent studies where single molecule methods have been used successfully to answer biological questions as well as some of the most common pitfalls associated with these methods. PMID:23578465

Persson, Fredrik; Barkefors, Irmeli; Elf, Johan



The importance of surfaces in single-molecule bioscience  

PubMed Central

The last ten years have witnessed an explosion of new techniques that can be used to probe the dynamic behavior of individual biological molecules, leading to discoveries that would not have been possible with more traditional biochemical methods. A common feature among these single-molecule approaches is the need for the biological molecules to be anchored to a solid support surface. This must be done under conditions that minimize nonspecific adsorption without compromising the biological integrity of the sample. In this review we highlight why surface attachments are a critical aspect of many single-molecule studies and we discuss current methods for anchoring biomolecules. Finally, we provide a detailed description of a new method developed by our laboratory for anchoring and organizing hundreds of individual DNA molecules on a surface, allowing “high-throughput” studies of protein–DNA interactions at the single-molecule level.

Visnapuu, Mari-Liis; Duzdevich, Daniel



Single-Molecule Fluorescence Imaging in Living Cells  

NASA Astrophysics Data System (ADS)

The transition of single-molecule fluorescence detection and imaging from in vitro to living cells has greatly enriched our knowledge on the behavior of single biomolecules in their native environments and their roles in cellular processes. Here we review recent advances of single-molecule biophysical approaches to live-cell studies based on fluorescence imaging. We start by discussing the practical considerations in designing single-molecule fluorescence imaging in cells, including the choice of fluorescent probes, labeling methods, instrumentation, and imaging techniques. We then describe representative examples in detail to illustrate the physicochemical parameters that can be obtained by imaging individually labeled biomolecules in cells and what can be learned from such characterizations.

Xia, Tie; Li, Nan; Fang, Xiaohong



Single molecule views of Nature's nano-machines  

NASA Astrophysics Data System (ADS)

We are interested in the perturbational analysis of biological molecules to better understand their mechanisms. Our readout is the fluorescence signal from individual biomolecules, mainly in the form of single molecule fluorescence resonance energy transfer (FRET). We are pioneering approaches to perturb and control biomolecular conformations using external force (combination of single molecule FRET and optical trap) or other biological motifs (DNA hybridization, G-quadruplex, aptamers,.). In this talk, I will present our latest results on mapping the conformational energy landscape of the Holliday junction through simultaneous fluorescence and force measurements. In addition, a new nanomechanical device called single molecule nano-metronome will be discussed with an outlook toward controlling protein conformations using nucleic acids motifs.

Ha, Taekjip



Single-molecule fluorescence characterization in native environment  

PubMed Central

Single-molecule detection (SMD) with fluorescence is a widely used microscopic technique for biomolecule structure and function characterization. The modern light microscope with high numerical aperture objective and sensitive CCD camera can image the brightly emitting organic and fluorescent protein tags with reasonable time resolution. Single-molecule imaging gives an unambiguous bottom-up biomolecule characterization that avoids the “missing information” problem characteristic of ensemble measurements. It has circumvented the diffraction limit by facilitating single-particle localization to ~1 nm. Probes developed specifically for SMD applications extend the advantages of single-molecule imaging to high probe density regions of cells and tissues. These applications perform under conditions resembling the native biomolecule environment and have been used to detect both probe position and orientation. Native, high density SMD may have added significance if molecular crowding impacts native biomolecule behavior as expected inside the cell.

Ajtai, Katalin



Confocal Single-Molecule FRET for Protein Conformational Dynamics.  


Single-molecule F?rster-type resonance energy transfer (smFRET) is a unique technique capable of following conformational motions of individual protein molecules. The direct observation of individual proteins provides rich information that would be washed away in ensemble measurements, hence opening up new avenues for establishing the protein structure-function relationships through dynamics. Retrieving dynamics information of biomolecular motions via smFRET, though, requires careful experiment design and rigorous treatment of single-molecule statistics. Here, we describe the rudimentary steps for an smFRET experiment, including sample preparation for the microscope, building of critical parts for single-molecule FRET detection, and a robust methodology for photon-by-photon data analysis. PMID:24061915

Tan, Yan-Wen; Hanson, Jeffrey A; Chu, Jhih-Wei; Yang, Haw



Exploring one-state downhill protein folding in single molecules  

PubMed Central

A one-state downhill protein folding process is barrierless at all conditions, resulting in gradual melting of native structure that permits resolving folding mechanisms step-by-step at atomic resolution. Experimental studies of one-state downhill folding have typically focused on the thermal denaturation of proteins that fold near the speed limit (ca. 106 s-1) at their unfolding temperature, thus being several orders of magnitude too fast for current single-molecule methods, such as single-molecule FRET. An important open question is whether one-state downhill folding kinetics can be slowed down to make them accessible to single-molecule approaches without turning the protein into a conventional activated folder. Here we address this question on the small helical protein BBL, a paradigm of one-state downhill thermal (un)folding. We decreased 200-fold the BBL folding-unfolding rate by combining chemical denaturation and low temperature, and carried out free-diffusion single-molecule FRET experiments with 50-?s resolution and maximal photoprotection using a recently developed Trolox-cysteamine cocktail. These experiments revealed a single conformational ensemble at all denaturing conditions. The chemical unfolding of BBL was then manifested by the gradual change of this unique ensemble, which shifts from high to low FRET efficiency and becomes broader at increasing denaturant. Furthermore, using detailed quantitative analysis, we could rule out the possibility that the BBL single-molecule data are produced by partly overlapping folded and unfolded peaks. Thus, our results demonstrate the one-state downhill folding regime at the single-molecule level and highlight that this folding scenario is not necessarily associated with ultrafast kinetics.

Liu, Jianwei; Campos, Luis A.; Cerminara, Michele; Wang, Xiang; Ramanathan, Ravishankar; English, Douglas S.; Munoz, Victor



Single Molecule Switches and Molecular Self-Assembly: Low Temperature STM Investigations and Manipulations  

SciTech Connect

This dissertation is devoted to single molecule investigations and manipulations of two porphyrin-based molecules, chlorophyll-a and Co-popphyrin. The molecules are absorbed on metallic substrates and studied at low temperatures using a scanning tunneling microscope. The electronic, structural and mechanical properties of the molecules are investigated in detail with atomic level precision. Chlorophyll-a is the key ingredient in photosynthesis processes while Co-porphyrin is a magnetic molecule that represents the recent emerging field of molecular spintronics. Using the scanning tunneling microscope tip and the substrate as electrodes, and the molecules as active ingredients, single molecule switches made of these two molecules are demonstrated. The first switch, a multiple and reversible mechanical switch, is realized by using chlorophyll-a where the energy transfer of a single tunneling electron is used to rotate a C-C bond of the molecule's tail on a Au(111) surface. Here, the det

Iancu, Violeta



Gating of single molecule transistors: Combining field-effect and chemical control  

NASA Astrophysics Data System (ADS)

Previously we have demonstrated that several structural features are crucial for the functionality of molecular field-effect transistors. The effect of additional structural aspects of molecular wires is explored. These include the type of, the thiol binding location on, and the chemical substitutions of a conjugated system. Pentacene, porphyrin, and the Tour-Reed devices are utilized as model systems. The thiol binding location is shown to have a varied effect on the transmission of a system depending on the molecular orbitals involved. Substitution by electron withdrawing and donating groups is illustrated to have a substantial effect on the transmission of single molecule devices. The substitution effect is either a simple energy shifting effect or a more complicated resonance effect, and can be used to effectively tune the electronic behavior of a single molecule field effect transistor.

Perrine, Trilisa M.; Smith, Ron G.; Marsh, Christopher; Dunietz, Barry D.



Single molecule processivity and dynamics of cAMP-dependent protein kinase (PKA)  

NASA Astrophysics Data System (ADS)

Using single-walled carbon nanotube (SWNT) transistors, we monitored the processivity and dynamics of single molecules of cAMP-dependent protein kinase (PKA). As PKA enzymatically phosphorylates its peptide substrate, it generates an electronic signal in the transistor that can be monitored continuously and with 20 ?s resolution. The electronic recording directly resolves substrate binding, ATP binding, and cooperative formation of PKA's catalytically functional, ternary complex. Statistical analysis of many events determines on- and off-rates for each of these events, as well as the full transistion probability matrix between them. Long duration monitoring further revealed minute-to-minute rate variability for a single molecule, and different mechanistic statistics for ATP binding than for substrate. The results depict a highly dynamic enzyme offering dramatic possibilities for regulated activity, an attribute that is useful for an enzyme that plays crucial roles in cell signaling.

Sims, Patrick C.; Choi, Yongki; Dong, Chengjun; Moody, Issa S.; Iftikhar, Mariam; Tolga Gul, O.; Weiss, Gregory A.; Collins, Philip G.



Rotation of a single molecule within a supramolecular bearing  


Experimental visualization and verification of a single-molecule rotor operating within a supramolecular bearing is reported. Using a scanning tunneling microscope, single molecules were observed to exist in one of two spatially defined states laterally separated by 0.26 nanometers. One was identified as a rotating state and the other as an immobilized state. Calculations of the energy barrier for rotation of these two states show that it is below the thermal energy at room temperature for the rotating state and above it for the immobilized state. PMID:9677189

Gimzewski; Joachim; Schlittler; Langlais; Tang; Johannsen



Structural dynamics of nucleosomes at single molecule resolution  

PubMed Central

The detailed mechanisms of how DNA that is assembled around a histone core can be accessed by DNA-binding proteins for transcription, replication, or repair, remain elusive nearly 40 years after Kornberg's nucleosome model was proposed. Uncovering the structural dynamics of nucleosomes is a crucial step in elucidating the mechanisms regulating genome accessibility. This requires the deconvolultion of multiple structural states within an ensemble. Recent advances in single molecule methods enable unprecedented efficiency in examining subpopulation dynamics. In this review, we summarize studies of nucleosome structure and dynamics from single molecule approaches and how they advance our understanding of the mechanisms that govern DNA transactions.

Choy, John S.; Lee, Tae-Hee



Scanning-probe Raman spectroscopy with single-molecule sensitivity  

NASA Astrophysics Data System (ADS)

Single-molecule vibrational Raman spectroscopy of malachite green adsorbed on planar metal surfaces is achieved by means of optical local-field enhancement provided by a scanning nanoscopic metallic tip. The single-molecule signature is evident from spectral diffusion and a discretization of Raman peak intensities. The optical tip-sample coupling gives rise to a localization of the response down to a sub- 10nm length scale and a Raman enhancement up to ˜5×109 . This combines vibrational spectroscopy with high resolution scanning-probe microscopy for ultrasensitive in situ analysis of individual molecules.

Neacsu, Catalin C.; Dreyer, Jens; Behr, Nicolas; Raschke, Markus B.



Multicolour single molecule imaging on cells using a supercontinuum source.  


Multicolour single molecule fluorescence imaging enables the study of multiple proteins in the membranes of living cells. We describe the use of a supercontinuum laser as the excitation source, show its comparability with multiplexed single-wavelength lasers and demonstrate that it can be used to study membrane proteins such as the ErbB receptor family. We discuss the benefits of white-light sources for single molecule fluorescence, in particular their ease of use and the freedom to use the most appropriate dye without being constrained by available laser wavelengths. PMID:22435089

Webb, Stephen E D; Zanetti-Domingues, Laura; Coles, Benjamin C; Rolfe, Daniel J; Wareham, Richard J; Martin-Fernandez, Marisa L



Detection and identification of single molecules in solution  

SciTech Connect

We have extended our recent experiments in the detection of single fluorescent molecules in solution to the exploration of spectroscopy at the single-molecule level. As a first step we have developed a technique that can efficiently distinguish between two species of dye molecules on the basis of differences in their emission spectra. We have also demonstrated that another spectroscopic property, fluorescence lifetime, can be accurately determined at the single-molecule level. Spectroscopic properties can be used to identify fluorescent molecules and to reveal static or slowly varying aspects of the microenvironment of each molecule, thereby yielding information unavailable from bulk studies.

Soper, S.A.; Davis, L.M.; Shera, E.B. (Los Alamos National Laboratory, Los Alamos, New Mexico 87545 (United States))



Interfacial electronic effects in functional biolayers integrated into organic field-effect transistors  

PubMed Central

Biosystems integration into an organic field-effect transistor (OFET) structure is achieved by spin coating phospholipid or protein layers between the gate dielectric and the organic semiconductor. An architecture directly interfacing supported biological layers to the OFET channel is proposed and, strikingly, both the electronic properties and the biointerlayer functionality are fully retained. The platform bench tests involved OFETs integrating phospholipids and bacteriorhodopsin exposed to 1–5% anesthetic doses that reveal drug-induced changes in the lipid membrane. This result challenges the current anesthetic action model relying on the so far provided evidence that doses much higher than clinically relevant ones (2.4%) do not alter lipid bilayers’ structure significantly. Furthermore, a streptavidin embedding OFET shows label-free biotin electronic detection at 10 parts-per-trillion concentration level, reaching state-of-the-art fluorescent assay performances. These examples show how the proposed bioelectronic platform, besides resulting in extremely performing biosensors, can open insights into biologically relevant phenomena involving membrane weak interfacial modifications.

Angione, Maria Daniela; Cotrone, Serafina; Magliulo, Maria; Mallardi, Antonia; Altamura, Davide; Giannini, Cinzia; Cioffi, Nicola; Sabbatini, Luigia; Fratini, Emiliano; Baglioni, Piero; Scamarcio, Gaetano; Palazzo, Gerardo; Torsi, Luisa



A Multi-State Single-Molecule Switch Actuated by Rotation of an Encapsulated Cluster within a Fullerene Cage  

SciTech Connect

We demonstrate a single-molecule switch based on tunneling electron-driven rotation of a triangular Sc?N cluster within an icosahedral C 80 fullerene cage among three pairs of enantiomorphic configura-tions. Scanning tunneling microscopy imaging of switching within single molecules and electronic structure theory identify the conformational isomers and their isomerization pathways. Bias-dependent actionspectra and modeling identify the antisymmetric stretch vibration of Sc 3N cluster to be the gateway for energy transfer from the tunneling electrons to the cluster rotation. Hierarchical switching of conductivity through the internal cluster motion among multiple stationary states while maintaining a constant shape, is advantageous for the integration of endohedral fullerene-based single-molecule memory and logic devices into parallel molecular computing arc.

Huang, Tian; Zhao, Jin; Feng, Min; Popov, Alexey A.; Yang, Shangfeng; Dunsch, Lothar; Petek, Hrvoje



Continuous base identification for single-molecule nanopore DNA sequencing  

Microsoft Academic Search

A single-molecule method for sequencing DNA that does not require fluorescent labelling could reduce costs and increase sequencing speeds. An exonuclease enzyme might be used to cleave individual nucleotide molecules from the DNA, and when coupled to an appropriate detection system, these nucleotides could be identified in the correct order. Here, we show that a protein nanopore with a covalently

Hai-Chen Wu; Lakmal Jayasinghe; Alpesh Patel; Stuart Reid; Hagan Bayley



Nano and Microfluidics for Single Molecule Biophysics Applications  

Microsoft Academic Search

The recently developed abilities to manipulate and visualize single deoxyribonucleic acid (DNA) molecules are making a significant impact on discoveries in cellular and molecular biology. Innovations in micro- and nanofluidics are an important enabler of single-molecule techniques. Two projects were pursued, one to develop microfluidic devices for optical tweezers applications and one to develop porous nanochannel devices for elongation of

Patrick Jurney


Single-molecule Studies of RNA Polymerase: Motoring Along  

PubMed Central

Single-molecule techniques have advanced our understanding of transcription by RNA polymerase. A new arsenal of approaches, including single-molecule fluorescence, atomic-force microscopy, magnetic tweezers, and optical traps have been employed to probe the many facets of the transcription cycle. These approaches supply fresh insights into the means by which RNA polymerase identifies a promoter; initiates transcription, translocates and pauses along the DNA template, proofreads errors, and ultimately terminates transcription. Results from single-molecule experiments complement knowledge gained from biochemical and genetic assays by facilitating the observation of states that are otherwise obscured by ensemble averaging, such as those resulting from heterogeneity in molecular structure, elongation rate, or pause propensity. Most studies to date have been performed with bacterial RNA polymerase, but work is also being carried out with eukaryotic polymerase (Pol II) and single-subunit polymerases from bacteriophages. We discuss recent progress achieved by single-molecule studies, highlighting some of the unresolved questions and ongoing debates.

Herbert, Kristina M.; Greenleaf, William J.; Block, Steven M.



Ten years of tension: single-molecule DNA mechanics  

Microsoft Academic Search

The basic features of DNA were elucidated during the half-century following the discovery of the double helix. But it is only during the past decade that researchers have been able to manipulate single molecules of DNA to make direct measurements of its mechanical properties. These studies have illuminated the nature of interactions between DNA and proteins, the constraints within which

Carlos Bustamante; Zev Bryant; Steven B. Smith



The dynamics of single-molecule DNA in flow  

Microsoft Academic Search

Within the last decade, fluorescence microscopy of single molecules of DNA in a plethora of flow fields has allowed an unprecedented examination of the dynamics of polymers in flow. As a result, new principles (e.g. “molecular individualism”) have been developed regarding these dynamics and old debates (e.g. conformational hysteresis of polymers in extensional flow) have received a fresh airing. The

Eric S. G. Shaqfeh



Fluorescence microscopy in superfluid helium: Single molecule imaging  

Microsoft Academic Search

A fluorescence microscope for the detection of spatially resolved single molecules at low temperature has been developed. The main part consists of a microscope objective which is inserted in superfluid helium. Two versions of the optical system are described. Both setups have a high numerical aperture and thus high collection efficiency. A video camera with an image intensifier is used

Jan Jasny; Jerzy Sepiol; Thomas Irngartinger; Markus Traber; Alois Renn; Urs P. Wild



Single-Molecule Biochemical Analysis Using Channel Current Cheminformatics  

NASA Astrophysics Data System (ADS)

A single nanometer-scale protein channel, residing in a bilayer, is used as a single-molecule measurement device. Single molecule kinetic information can be directly obtained with this approach via observation of single-molecule channel current blockades. A nanopore-based detector can also measure molecular characteristics indirectly, by changes in the blockades resulting from a changing bound-molecule complex. In essence, the heart of chemistry - the nature of the chemical bond - is now accessible via a new, computationally intensive, single-molecule observation method. In this work: (i) analysis of blockade signals is done using a variety of bioinformatics and machine learning tools; (ii) antibody blockade signals are examined and preliminary data on the characterization of antibody-antigen binding is briefly explored; and (iii) aptamer-based drug-discovery screening prospects are explored. The initial feature identification and extraction of blockade signals involves HMMs for level identification, HMM-EM for level projection, and time-domain FSAs for processing of the level-projected waveform. HMMs are then used for feature extraction and an SVM decision tree for multiclass discrimination. A new family of SVM variants is used, based on regularized-divergence kernels, and restriction is also made to feature vectors that can be interpreted as probability vectors. A web interface to the Channel Current Cheminformatics tools (unoCCC) and the Support Vector Machine classifier (unoSVM) will also be described.

Winters-Hilt, Stephen



Interlaced Optical Force-Fluorescence Measurements for Single Molecule Biophysics  

Microsoft Academic Search

Combining optical tweezers with single molecule fluorescence offers a powerful technique to study the biophysical properties of single proteins and molecules. However, such integration into a combined, coincident arrangement has been severely limited by the dramatic reduction in fluorescence longevity of common dyes under simultaneous exposure to trapping and fluorescence excitation beams. We present a novel approach to overcome this

Ricardo R. Brau; Peter B. Tarsa; Jorge M. Ferrer; Peter Lee; Matthew J. Lang



An RNA toolbox for single-molecule force spectroscopy studies  

PubMed Central

Precise, controllable single-molecule force spectroscopy studies of RNA and RNA-dependent processes have recently shed new light on the dynamics and pathways of RNA folding and RNA-enzyme interactions. A crucial component of this research is the design and assembly of an appropriate RNA construct. Such a construct is typically subject to several criteria. First, single-molecule force spectroscopy techniques often require an RNA construct that is longer than the RNA molecules used for bulk biochemical studies. Next, the incorporation of modified nucleotides into the RNA construct is required for its surface immobilization. In addition, RNA constructs for single-molecule studies are commonly assembled from different single-stranded RNA molecules, demanding good control of hybridization or ligation. Finally, precautions to prevent RNase- and divalent cation-dependent RNA digestion must be taken. The rather limited selection of molecular biology tools adapted to the manipulation of RNA molecules, as well as the sensitivity of RNA to degradation, make RNA construct preparation a challenging task. We briefly illustrate the types of single-molecule force spectroscopy experiments that can be performed on RNA, and then present an overview of the toolkit of molecular biology techniques at one's disposal for the assembly of such RNA constructs. Within this context, we evaluate the molecular biology protocols in terms of their effectiveness in producing long and stable RNA constructs.

Vilfan, Igor D.; Kamping, Wiecher; van den Hout, Michiel; Candelli, Andrea; Hage, Susanne; Dekker, Nynke H.



Simultaneous single molecule atomic force and fluorescence lifetime imaging  

NASA Astrophysics Data System (ADS)

The combination of atomic force microscopy (AFM) with single-molecule-sensitive confocal fluorescence microscopy enables a fascinating investigation into the structure, dynamics and interactions of single biomolecules or their assemblies. AFM reveals the structure of macromolecular complexes with nanometer resolution, while fluorescence can facilitate the identification of their constituent parts. In addition, nanophotonic effects, such as fluorescence quenching or enhancement due to the AFM tip, can be used to increase the optical resolution beyond the diffraction limit, thus enabling the identification of different fluorescence labels within a macromolecular complex. We present a novel setup consisting of two commercial, state-of-the-art microscopes. A sample scanning atomic force microscope is mounted onto an objective scanning confocal fluorescence lifetime microscope. The ability to move the sample and objective independently allows for precise alignment of AFM probe and laser focus with an accuracy down to a few nanometers. Time correlated single photon counting (TCSPC) gives us the opportunity to measure single-molecule fluorescence lifetimes. We will be able to study molecular complexes in the vicinity of an AFM probe on a level that has yet to be achieved. With this setup we simultaneously obtained single molecule sensitivity in the AFM topography and fluorescence lifetime imaging of YOYO-1 stained lambda-DNA samples and we showed silicon tip induced single molecule quenching on organic fluorophores.

Schulz, Olaf; Koberling, Felix; Walters, Deron; Koenig, Marcelle; Viani, Jacob; Ros, Robert



CHEMICAL PHYSICS: Single-Molecule Spectroscopy Comes of Age  

NSDL National Science Digital Library

Access to the article is free, however registration and sign-in are required. In their Perspective, Kelley et al. report from a recent symposium on single-molecule spectroscopy. The symposium demonstrates that the field has matured and is now providing unprecedented insights in biology and materials science.

Anne Myers Kelley (Kansas State University;Department of Chemistry); Xavier Michalet (Lawrence Berkeley National Laboratory;Materials Sciences and Biophysical Sciences Divisions); Shimon Weiss (Lawrence Berkeley National Laboratory;Materials Sciences and Biophysical Sciences Divisions)



Stretching polysaccharides on live cells using single molecule force spectroscopy  

Microsoft Academic Search

The knowledge of molecular mechanisms underlying the adhesive and mechanical properties of cell surface-associated molecules is a key to understanding their functions. In this context, single-molecule force spectroscopy (SMFS) has recently offered new opportunities for probing the adhesion and mechanics of polysaccharides and proteins on live cells. Here we present a protocol that we have used to analyze polysaccharide chains

Grégory Francius; David Alsteens; Vincent Dupres; Sarah Lebeer; Sigrid De Keersmaecker; Jos Vanderleyden; Hermann J Gruber; Yves F Dufrêne



Label-free biosensing with single-molecule force spectroscopy.  


We demonstrate here a novel single-molecule, label-free bioanalytical system capable of sensing the presence of specific ssDNA oligomer sequences and proteins with high selectivity and sensitivity. An ssDNA concentration of 1 nM and a Lyz concentration of 0.65 nM could be detected. PMID:23486781

Wei, Gang; Steckbeck, Sascha; Köppen, Susan; Colombi Ciacchi, Lucio



The Single Molecule Imaging Approach to Membrane Protein Stoichiometry  

PubMed Central

Recent technical advances have enabled the imaging of single fluorescent molecules. The application of single molecule visualization techniques has opened up new avenues of experimentation in biology at the molecular level. In this article, we review the application of single fluorescent molecule visualization and analysis to an important problem, that of sub-unit stoichiometry in membrane proteins, with particular emphasis on our approach. Single fluorescent molecules, coupled to fluorescent proteins, are localized in the membranes of cells. The molecules are then exposed to continuous low-level excitation until their fluorescence emissions reach background levels. The high sensitivity of modern instrumentation has enabled direct observations of discrete step decreases in the fluorescence of single molecules, which represent the bleaching of single fluorophores. By counting the number of steps over a large number of single molecules, an average step count is determined, from which the stoichiometry is deduced using a binomial model. We examined the stoichiometry of a protein, prestin, that is central to mammalian hearing. We discuss how we prepared, identified and imaged single molecules of prestin. The methodological considerations behind our approach are described and compared to similar procedures in other laboratories.

Hallworth, Richard; Nichols, Michael G.



Modeling single molecule detection probabilities in microdroplets. Final report  

SciTech Connect

Numerical tools for modeling the fluorescence collected from a single molecule within a microsphere as a function of it`s position and orientation, the size of the droplet, the numerical aperture of the lens, the detection geometry, type of illumination, and the linewidth of the emitting molecule, are described.

Hill, S.C.



Interfacial Charge Transport in Organic Electronic Materials: the Key to a New Electronics Technology  

SciTech Connect

This is the final report of a three-year, Laboratory Directed Research and Development (LDRD) project at Los Alamos National Laboratory (LANL). The primary aim of this project is to obtain a basic scientific understanding of electrical transport processes at interfaces that contain an organic electronic material. Because of their processing advantages and the tunability of their electronic properties, organic electronic materials are revolutionizing major technological areas such as information display. We completed an investigation of the fundamental electronic excitation energies in the prototype conjugated polymer MEH-PPV. We completed a combined theoretical/experimental study of the energy relation between charged excitations in a conjugated polymer and the metal at a polymer/metal interface. We developed a theoretical model that explains injection currents at polymer/metal interfaces. We have made electrical measurements on devices fabricated using the conjugated polymer MEH-PPV a nd a series of metals.

Smith, D.L.; Campbell, I.H.; Davids, P.S.; Heller, C.M.; Laurich, B.K.; Crone, B.K.; Saxena, A.; Bishop, A.R.; Ferraris, J.P.; Yu, Z.G.



Temperature Independent Porous Nanocontainers for Single Molecule Fluorescence Studies  

PubMed Central

In this work, we demonstrate the capability of using lipid vesicles biofunctionalized with protein channels to perform single molecule fluorescence measurements over a biologically relevant temperature range. Lipid vesicles can serve as an ideal nanocontainer for single molecule fluorescence measurements of bio-macromolecules. One serious limitation of the vesicle encapsulation method has been that the lipid membrane is practically impermeable to most ions and small molecules, limiting its application to observing reactions in equilibrium with the initial buffer condition. In order to permeabilize the barrier, Staphylococcal aureus toxin ?-hemolysin (aHL) channels have been incorporated into the membrane. These aHL channels have been characterized using single molecule fluorescence resonance energy transfer (smFRET) signals from vesicle encapsulated guanine-rich DNA that folds in G-quadruplex motif as well as from Rep helicase-DNA system. We show that these aHL channels are permeable to monovalent ions and small molecules, such as ATP, over the biologically relevant temperature range (17–37 °C). Ions can efficiently pass through preformed aHL channels in order to initiate DNA folding without any detectable delay. With addition of the cholesterol to the membrane, we also report 35-fold improvement in aHL channel formation efficiency making this approach more practical for wider applications. Finally, the temperature dependent single molecule enzymatic study inside these nanocontainers is demonstrated by measuring Rep helicase repetitive shuttling dynamic along a single stranded DNA at various temperature values. The permeability of the biofriendly nanocontainer over a wide range of temperature would be effectively applied to other surface-based high throughput measurements and sensors beyond the single molecule fluorescence measurements.

Ishitsuka, Yuji; Okumus, Burak; Arslan, Sinan; Chen, Kok Hao; Ha, Taekjip



Heterogeneous nucleation of organic crystals mediated by single-molecule templates.  


Fundamental understanding of how crystals of organic molecules nucleate on a surface remains limited because of the difficulty of probing rare events at the molecular scale. Here we show that single-molecule templates on the surface of carbon nanohorns can nucleate the crystallization of two organic compounds from a supersaturated solution by mediating the formation of disordered and mobile molecular nanoclusters on the templates. Single-molecule real-time transmission electron microscopy indicates that each nanocluster consists of a maximum of approximately 15 molecules, that there are fewer nanoclusters than crystals in solution, and that in the absence of templates physisorption, but not crystal formation, occurs. Our findings suggest that template-induced heterogeneous nucleation mechanistically resembles two-step homogeneous nucleation. PMID:22983432

Harano, Koji; Homma, Tatsuya; Niimi, Yoshiko; Koshino, Masanori; Suenaga, Kazu; Leibler, Ludwik; Nakamura, Eiichi



Anchoring of rare-earth-based single-molecule magnets on single-walled carbon nanotubes.  


A new heteroleptic bis(phthalocyaninato) terbium(III) complex 1, bearing a pyrenyl group, exhibits temperature and frequency dependence of ac magnetic susceptibility, typical of single-molecule magnets. The complex was successfully attached to single-walled carbon nanotubes (SWNTs) using pi-pi interactions, yielding a 1-SWNT conjugate. The supramolecular grafting of 1 to SWNTs was proven qualitatively and quantitatively by high-resolution transmission electron microscopy, emission spectroscopy, and atomic force spectroscopy. Giving a clear magnetic fingerprint, the anisotropy energy barrier and the magnetic relaxation time of the 1-SWNT conjugate are both increased in comparison with the pure crystalline compound 1, likely due to the suppression of intermolecular interactions. The obtained results propose the 1-SWNT conjugate as a promising constituent unit in magnetic single-molecule measurements using molecular spintronics devices. PMID:19799421

Kyatskaya, Svetlana; Mascarós, José Ramón Galán; Bogani, Lapo; Hennrich, Frank; Kappes, Manfred; Wernsdorfer, Wolfgang; Ruben, Mario




PubMed Central

Owing to its high quantum efficiency, the charge-coupled device (CCD) is an important imaging tool employed in biological applications such as single molecule microscopy. Under extremely low light conditions, however, a CCD is generally unsuitable because its readout noise can easily overwhelm the weak signal. Instead, an electron-multiplying charge-coupled device (EMCCD), which stochastically amplifies the acquired signal to drown out the readout noise, can be used. We have previously proposed a framework for calculating the Fisher information, and hence the Cramer-Rao lower bound, for estimating parameters (e.g., single molecule location) from the images produced by an optical microscope. Here, we develop the theory that is needed for deriving, within this framework, performance measures pertaining to the estimation of parameters from an EMCCD image. Our results allow the comparison of a CCD and an EMCCD in terms of the best accuracy with which parameters can be estimated from their acquired images.

Chao, Jerry; Ward, E. Sally; Ober, Raimund J.



Electronic band structures and optical properties of type-II superlattice photodetectors with interfacial effect.  


The electronic band structures and optical properties of type-II superlattice (T2SL) photodetectors in the mid-infrared (IR) range are investigated. We formulate a rigorous band structure model using the 8-band k · p method to include the conduction and valence band mixing. After solving the 8 × 8 Hamiltonian and deriving explicitly the new momentum matrix elements in terms of envelope functions, optical transition rates are obtained through the Fermi's golden rule under various doping and injection conditions. Optical measurements on T2SL photodetectors are compared with our model and show good agreement. Our modeling results of quantum structures connect directly to the device-level design and simulation. The predicted doping effect is readily applicable to the optimization of photodetectors. We further include interfacial (IF) layers to study the significance of their effect. Optical properties of T2SLs are expected to have a large tunable range by controlling the thickness and material composition of the IF layers. Our model provides an efficient tool for the designs of novel photodetectors. PMID:22330471

Qiao, Peng-Fei; Mou, Shin; Chuang, Shun Lien



Quantum Optics: Colloidal Fluorescent Semiconductor Nanocrystals (Quantum Dots) in Single-Molecule Detection and Imaging  

NASA Astrophysics Data System (ADS)

Coming from the electronic material sciences, semiconductor nanocrystals, called quantum dots (QDs), have emerged as new powerful fluorescent probes for in vitro and in vivo biological labeling and single-molecule experiments. QDs possess several unique optical properties that make them very attractive over conventional fluorescent dyes and genetically encoded proteins technologies. They have precise emission color tunability by size due to quantum confinement effects, better photostability and brightness, wide absorption band and very narrow emission band for multiplexing, and increased fluorescence lifetimes. These characteristics, combined with some dramatic progresses achieved in surface chemistry, biocompatibility and targeting strategies have allowed their recent advances in the field of single-molecule detection and imaging using diverse microscope geometries like confocal microscopy, total internal reflection (TIR) microscopy or basic wide-field epifluorescence microscopy. This chapter reviews the basic principles of QDs' electronic structure necessary to understand their fundamental optical and physical properties and goes on to present recent QDs' uses in biological imaging with an emphasis on single-molecule detection.

Bentolila, Laurent A.; Michalet, Xavier; Weiss, Shimon


Inverse acoustic Faraday effect in single molecule magnets  

NASA Astrophysics Data System (ADS)

A theoretical study into the effect of stationary magnetization induced in a paramagnetic medium by propagation of a circularly-polarized acoustic wave is reported. We termed this magnetoacoustic phenomenon "the inverse acoustic Faraday effect" for its obvious similarity to the inverse Faraday effect in electrodynamics (magnetooptics, in particular). A phenomenological relation is established between the inverse acoustic Faraday effect and the paramagnetic acoustic Faraday rotation widely known as the acoustic Faraday effect. This relation is demonstrated for single molecule magnets. We estimated the magnetization induced by the inverse acoustic Faraday effect in single molecule magnets Mn12-Ac, for the case of a hypersonic wave. This value proved sufficient for the effect to be observed experimentally.

Davidovich Tokman, Iosif; Il'inichna Pozdnyakova, Vera



Probing DNA clamps with single-molecule force spectroscopy.  


Detailed mechanisms of DNA clamps in prokaryotic and eukaryotic systems were investigated by probing their mechanics with single-molecule force spectroscopy. Specifically, the mechanical forces required for the Escherichia coli and Saccharomyces cerevisiae clamp opening were measured at the single-molecule level by optical tweezers. Steered molecular dynamics simulations further examined the forces involved in DNA clamp opening from the perspective of the interface binding energies associated with the clamp opening processes. In combination with additional molecular dynamics simulations, we identified the contact networks between the clamp subunits that contribute significantly to the interface stability of the S.cerevisiae and E. coli clamps. These studies provide a vivid picture of the mechanics and energy landscape of clamp opening and reveal how the prokaryotic and eukaryotic clamps function through different mechanisms. PMID:23783571

Wang, Lin; Xu, Xiaojun; Kumar, Ravindra; Maiti, Buddhadev; Liu, C Tony; Ivanov, Ivaylo; Lee, Tae-Hee; Benkovic, Stephen J



Probing DNA clamps with single-molecule force spectroscopy  

PubMed Central

Detailed mechanisms of DNA clamps in prokaryotic and eukaryotic systems were investigated by probing their mechanics with single-molecule force spectroscopy. Specifically, the mechanical forces required for the Escherichia coli and Saccharomyces cerevisiae clamp opening were measured at the single-molecule level by optical tweezers. Steered molecular dynamics simulations further examined the forces involved in DNA clamp opening from the perspective of the interface binding energies associated with the clamp opening processes. In combination with additional molecular dynamics simulations, we identified the contact networks between the clamp subunits that contribute significantly to the interface stability of the S.cerevisiae and E. coli clamps. These studies provide a vivid picture of the mechanics and energy landscape of clamp opening and reveal how the prokaryotic and eukaryotic clamps function through different mechanisms.

Wang, Lin; Xu, Xiaojun; Kumar, Ravindra; Maiti, Buddhadev; Liu, C. Tony; Ivanov, Ivaylo; Lee, Tae-Hee; Benkovic, Stephen J.



Applications of optical trapping to single molecule DNA  

SciTech Connect

This is the final report of a three-year, Laboratory Directed Research and Development (LDRD) project at the Los Alamos National Laboratory (LANL). The project focused on the methodologies required to integrate optical trapping with single molecule detection (SMD) so as to demonstrate high speed sequencing through optical micromanipulation of host substrates, nucleotide cleavage, and single molecule detection. As part of this effort, the new technology of optical tweezers was applied to the confinement and manipulation of microsphere handles containing attached DNA fragments. The authors demonstrated substrate optical trapping in rapid flow streams, the fluorescence excitation and detection of fluorescently labeled nucleotides in an optical trapping system, and the epifluorescent imaging of DNA fragments in flow streams. They successfully demonstrated optical trapping in laminar flow streams and completely characterized the trapping process as functions of fluid flow velocity, chamber dimension, trapping depth, incident laser power, and fluorescence measurement geometry.

Sonek, G.J.; Berns, M.W. [Univ. of California, Irvine, CA (United States). Beckman Laser Inst. and Medical Clinic; Keller, R.A. [Los Alamos National Lab., NM (United States). Chemical Science and Technology Div.



Light Sheet Microscopy for Single Molecule Tracking in Living Tissue  

PubMed Central

Single molecule observation in cells and tissue allows the analysis of physiological processes with molecular detail, but it still represents a major methodological challenge. Here we introduce a microscopic technique that combines light sheet optical sectioning microscopy and ultra sensitive high-speed imaging. By this approach it is possible to observe single fluorescent biomolecules in solution, living cells and even tissue with an unprecedented speed and signal-to-noise ratio deep within the sample. Thereby we could directly observe and track small and large tracer molecules in aqueous solution. Furthermore, we demonstrated the feasibility to visualize the dynamics of single tracer molecules and native messenger ribonucleoprotein particles (mRNPs) in salivary gland cell nuclei of Chironomus tentans larvae up to 200 µm within the specimen with an excellent signal quality. Thus single molecule light sheet based fluorescence microscopy allows analyzing molecular diffusion and interactions in complex biological systems.

Ritter, Jorg Gerhard; Veith, Roman; Veenendaal, Andreas; Siebrasse, Jan Peter; Kubitscheck, Ulrich



Single-Molecule Studies of DNA Replisome Function  

PubMed Central

Fast and accurate replication of DNA is accomplished by the interactions of multiple proteins in the dynamic DNA replisome. The DNA replisome effectively coordinates the leading and lagging strand synthesis of DNA. These complex, yet elegantly organized, molecular machines have been studied extensively by kinetic and structural methods to provide an in-depth understanding of the mechanism of DNA replication. Owing to averaging of observables, unique dynamic information of the biochemical pathways and reactions are concealed in conventional ensemble methods. However, recent advances in the rapidly expanding field of single-molecule analyses to study single biomolecules offer opportunities to probe and understand the dynamic processes involved in large biomolecular complexes such as replisomes. This review will focus on the recent developments in the biochemistry and biophysics of DNA replication employing single-molecule techniques and the insights provided by these methods towards a better understanding of the intricate mechanisms of DNA replication.

Perumal, Senthil K.; Yue, Hongjun; Hu, Zhenxin; Spiering, Michelle M.; Benkovic, Stephen J.



Three-dimensional Molecular Modeling with Single Molecule FRET  

PubMed Central

Single molecule fluorescence energy transfer experiments enable investigations of macromolecular conformation and folding by the introduction of fluorescent dyes at specific sites in the macromolecule. Multiple such experiments can be performed with different labeling site combinations in order to map complex conformational changes or interactions between multiple molecules. Distances that are derived from such experiments can be used for determination of the fluorophore positions by triangulation. When combined with a known structure of the macromolecule(s) to which the fluorophores are attached, a three-dimensional model of the system can be determined. However, care has to be taken to properly derive distance from fluorescence energy transfer efficiency and to recognize the systematic or random errors for this relationship. Here we review the experimental and computational methods used for three-dimensional modeling based on single molecule fluorescence resonance transfer, and describe recent progress in pushing the limits of this approach to macromolecular complexes.

Brunger, Axel T.; Strop, Pavel; Vrljic, Marija; Chu, Steven; Weninger, Keith R.



What is not required to make a single molecule magnet.  


The widely accepted assumption that the development of more efficient single molecule magnets must involve ever higher total spin values has so far driven synthetic efforts towards molecular clusters of increasing nuclearity. In the present paper it is suggested that it might be worthwhile to reconsider this approach. There is evidence from theory and experiment to suggest that the race for multinuclear complexes with higher total spin might not necessarily be fruitful as a strategy for maximizing the magnetic relaxation barrier. Instead, we propose that more effort should be directed in understanding the parameters involved in maximizing the anisotropy of small, perhaps even mononuclear, molecules. Using multi-reference ab initio calculations we demonstrate the theory that can be applied and the principles of the computational approach for representative mononuclear complexes. Such small units may subsequently be employed as building blocks for the controlled assembly of larger and maximally anisotropic single molecule magnets. PMID:21322486

Neese, Frank; Pantazis, Dimitrios A




NASA Astrophysics Data System (ADS)

The fascinating advances in single atom/molecule manipulation with a scanning tunneling microscope (STM) tip allow scientists to fabricate atomic-scale structures or to probe chemical and physical properties of matters at an atomic level. Owing to these advances, it has become possible for the basic chemical reaction steps, such as dissociation, diffusion, adsorption, readsorption, and bond-formation processes, to be performed by using the STM tip. Complete sequences of chemical reactions are able to induce at a single-molecule level. New molecules can be constructed from the basic molecular building blocks on a one-molecule-at-a-time basis by using a variety of STM manipulation schemes in a systematic step-by-step manner. These achievements open up entirely new opportunities in nanochemistry and nanochemical technology. In this review, various STM manipulation techniques useful in the single-molecule reaction process are reviewed, and their impact on the future of nanoscience and technology are discussed.

Hla, Saw-Wai; Rieder, Karl-Heinz



High-Resolution, Single-Molecule Measurements of Biomolecular Motion  

PubMed Central

Many biologically important macromolecules undergo motions that are essential to their function. Biophysical techniques can now resolve the motions of single molecules down to the nanometer scale or even below, providing new insights into the mechanisms that drive molecular movements. This review outlines the principal approaches that have been used for high-resolution measurements of single-molecule motion, including centroid tracking, fluorescence resonance energy transfer, magnetic tweezers, atomic force microscopy, and optical traps. For each technique, the principles of operation are outlined, the capabilities and typical applications are examined, and various practical issues for implementation are considered. Extensions to these methods are also discussed, with an eye toward future application to outstanding biological problems.

Greenleaf, William J.; Woodside, Michael T.; Block, Steven M.



A Single-Molecule Study of RNA Catalysis and Folding  

NASA Astrophysics Data System (ADS)

Using fluorescence microscopy, we studied the catalysis by and folding of individual Tetrahymena thermophila ribozyme molecules . The dye-labeled and surface-immobilized ribozymes used were shown to be functionally indistinguishable from the unmodified free ribozyme in solution. A reversible local folding step in which a duplex docks and undocks from the ribozyme core was observed directly in single-molecule time trajectories, allowing the determination of the rate constants and characterization of the transition state. A rarely populated docked state, not measurable by ensemble methods, was observed. In the overall folding process, intermediate folding states and multiple folding pathways were observed. In addition to observing previously established folding pathways, a pathway with an observed folding rate constant of 1 per second was discovered. These results establish single-molecule fluorescence as a powerful tool for examining RNA folding.

Zhuang, Xiaowei; Bartley, Laura E.; Babcock, Hazen P.; Russell, Rick; Ha, Taekjip; Herschlag, Daniel; Chu, Steven



Quantum Spin Dynamics in Single-Molecule Magnets  

Microsoft Academic Search

This thesis contains a thorough investigation of the quantum spin dynamics in Mn12-ac and Mn6 Single-molecule magnets. In particular, we have investigated the interplay between quantum tunneling of magnetization and nuclear spin dynamics in Mn12-ac by ultra-low temperature NMR experiments. We discuss the effect of quantum tunneling on the nuclear spin-lattice relaxation, the nuclear spin diffusion, the thermalization of the

Andrea Morello



Ultrasensitive laser spectroscopy in solids: Single-molecule detection  

NASA Astrophysics Data System (ADS)

In spite of detection intensity constraints necessary to avoid power broadening, the optical absorption spectrum of single molecules of pentacene in p-terphenyl crystals can be measured by using laser FM spectroscopy combined with Stark and/or ultrasonic double modulation (to remove residual amplitude modulation) and recording spectra far out in the wings of the inhomogeneous line to reduce the number of molecules in resonance to one.

Moerner, W. E.; Ambrose, W. P.; Kador, L.



Single Molecule Studies Of Spliceosomal Snrnas U2-U6  

Microsoft Academic Search

Spliceosomes catalyze the maturation of precursor mRNAs in organisms ranging\\u000afrom yeast to humans. Their catalytic core comprises three small nuclear RNAs (U2, U5\\u000aand U6) involved in substrate positioning and catalysis. It has been postulated, but never\\u000ashown experimentally, that the U2-U6 complex adopts at least two conformations that\\u000areflect different activation states. We have used single-molecule fluorescence to

Zhuojun Guo



Single Molecule Detection Using Surface-Enhanced Raman Scattering (SERS)  

Microsoft Academic Search

By exploiting the extremely large effective cross sections ( 10-17-10-16 cm2\\/molecule) available from surface-enhanced Raman scattering (SERS), we achieved the first observation of single molecule Raman scattering. Measured spectra of a single crystal violet molecule in aqueous colloidal silver solution using one second collection time and about 2×105 W\\/cm2 nonresonant near-infrared excitation show a clear ``fingerprint'' of its Raman features

Katrin Kneipp; Yang Wang; Harald Kneipp; Lev T. Perelman; Irving Itzkan; Ramachandra R. Dasari; Michael S. Feld



Single-Molecule Analysis of Cell-Virus Binding Interactions  

Microsoft Academic Search

\\u000a Adhesion assays based on single molecule interactions are a useful option when discerning between avidity and affinity in\\u000a complex systems. This is especially true for viral adhesion to living cells which typically involves a complex system of proteins\\u000a working together to lead to productive infection. Here, we discuss assays that have been used to quantitatively study the\\u000a adhesion of viral

Terrence M. Dobrowsky; Denis Wirtz


Identification of Binding Mechanisms in Single Molecule–DNA Complexes  

Microsoft Academic Search

Changes in the elastic properties of single deoxyribonucleic acid (DNA) molecules in the presence of different DNA-binding agents are identified using atomic force microscope single molecule force spectroscopy. We investigated the binding of poly(dG-dC) dsDNA with the minor groove binder distamycin A, two supposed major groove binders, an ?-helical and a 310-helical peptide, the intercalants daunomycin, ethidium bromide and YO,

Rainer Eckel; Robert Ros; Alexandra Ros; Sven David Wilking; Norbert Sewald; Dario Anselmetti



Microfluidic device for single-molecule experiments with enhanced photostability.  


A microfluidic device made of polydimethylsiloxane (PDMS) addresses key limitations in single-molecule fluorescence experiments by providing high dye photostability and low sample sticking. Photobleaching is dramatically reduced by deoxygenation via gas diffusion through porous channel walls. Rapid buffer exchange in a laminar sheath flow followed by optical interrogation minimizes surface-sample contacts and allows the in situ addition and combination of other reagents. PMID:19772358

Lemke, Edward A; Gambin, Yann; Vandelinder, Virginia; Brustad, Eric M; Liu, Hsiao-Wei; Schultz, Peter G; Groisman, Alex; Deniz, Ashok A



Mechanical Single Molecule Investigations of SNARE Protein Interactions  

NASA Astrophysics Data System (ADS)

We used an Atomic Force Microscope (AFM) to perform single molecule investigations of the SNARE (soluble N-ethyl maleimide-sensitive fusion protein attachment protein receptors) proteins, syntaxin, synaptobrevin and SNAP 25. These proteins are involved in the docking and release of neurotransmitters. The rupture force and extension of the interactions were measured. Chemical reaction rate theory was applied to obtain the energy barrier width and lifetime. Their temperature dependence was also explored.

Liu, Wei; Montana, Vedrana; Parpura, Vladimir; Mohideen, Umar



Exchange bias in Ni 4 single-molecule magnets  

Microsoft Academic Search

The syntheses and physical properties are reported for three single-molecule magnets (SMMs) with the composition [Ni(hmp)(ROH)Cl]4, where R is CH3 (complex 1), CH2CH3 (complex 2) or CH2CH2C(CH3)3 (complex 3) and hmp? is the monoanion of 2-hydroxymethylpyridine. The core of each complex is a distorted cube formed by four NiII ions and four alkoxide hmp? oxygen atoms at alternating corners. Ferromagnetic

En-Che Yang; Wolfgang Wernsdorfer; Stephen Hill; Rachel S. Edwards; Motohiro Nakano; S. Maccagnano; Lev N. Zakharov; Arnold L. Rheingold; George Christou; David N. Hendrickson



Single-molecule fluorescence spectroscopy in (bio)catalysis.  


The ever-improving time and space resolution and molecular detection sensitivity of fluorescence microscopy offer unique opportunities to deepen our insights into the function of chemical and biological catalysts. Because single-molecule microscopy allows for counting the turnover events one by one, one can map the distribution of the catalytic activities of different sites in solid heterogeneous catalysts, or one can study time-dependent activity fluctuations of individual sites in enzymes or chemical catalysts. By experimentally monitoring individuals rather than populations, the origin of complex behavior, e.g., in kinetics or in deactivation processes, can be successfully elucidated. Recent progress of temporal and spatial resolution in single-molecule fluorescence microscopy is discussed in light of its impact on catalytic assays. Key concepts are illustrated regarding the use of fluorescent reporters in catalytic reactions. Future challenges comprising the integration of other techniques, such as diffraction, scanning probe, or vibrational methods in single-molecule fluorescence spectroscopy are suggested. PMID:17664433

Roeffaers, Maarten B J; De Cremer, Gert; Uji-i, Hiroshi; Muls, Benîot; Sels, Bert F; Jacobs, Pierre A; De Schryver, Frans C; De Vos, Dirk E; Hofkens, Johan



Visualizing and controlling vibrational wave packets of single molecules.  


The active steering of the pathways taken by chemical reactions and the optimization of energy conversion processes provide striking examples of the coherent control of quantum interference through the use of shaped laser pulses. Experimentally, coherence is usually established by synchronizing a subset of molecules in an ensemble with ultra-short laser pulses. But in complex systems where even chemically identical molecules exist with different conformations and in diverse environments, the synchronized subset will have an intrinsic inhomogeneity that limits the degree of coherent control that can be achieved. A natural-and, indeed, the ultimate-solution to overcoming intrinsic inhomogeneities is the investigation of the behaviour of one molecule at a time. The single-molecule approach has provided useful insights into phenomena as diverse as biomolecular interactions, cellular processes and the dynamics of supercooled liquids and conjugated polymers. Coherent state preparation of single molecules has so far been restricted to cryogenic conditions, whereas at room temperature only incoherent vibrational relaxation pathways have been probed. Here we report the observation and manipulation of vibrational wave-packet interference in individual molecules at ambient conditions. We show that adapting the time and phase distribution of the optical excitation field to the dynamics of each molecule results in a high degree of control, and expect that the approach can be extended to achieve single-molecule coherent control in other complex inhomogeneous systems. PMID:20559383

Brinks, Daan; Stefani, Fernando D; Kulzer, Florian; Hildner, Richard; Taminiau, Tim H; Avlasevich, Yuri; Müllen, Klaus; van Hulst, Niek F



Ensemble and Single-Molecule Studies on Fluorescence Quenching in Transition Metal Bipyridine-Complexes  

PubMed Central

Beyond their use in analytical chemistry fluorescent probes continuously gain importance because of recent applications of single-molecule fluorescence spectroscopy to monitor elementary reaction steps. In this context, we characterized quenching of a fluorescent probe by different metal ions with fluorescence spectroscopy in the bulk and at the single-molecule level. We apply a quantitative model to explain deviations from existing standard models for fluorescence quenching. The model is based on a reversible transition from a bright to a dim state upon binding of the metal ion. We use the model to estimate the stability constants of complexes with different metal ions and the change of the relative quantum yield of different reporter dye labels. We found ensemble data to agree widely with results from single-molecule experiments. Our data indicates a mechanism involving close molecular contact of dye and quenching moiety which we also found in molecular dynamics simulations. We close the manuscript with a discussion of possible mechanisms based on Förster distances and electrochemical potentials which renders photo-induced electron transfer to be more likely than Förster resonance energy transfer.

Brox, Dominik; Kiel, Alexander; Worner, Svenja Johanna; Pernpointner, Markus; Comba, Peter; Martin, Bodo; Herten, Dirk-Peter



Information theory resolution limits and hidden Markov model analysis of single molecule fluorescence  

NASA Astrophysics Data System (ADS)

Time correlated single photon counting determines luminescence lifetime information on a single molecule level. This paper develops a formalism to allow information-theory analysis of the ability of luminescence lifetime measurements to resolve states in a single molecule. It analyzes experimental losses of information due to instrument response, digitization, and background. This paper shows how to use the information theoretical formalism to evaluate the number of photons required to distinguish dyes that differ only by lifetime, by electron transfer quenching, or by FRET. It shows how the differences between the lifetime of signal and background can help distinguish the dye position in an excitation beam. Many systems follow phenomenological kinetics where discrete states are connected by rate equations. However systems with low energetic barriers and multiple interchanging structures are not as amenable to this approach. Such continuous state spaces are best described by Langevin dynamics (LD) and an appropriate Fokker-Planck equation (FPE). This paper develops hidden Markov models (HMMs) for LD and the FPE. It shows how molecular friction and activation barrier along an effective coordinate can be estimated. It utilizes the models to guide the design of single molecule experiments.

Talaga, David



Ensemble and single-molecule studies on fluorescence quenching in transition metal bipyridine-complexes.  


Beyond their use in analytical chemistry fluorescent probes continuously gain importance because of recent applications of single-molecule fluorescence spectroscopy to monitor elementary reaction steps. In this context, we characterized quenching of a fluorescent probe by different metal ions with fluorescence spectroscopy in the bulk and at the single-molecule level. We apply a quantitative model to explain deviations from existing standard models for fluorescence quenching. The model is based on a reversible transition from a bright to a dim state upon binding of the metal ion. We use the model to estimate the stability constants of complexes with different metal ions and the change of the relative quantum yield of different reporter dye labels. We found ensemble data to agree widely with results from single-molecule experiments. Our data indicates a mechanism involving close molecular contact of dye and quenching moiety which we also found in molecular dynamics simulations. We close the manuscript with a discussion of possible mechanisms based on Förster distances and electrochemical potentials which renders photo-induced electron transfer to be more likely than Förster resonance energy transfer. PMID:23483966

Brox, Dominik; Kiel, Alexander; Wörner, Svenja Johanna; Pernpointner, Markus; Comba, Peter; Martin, Bodo; Herten, Dirk-Peter



Single-molecule DNA detection with an engineered MspA protein nanopore  

PubMed Central

Nanopores hold great promise as single-molecule analytical devices and biophysical model systems because the ionic current blockades they produce contain information about the identity, concentration, structure, and dynamics of target molecules. The porin MspA of Mycobacterium smegmatis has remarkable stability against environmental stresses and can be rationally modified based on its crystal structure. Further, MspA has a short and narrow channel constriction that is promising for DNA sequencing because it may enable improved characterization of short segments of a ssDNA molecule that is threaded through the pore. By eliminating the negative charge in the channel constriction, we designed and constructed an MspA mutant capable of electronically detecting and characterizing single molecules of ssDNA as they are electrophoretically driven through the pore. A second mutant with additional exchanges of negatively-charged residues for positively-charged residues in the vestibule region exhibited a factor of ?20 higher interaction rates, required only half as much voltage to observe interaction, and allowed ssDNA to reside in the vestibule ?100 times longer than the first mutant. Our results introduce MspA as a nanopore for nucleic acid analysis and highlight its potential as an engineerable platform for single-molecule detection and characterization applications.

Butler, Tom Z.; Pavlenok, Mikhail; Derrington, Ian M.; Niederweis, Michael; Gundlach, Jens H.



Single-molecule studies of nucleocytoplasmic transport: from one dimension to three dimensions  

PubMed Central

In eukaryotic cells, the bidirectional trafficking of proteins and genetic materials across the double-membrane nuclear envelope is mediated by nuclear pore complexes (NPCs). A highly selective barrier formed by the phenylalanine–glycine (FG)-nucleoporin (Nup) in the NPC allows for two transport modes: passive diffusion and transport receptor-facilitated translocation. Strict regulation of nucleocytoplasmic transport is crucial for cell survival, differentiation, growth and other essential activities. However, due to the limited knowledge of the native configuration of the FG-Nup barrier and the interactions between the transiting molecules and the barrier in the NPC, the precise nucleocytoplasmic transport mechanism remains unresolved. To refine the transport mechanism, single-molecule fluorescence microscopy methods have been employed to obtain the transport kinetics of individual fluorescent molecules through the NPC and to map the interactions between transiting molecules and the FG-Nup barrier. Important characteristics of nucleocytoplasmic transport, such as transport time, transport efficiency and spatial distribution of single transiting molecules in the NPC, have been obtained that could not be measured by either ensemble average methods or conventional electron microscopy. In this critical review, we discuss the development of various single-molecule techniques and their application to nucleocytoplasmic transport in vitro and in vivo. In particular, we highlight a recent advance from one-dimensional to three-dimensional single-molecule characterization of transport through the NPC and present a comprehensive understanding of the nucleocytoplasmic transport mechanism obtained by this new technical development (105 references).

Goryaynov, Alexander; Ma, Jiong; Yang, Weidong



Simultaneous spectroscopic and topographic near-field imaging of TiO2 single surface states and interfacial electronic coupling.  


We have probed single surface states and the involved interfacial charge transfer coupling on the TiO(2) surface using confocal as well as tip-enhanced near-field topographic-spectroscopic imaging analysis on a niobium-doped rutile TiO(2)(110) surface. The confocal images excited with a radially polarized donut mode render ring-shaped excitation patterns typical for quantum systems with two perpendicular transition dipole moments. The tip-enhanced near-field optical images of single surface states are visualized by the strong exciton plasmon-polariton coupling localized at the subdomain boundaries with a spatial resolution of ?15 nm (far beyond the optical diffraction limit). We suggest that the abundant surface states in the doped TiO(2) generate excitons under laser excitation which are strongly coupled to the surface plasmon-polaritons of the Au tip. Moreover, the interfacial electronic molecule-substrate coupling has been characterized by probing the molecule-perturbed surface states distribution and the associated specific Raman vibrational modes. The imaging and characterization of the surface states and their distributions on TiO(2) surfaces at nanoscale are critically relevant to a deep understanding of interfacial electron transfer dynamics and energetics involving in solar energy conversion, photocatalysis, and mechanistic understanding of surface-enhanced Raman scattering spectroscopy. PMID:21375338

Sevinc, Papatya C; Wang, Xiao; Wang, Yuanmin; Zhang, Dai; Meixner, Alfred J; Lu, H Peter



Protein mechanics: from single molecules to functional biomaterials.  


Elastomeric proteins act as the essential functional units in a wide variety of biomechanical machinery and serve as the basic building blocks for biological materials that exhibit superb mechanical properties. These proteins provide the desired elasticity, mechanical strength, resilience, and toughness within these materials. Understanding the mechanical properties of elastomeric protein-based biomaterials is a multiscale problem spanning from the atomistic/molecular level to the macroscopic level. Uncovering the design principles of individual elastomeric building blocks is critical both for the scientific understanding of multiscale mechanics of biomaterials and for the rational engineering of novel biomaterials with desirable mechanical properties. The development of single-molecule force spectroscopy techniques has provided methods for characterizing mechanical properties of elastomeric proteins one molecule at a time. Single-molecule atomic force microscopy (AFM) is uniquely suited to this purpose. Molecular dynamic simulations, protein engineering techniques, and single-molecule AFM study have collectively revealed tremendous insights into the molecular design of single elastomeric proteins, which can guide the design and engineering of elastomeric proteins with tailored mechanical properties. Researchers are focusing experimental efforts toward engineering artificial elastomeric proteins with mechanical properties that mimic or even surpass those of natural elastomeric proteins. In this Account, we summarize our recent experimental efforts to engineer novel artificial elastomeric proteins and develop general and rational methodologies to tune the nanomechanical properties of elastomeric proteins at the single-molecule level. We focus on general design principles used for enhancing the mechanical stability of proteins. These principles include the development of metal-chelation-based general methodology, strategies to control the unfolding hierarchy of multidomain elastomeric proteins, and the design of novel elastomeric proteins that exhibit stimuli-responsive mechanical properties. Moving forward, we are now exploring the use of these artificial elastomeric proteins as building blocks of protein-based biomaterials. Ultimately, we would like to rationally tailor mechanical properties of elastomeric protein-based materials by programming the molecular sequence, and thus nanomechanical properties, of elastomeric proteins at the single-molecule level. This step would help bridge the gap between single protein mechanics and material biomechanics, revealing how the mechanical properties of individual elastomeric proteins are translated into the properties of macroscopic materials. PMID:20669937

Li, Hongbin; Cao, Yi



Single-molecule studies of unconventional motor protein myosin VI  

NASA Astrophysics Data System (ADS)

Myosin VI is one of the myosin superfamily members that are actin-based molecular motors. It has received special attention due to its distinct features as compared to other myosins, such as its opposite directionality and a much larger step size than expected given the length of its "leg". This dissertation presents the author.s graduate work of several single-molecule studies on myosin VI. Special attention was paid to some of myosin VI.s tail domains that consist of proximal tail (PT), medial tail (MT), distal tail (DT) domains and cargo-binding domain (CBD). The functional form of myosin VI in cells is still under debate. Although full length myosin VI proteins in cytosolic extracts of cells were monomers from earlier studies, there are several reasons why it is now believed that myosin VI could exist as a dimer. If this is true and dimerization occurs, the next logical question would be which parts of myosin VI are dimerization regions? One model claimed that the CBD is the sole dimerization region. A competing model claimed that there must be another region that could be involved in dimerization, based on their observation that a construct without the CBD could still dimerize. Our single-molecule experiment with progressively truncated myosin VI constructs showed that the MT domain is a dimerization region, supporting the latter model. Additional single-molecule experiments and molecular dynamics (MD) simulation done with our collaborators suggest that electrostatic salt bridges formed between positive and negative amino acid residues are mainly responsible for the MT domain dimerization. After resolving this, we are left with another important question which is how myosin VI can take such a large step. Recent crystal structure showed that one of the tail domains preceding the MT domain, called the PT domain, is a three-helix bundle. The most easily conceivable way might be an unfolding of the three-helix bundle upon dimerization, allowing the protein to stretch and reach a larger distance. The single-molecule stepping data with mutant full-length construct that lacks two helices out of three in the PT domain tell that it is indeed the case. In this dissertation, more details of myosin VI PT/MT domain experiments will be explored along with background information on the single-molecule experiment methods used in these studies.

Kim, HyeongJun


A single-molecule force spectroscopy study of the interactions between lectins and carbohydrates on cancer and normal cells  

NASA Astrophysics Data System (ADS)

The interaction forces between carbohydrates and lectins were investigated by single-molecule force spectroscopy on both cancer and normal cells. The binding kinetics was also studied, which shows that the carbohydrate-lectin complex on cancer cells is less stable than that on normal cells.The interaction forces between carbohydrates and lectins were investigated by single-molecule force spectroscopy on both cancer and normal cells. The binding kinetics was also studied, which shows that the carbohydrate-lectin complex on cancer cells is less stable than that on normal cells. Electronic supplementary information (ESI) available: Experimental details. See DOI: 10.1039/c3nr00553d

Zhao, Weidong; Cai, Mingjun; Xu, Haijiao; Jiang, Junguang; Wang, Hongda



Nanoscale characterization of interfacial electronic properties and degradation mechanisms of organic thin films for electroluminescence displays  

NASA Astrophysics Data System (ADS)

For the first time, the promising organic light-emitting diode (OLED), indium tin oxide (ITO)/copper phthalocyanine (CuPc)/N,N '-di(naphthalene-l-yl)-N,N'-diphthalbenzidine (NPB)/tris(8-quinolinolatoline) aluminum (Alq3) (ITO/CuPc/NPB/Alq 3), has been selected for investigating its interfacial properties, and morphological degradation mechanisms induced by moisture and temperature through the systematic visualization by the state-of-the-art scanning probe microscopy (SPM). The motivation of this thesis lies in the importance of the OLED interfaces associated with the OLED's quantum efficiency, and morphological alteration in respect to device lifetime and luminance. Variable-temperature ultrahigh vacuum scanning tunnelling microscopy and spectroscopy (VT-UHV/STM and STS) are used to investigate the interfacial electronic properties at the Au(111)/CuPc interface. The energy band gap of CuPc ultrathin films on Au(111) measured by the charge injection from the STM tip into the highest occupied molecular orbitals (HOMO) and the lowest unoccupied molecular orbitals (LUMO) levels of CuPc is ˜1.9--2.1 eV, which is comparable to the optical band gap (˜1.7 eV). This result indicates that a dipole layer and/or image forces is present right at the Au(111)/CuPc interface, and confirms that the common vacuum level alignment at the interface is invalid. Also, it is found that the morphological features probably affect the tunnelling current. To scrutinize the degradation associated with morphological variations of the Alga-based OLED structures, a variable-temperature tapping mode atomic force microscope (VT-AFM) and other complementary techniques such as thickness-shear mode (TSM) acoustic wave mass sensor and micro-Raman spectroscopy have been employed to sift the degradation mechanisms induced by ambient atmosphere and Joule heating. A volatile species released from the Alq3 thin films under exposure to moisture has been traced by the TSM acoustic wave mass sensor. This volatile species is most likely to be the freed 8-quinolinolatoline (8-Hq), a product of the reaction by Alq3 with water. As a result, the degradation mechanisms associated with morphological change of Alq 3 thin film by moisture are the crystallization of Alq3, the hydration of Alq3, and the reaction by Alq3 complex with moisture. Moreover, it has been visualized that the hydrated Alq 3 can be transformed into crystalline Alq3 structures and dark holes by the captioned reactions. Similarly, the ambient-induced morphological changes of ITO/CuPc, ITO/CuPc/NPB and ITO/CuPc/NPB/Alq3 structures have also been investigated. It is found that indium diffusion from the ITO substrate into organic layers possibly exists. After stored in an ambient atmosphere, crystallization of NPB thin films takes place. Photoluminescence (PL) investigation and local current map of the various degrading/degraded thin films suggest that the morphological changes, e.g., the formation of dark holes and Alq3 crystalline structures, essentially affect the PL and electrical characteristics. (Abstract shortened by UMI.)

Xu, Mingsheng


A functional single-molecule binding assay via force spectroscopy  

PubMed Central

Protein–ligand interactions, including protein–protein interactions, are ubiquitously essential in biological processes and also have important applications in biotechnology. A wide range of methodologies have been developed for quantitative analysis of protein–ligand interactions. However, most of them do not report direct functional/structural consequence of ligand binding. Instead they only detect the change of physical properties, such as fluorescence and refractive index, because of the colocalization of protein and ligand, and are susceptible to false positives. Thus, important information about the functional state of protein–ligand complexes cannot be obtained directly. Here we report a functional single-molecule binding assay that uses force spectroscopy to directly probe the functional consequence of ligand binding and report the functional state of protein–ligand complexes. As a proof of principle, we used protein G and the Fc fragment of IgG as a model system in this study. Binding of Fc to protein G does not induce major structural changes in protein G but results in significant enhancement of its mechanical stability. Using mechanical stability of protein G as an intrinsic functional reporter, we directly distinguished and quantified Fc-bound and Fc-free forms of protein G on a single-molecule basis and accurately determined their dissociation constant. This single-molecule functional binding assay is label-free, nearly background-free, and can detect functional heterogeneity, if any, among protein–ligand interactions. This methodology opens up avenues for studying protein–ligand interactions in a functional context, and we anticipate that it will find broad application in diverse protein–ligand systems.

Cao, Yi; Balamurali, M. M.; Sharma, Deepak; Li, Hongbin



Detection of pathogenic DNA at the single-molecule level  

NASA Astrophysics Data System (ADS)

We demonstrate ultrasensitive detection of pathogenic DNA in a homogeneous assay at the single-molecule level applying two-color coincidence analysis. The target molecule we quantify is a 100 nucleotide long synthetic single-stranded oligonucleotide adapted from Streptococcus pneumoniae, a bacterium causing lower respiratory tract infections. Using spontaneous hybridization of two differently fluorescing Molecular Beacons we demonstrate a detection sensitivity of 100 fM (10-13M) in 30 seconds applying a simple microfluidic device with a 100 ?m channel and confocal two-color fluorescence microscopy.

Yahiatène, Idir; Klamp, Tobias; Schüttpelz, Mark; Sauer, Markus



Single molecule studies on dynamics in liquid crystals.  


Single molecule (SM) methods are able to resolve structure related dynamics of guest molecules in liquid crystals (LC). Highly diluted small dye molecules on the one hand explore structure formation and LC dynamics, on the other hand they report about a distortion caused by the guest molecules. The anisotropic structure of LC materials is used to retrieve specific conformation related properties of larger guest molecules like conjugated polymers. This in particular sheds light on organization mechanisms within biological cells, where large molecules are found in nematic LC surroundings. This review gives a short overview related to the application of highly sensitive SM detection schemes in LC. PMID:24077123

Täuber, Daniela; von Borczyskowski, Christian



Optical detection and spectroscopy of single molecules in a solid  

NASA Astrophysics Data System (ADS)

Using two different double-modulation techniques, we have observed the optical-absorption spectrum of single dopant molecules of pentacene in a p-terphenyl host crystal at liquid-helium temperatures. To achieve this, frequency-modulation spectroscopy was combined either with Stark or ultrasonic modulation to remove interfering background signals from residual amplitude modulation, and the number of molecules in resonance was reduced to one by operating in the wings of the inhomogeneous line. Triplet bottleneck saturation appears to be suppressed in the single-molecule regime.

Moerner, W. E.; Kador, L.



Tip-induced spectral dynamics of single molecules  

NASA Astrophysics Data System (ADS)

Manipulation of spectral dynamics of single molecules (SM) by a metallized scanning probe tip is demonstrated. The Stark effect of the zero-phonon lines of single pentacene molecules in a p-terphenyl host at 1.8 K is investigated by applying a voltage to the tip in contact with the sample. The measured Stark shifts exhibit a plateau and the line widths depend on the electric field. These anomalies are explained by a model based on two-level systems with field-dependent double-well potentials. The experimental data show that the two-level systems are induced by the tip.

Segura, J.-M.; Zumofen, G.; Renn, A.; Hecht, B.; Wild, U. P.



Single-molecule super-resolution imaging in bacteria.  


Bacteria have evolved complex, multi-component cellular machineries to carry out fundamental cellular processes such as cell division/separation, locomotion, protein secretion, DNA transcription/replication, or conjugation/competence. Diffraction of light has so far restricted the use of conventional fluorescence microscopy to reveal the composition, internal architecture and dynamics of these important machineries. This review describes some of the more recent advances on single-molecule super-resolution microscopy methods applied to bacteria and highlights their application to chemotaxis, cell division, DNA segregation, and DNA transcription machineries. Finally, we discuss some of the lessons learned from this approach, and future perspectives. PMID:23142583

Cattoni, D I; Fiche, J B; Nöllmann, M



The statistics of single molecule detection: An overview  

SciTech Connect

An overview of our recent results in modeling single molecule detection in fluid flow is presented. Our mathematical approach is based on a path integral representation. The model accounts for all experimental details, such as light collection, laser excitation, hydrodynamics and diffusion, and molecular photophysics. Special attention is paid to multiple molecule crossings through the detection volume. Numerical realization of the theory is discussed. Measurements of burst size distributions in single B-phycoerythrin molecule detection experiments are presented and compared with theoretical predictions.

Enderlein, J.; Robbins, D.L.; Ambrose, W.P. [and others



Multiphoton cascade absorption in single molecule fluorescence saturation spectroscopy.  


Saturation spectroscopy is a relevant method to investigate photophysical parameters of single fluorescent molecules. Nevertheless, the impact of a gradual increase, over a broad range, of the laser excitation on the intramolecular dynamics is not completely understood, particularly concerning their fluorescence emission (the so-called brightness). Thus, we propose a comprehensive theoretical and experimental study to interpret the unexpected evolution of the brightness with the laser power taking into account the cascade absorption of two and three photons. Furthermore, we highlight the key role played by the confocal observation volume in fluorescence saturation spectroscopy of single molecules in solution. PMID:23521543

Winckler, Pascale; Jaffiol, Rodolphe



Graphene Nanopores for Single-Molecule DNA Sequencing  

NASA Astrophysics Data System (ADS)

We fabricate a nanopore in a suspended single-layer graphene membrane, which serves as a barrier between two aqueous DNA reservoirs. This nanopore device can detect the electrophoretic passage of single or double stranded DNA through transient ionic current blockades caused by DNA obstruction of the pore. Furthermore, a graphene pore, which has atomic thickness, should allow discrimination of different DNA base pairs by ionic current measurements alone. This base discrimination can become the basis of a single-molecule, ultrafast DNA sequencing scheme. We demonstrate the fabrication and evaluate the performance of these graphene nanopore devices.

Kuan, Aaron; Hoogerheide, David; Xie, Ping; Branton, Daniel; Golovchenko, Jene



Enzyme-immobilized SiO2-Si electrode: Fast interfacial electron transfer with preserved enzymatic activity  

NASA Astrophysics Data System (ADS)

The enzyme, glucose oxidase (GOx), is immobilized using electrostatic interaction on the native oxide of heavily doped n-type silicon. Voltammetric measurement shows that the immobilized GOx gives rise to a very fast enzyme-silicon interfacial electron transfer rate constant of 7.9 s-1. The measurement also suggests that the enzyme retains its native conformation when immobilized on the silicon surface. The preserved native conformation of GOx is further confirmed by testing the enzymatic activity of the immobilized GOx using glucose. The GOx-immobilized silicon is shown to behave as a glucose sensor that detects glucose with concentrations as low as 50 ?M.

Wang, Gang; Yau, Siu-Tung



Single-molecule studies of bacterial protein translocation.  


In prokaryotes, a large share of the proteins are secreted from the cell through a process that requires their translocation across the cytoplasmic membrane. This process is mediated by the universally conserved Sec system with homologues in the endoplasmic reticulum and thylakoid membranes of eukaryotes. The Sec system also facilitates the membrane insertion of integral membrane proteins, an essential step along their folding pathway. In bacteria, the Sec system consists of the protein-conducting channel (SecYEG) that associates with soluble components, such as the motor protein SecA or translating ribosomes, and with integral membrane proteins, such as the heterotrimeric complex termed SecDFyajC and the YidC insertase. Over the past three decades, biochemical and structural studies have provided a comprehensive view of protein translocation, but the exact mechanistic details of this process remain to be resolved. For a number of other biomolecular systems, single-molecule biophysical analysis has efficiently complemented the conventional biochemical studies conducted in bulk, with high-sensitivity measurements probing the structure and dynamics of individual molecules in vitro and in vivo. Here, we review recent advances in studies of protein translocation employing single-molecule techniques with the aim of resolving molecular mechanisms, thereby providing a new and detailed view of the process. PMID:24024480

Kedrov, Alexej; Kusters, Ilja; Driessen, Arnold J M



Ultrabright bowtie nanoaperture antenna probes studied by single molecule fluorescence.  


We report on a novel design for the fabrication of ultrabright bowtie nanoaperture antenna (BNA) probes to breach the intrinsic trade-off between power transmission and field confinement of circular nanoapertures as in near-field scanning optical microscopy (NSOM) or planar zero mode waveguides. The approach relies on the nanofabrication of BNAs at the apex of tapered optical fibers tuned to diameters close to their cutoff region, resulting in 10(3)× total improvement in throughput over conventional NSOM probes of similar confinement area. By using individual fluorescence molecules as optical nanosensors, we show for the first time nanoimaging of single molecules using BNA probes with an optical confinement of 80 nm, measured the 3D near-field emanating from these nanostructures and determined a ~6-fold enhancement on the single molecule signal. The broadband field enhancement, nanoscale confinement, and background free illumination provided by these nanostructures offer excellent perspectives as ultrabright optical nanosources for a full range of applications, including cellular nanoimaging, spectroscopy, and biosensing. PMID:23098104

Mivelle, Mathieu; van Zanten, Thomas S; Neumann, Lars; van Hulst, Niek F; Garcia-Parajo, Maria F



Single-Molecule Electrical Random Resequencing of DNA and RNA  

NASA Astrophysics Data System (ADS)

Two paradigm shifts in DNA sequencing technologies--from bulk to single molecules and from optical to electrical detection--are expected to realize label-free, low-cost DNA sequencing that does not require PCR amplification. It will lead to development of high-throughput third-generation sequencing technologies for personalized medicine. Although nanopore devices have been proposed as third-generation DNA-sequencing devices, a significant milestone in these technologies has been attained by demonstrating a novel technique for resequencing DNA using electrical signals. Here we report single-molecule electrical resequencing of DNA and RNA using a hybrid method of identifying single-base molecules via tunneling currents and random sequencing. Our method reads sequences of nine types of DNA oligomers. The complete sequence of 5'-UGAGGUA-3' from the let-7 microRNA family was also identified by creating a composite of overlapping fragment sequences, which was randomly determined using tunneling current conducted by single-base molecules as they passed between a pair of nanoelectrodes.

Ohshiro, Takahito; Matsubara, Kazuki; Tsutsui, Makusu; Furuhashi, Masayuki; Taniguchi, Masateru; Kawai, Tomoji



Quantification of the yeast transcriptome by single-molecule sequencing.  


We present single-molecule sequencing digital gene expression (smsDGE), a high-throughput, amplification-free method for accurate quantification of the full range of cellular polyadenylated RNA transcripts using a Helicos Genetic Analysis system. smsDGE involves a reverse-transcription and polyA-tailing sample preparation procedure followed by sequencing that generates a single read per transcript. We applied smsDGE to the transcriptome of Saccharomyces cerevisiae strain DBY746, using 6 of the available 50 channels in a single sequencing run, yielding on average 12 million aligned reads per channel. Using spiked-in RNA, accurate quantitative measurements were obtained over four orders of magnitude. High correlation was demonstrated across independent flow-cell channels, instrument runs and sample preparations. Transcript counting in smsDGE is highly efficient due to the representation of each transcript molecule by a single read. This efficiency, coupled with the high throughput enabled by the single-molecule sequencing platform, provides an alternative method for expression profiling. PMID:19581875

Lipson, Doron; Raz, Tal; Kieu, Alix; Jones, Daniel R; Giladi, Eldar; Thayer, Edward; Thompson, John F; Letovsky, Stan; Milos, Patrice; Causey, Marie



Modeling single molecule detection probabilities in microdroplets. Final report  

SciTech Connect

Optimization of molecular detection efficiencies is important for analytical applications of single molecule detection methods. In microdroplets some experimental limitations can be reduced, primarily because the molecule cannot diffuse away from the excitation and collection volume. Digital molecular detection using a stream of microdroplets has been proposed as a method of reducing concentration detection limits by several orders of magnitude relative to conventional measurements. However, the bending and reflection of light at the microdroplet`s liquid-air interface cause the illumination intensity and fluorescence intensity collected to be strongly dependent on the position of the molecule within the droplet. The goal is to model the detection of single molecules in microdroplets so that one can better understand and optimize detection efficiencies. In the first year of this modeling effort the authors studied the collection of fluorescence from unit-amplitude dipoles inside of spheres. In this second year they modified their analysis to accurately model the effects of excitation inhomogeneities, including effects of molecular saturation, motion of the droplet, and phase variations between the two counter-propagating waves that illuminate the droplet. They showed that counter-propagating plane wave illumination can decrease the variations in the intensity which excites the molecules. Also in this second year they simulated (using a Monte Carlo method) the detection of fluorescence from many droplets, each of which may contain zero, or one (or at higher concentrations, a few) fluorescent molecules.

Hill, S.C.



High sensitivity optical microscope for single molecule spectroscopy studies  

NASA Astrophysics Data System (ADS)

We describe the setup and apply two algorithms for fast imaging in a sample raster scanning two photon microscope. Imaging can be performed at a rate of 1-100 lines per second with a closed loop piezo actuator, and the detection is performed via avalanche photodiodes. This allows to investigate single molecule emission with 50 ms time resolution. In a slow scanning algorithm we have implemented fluorescence fluctuation analysis by computing the photon counting histogram (PCH) on each pixel of the image. In a fast-scan acquistion method the image acquistion rate is 5 lines per second on a large field of view and high resolution(50 nm scanning step, 100×100 ?m2 field of view) and ?100 lines per second on smaller field of views with optically limited resolution (200 nm scanning step, 20×20 ?m2 field of view). This figure, which is lower than the typical value for normal confocal scanning imaging (?500 lines per second), allows nevertheless to perform imaging studies of extended samples in reasonable times for intracellular kinetics and interactions. With this setup and by means of the PCH analysis we are able to discriminate between local concentration and molecular brightness on extended samples also at the level of the single molecule.

Malengo, Gabriele; Milani, Roberto; Cannone, Fabio; Krol, Silke; Diaspro, Alberto; Chirico, Giuseppe



Mechanobiology of Short DNA Molecules: A Single Molecule Perspective  

NASA Astrophysics Data System (ADS)

Mechanical properties of DNA are known to play a significant role in several biological processes like wrapping of DNA around histones and looping. Most of these cellular events occur on a DNA length scale of a few hundred basepairs. Single molecule methods have been highly successful in directly investigating heterogeneity in different biomolecular systems and serve as ideal tools to study the mechanical properties of DNA. However, their use in studying DNA of contour lengths less than a kilobase are fraught with experimental difficulties. The research presented in this thesis explores the behavior of short stretches of DNA (? 500bp) using existing and novel single molecule methods. We have quantified the variation in persistence lengths between sequences having different elasticity using a constant force axial optical tweezers. Our experiments have also revealed that this difference in persistence lengths manifests itself as a difference in looping lifetimes of lac repressor, in sequences having the aforementioned constructs as the intervening sequence between the operator sites. We have also developed a system to probe DNA dynamics in vivo. We have found that the active processes in the cell have distinct effects on dynamics of DNA and eliminating the active processes causes a 'phase transition' like behavior in the inside the cell. We are currently extending this technique to understand DNA dynamics inside bacterial systems. Our results provide vital insights into mechanical properties of DNA and the effect of athermal fluctuations on DNA dynamics.

Raghunathan, Krishnan


Single molecule sensitivity in near field tip enhanced Raman scattering  

NASA Astrophysics Data System (ADS)

The local-field enhancement at a sharp metallic tip in combination with resonance Raman spectroscopy provides an optical scanning probe method with ultrahigh spatial resolution. Here we report on achieving sensitivity down to the single molecule level. Illuminating the apex of a Au wire tip at variable tip-sample distances down to nm proximity results in a strong field confinement and near field coupling. This provides a highly localized light source and a controlled degree of field enhancement for Raman scattering. In the tip-scattered resonance Raman response of malachite green and rhodamine 6G molecules spectral line narrowing compared to the ensemble average and spectral diffusion are seen. Temporal fluctuations of spectral position and relative peak intensities as well as transient line splitting in time series of sequentially recorded spectra are observed. The results illustrate that single molecule Raman spectroscopy can be achieved in scattering-type near-field microscopy. This approach provides the degrees of freedom necessary for a systematic investigation and understanding of the underlying mechanisms of surface-enhanced Raman spectroscopy.

Neacsu, Catalin C.



Direct characterization of amyloidogenic oligomers by single-molecule fluorescence  

PubMed Central

A key issue in understanding the pathogenic conditions associated with the aberrant aggregation of misfolded proteins is the identification and characterization of species formed during the aggregation process. Probing the nature of such species has, however, proved to be extremely challenging to conventional techniques because of their transient and heterogeneous character. We describe here the application of a two-color single-molecule fluorescence technique to examine the assembly of oligomeric species formed during the aggregation of the SH3 domain of PI3 kinase. The single-molecule experiments show that the species formed at the stage of the reaction where aggregates have previously been found to be maximally cytotoxic are a heterogeneous ensemble of oligomers with a median size of 38 ± 10 molecules. This number is remarkably similar to estimates from bulk measurements of the critical size of species observed to seed ordered fibril formation and of the most infective form of prion particles. Moreover, although the size distribution of the SH3 oligomers remains virtually constant as the time of aggregation increases, their stability increases substantially. These findings together provide direct evidence for a general mechanism of amyloid aggregation in which the stable cross-? structure emerges via internal reorganization of disordered oligomers formed during the lag phase of the self-assembly reaction.

Orte, Angel; Birkett, Neil R.; Clarke, Richard W.; Devlin, Glyn L.; Dobson, Christopher M.; Klenerman, David



Single molecule study of a processivity clamp sliding on DNA  

SciTech Connect

Using solution based single molecule spectroscopy, we study the motion of the polIII {beta}-subunit DNA sliding clamp ('{beta}-clamp') on DNA. Present in all cellular (and some viral) forms of life, DNA sliding clamps attach to polymerases and allow rapid, processive replication of DNA. In the absence of other proteins, the DNA sliding clamps are thought to 'freely slide' along the DNA; however, the abundance of positively charged residues along the inner surface may create favorable electrostatic contact with the highly negatively charged DNA. We have performed single-molecule measurements on a fluorescently labeled {beta}-clamp loaded onto freely diffusing plasmids annealed with fluorescently labeled primers of up to 90 bases. We find that the diffusion constant for 1D diffusion of the {beta}-clamp on DNA satisfies D {le} 10{sup -14} cm{sup 2}/s, much slower than the frictionless limit of D = 10{sup -10} cm{sup 2}/s. We find that the {beta} clamp remains at the 3-foot end in the presence of E. coli single-stranded binding protein (SSB), which would allow for a sliding clamp to wait for binding of the DNA polymerase. Replacement of SSB with Human RP-A eliminates this interaction; free movement of sliding clamp and poor binding of clamp loader to the junction allows sliding clamp to accumulate on DNA. This result implies that the clamp not only acts as a tether, but also a placeholder.

Laurence, T A; Kwon, Y; Johnson, A; Hollars, C; O?Donnell, M; Camarero, J A; Barsky, D



Biophysical characterization of DNA binding from single molecule force measurements  

PubMed Central

Single molecule force spectroscopy is a powerful method that uses the mechanical properties of DNA to explore DNA interactions. Here we describe how DNA stretching experiments quantitatively characterize the DNA binding of small molecules and proteins. Small molecules exhibit diverse DNA binding modes, including binding into the major and minor grooves and intercalation between base pairs of double-stranded DNA (dsDNA). Histones bind and package dsDNA, while other nuclear proteins such as high mobility group proteins bind to the backbone and bend dsDNA. Single-stranded DNA (ssDNA) binding proteins slide along dsDNA to locate and stabilize ssDNA during replication. Other proteins exhibit binding to both dsDNA and ssDNA. Nucleic acid chaperone proteins can switch rapidly between dsDNA and ssDNA binding modes, while DNA polymerases bind both forms of DNA with high affinity at distinct binding sites at the replication fork. Single molecule force measurements quantitatively characterize these DNA binding mechanisms, elucidating small molecule interactions and protein function.

Chaurasiya, Kathy R.; Paramanathan, Thayaparan; McCauley, Micah J.; Williams, Mark C.



Single-molecule electrical random resequencing of DNA and RNA.  


Two paradigm shifts in DNA sequencing technologies-from bulk to single molecules and from optical to electrical detection-are expected to realize label-free, low-cost DNA sequencing that does not require PCR amplification. It will lead to development of high-throughput third-generation sequencing technologies for personalized medicine. Although nanopore devices have been proposed as third-generation DNA-sequencing devices, a significant milestone in these technologies has been attained by demonstrating a novel technique for resequencing DNA using electrical signals. Here we report single-molecule electrical resequencing of DNA and RNA using a hybrid method of identifying single-base molecules via tunneling currents and random sequencing. Our method reads sequences of nine types of DNA oligomers. The complete sequence of 5'-UGAGGUA-3' from the let-7 microRNA family was also identified by creating a composite of overlapping fragment sequences, which was randomly determined using tunneling current conducted by single-base molecules as they passed between a pair of nanoelectrodes. PMID:22787559

Ohshiro, Takahito; Matsubara, Kazuki; Tsutsui, Makusu; Furuhashi, Masayuki; Taniguchi, Masateru; Kawai, Tomoji



Localizing single molecules in three dimensions - a brief review.  


Single molecule tracking in three dimensions (3D) in a live cell environment holds the promise of revealing important new biological insights. However, conventional microscopy based imaging techniques are not well suited for fast 3D tracking of single molecules in cells. Previously we developed an imaging modality multifocal plane microscopy (MUM) to image fast intracellular dynamics in 3D in live cells. Recently, we have reported an algorithm, the MUM localization algorithm (MUMLA), for the 3D localization of point sources that are imaged using MUM. Here, we present a review of our results on MUM and MUMLA. We have validated MUMLA through simulated and experimental data and have shown that the 3D-position of quantum dots (QDs) can be determined with high spatial accuracy over a wide spatial range. We have calculated the Cramer-Rao lower bound for the problem of determining the 3D location of point sources from MUM and from conventional microscopes. Our analyses shows that MUM overcomes the poor depth discrimination of the conventional microscope, and thereby paves the way for high accuracy tracking of nanoparticles in a live cell environment. We have also shown that the performance of MUMLA comes consistently close to the Cramer-Rao lower bound. PMID:21887405

Ram, Sripad; Prabhat, Prashant; Chao, Jerry; Abraham, Anish V; Ward, E Sally; Ober, Raimund J



Localizing single molecules in three dimensions - a brief review  

PubMed Central

Single molecule tracking in three dimensions (3D) in a live cell environment holds the promise of revealing important new biological insights. However, conventional microscopy based imaging techniques are not well suited for fast 3D tracking of single molecules in cells. Previously we developed an imaging modality multifocal plane microscopy (MUM) to image fast intracellular dynamics in 3D in live cells. Recently, we have reported an algorithm, the MUM localization algorithm (MUMLA), for the 3D localization of point sources that are imaged using MUM. Here, we present a review of our results on MUM and MUMLA. We have validated MUMLA through simulated and experimental data and have shown that the 3D-position of quantum dots (QDs) can be determined with high spatial accuracy over a wide spatial range. We have calculated the Cramer-Rao lower bound for the problem of determining the 3D location of point sources from MUM and from conventional microscopes. Our analyses shows that MUM overcomes the poor depth discrimination of the conventional microscope, and thereby paves the way for high accuracy tracking of nanoparticles in a live cell environment. We have also shown that the performance of MUMLA comes consistently close to the Cramer-Rao lower bound.

Ram, Sripad; Prabhat, Prashant; Chao, Jerry; Abraham, Anish V.; Ward, E. Sally



Interfacial charge transfer mechanism in nanostructured TiO2-ZnS coupled network for single electron device applications  

NASA Astrophysics Data System (ADS)

We report on the production of nanoporous TiO2 network sensitized by ZnS nanospheres as an idealized scheme to facilitate interfacial charge transfer effects. The nanoporous TiO2 system was fabricated on the 0.1 ?m thick Al substrate from titanium isopropoxide [Ti(i-OC3H7)] and 1-butanol (C4H9OH) as requisite precursor. The Zn++ ions are internally adsorbed to provide heterogeneous coupled TiO2-ZnS nanosystem. The I-V response shows transistor characteristics which suggests sharp rise in current with forward biasing voltage before attaining saturation. It is expected that with the increase in signal frequency more number of trap carriers being able to follow signal assist higher carrier transfer rate across the interface in the coupled system and hence saturation current (IS) increases. However, in all the cases saturation occurs around finite biasing voltage, i.e., 3.6 V. This ensures that the surface states (which normally lie within the forbidden gap and below the conduction bands for electrons) mainly participate in carrier transfer mechanism within the device. A phenomenon in understanding highly controlled interfacial carrier transport process would find potential in nanoelectronics, e.g., single electron transistor and other single electron devices.

Mohanta, D.; Deka, M.; Choudhury, A.



Interpreting the catalytic voltammetry of an adsorbed enzyme by considering substrate mass transfer, enzyme turnover, and interfacial electron transport.  


Redox active enzymes can be adsorbed onto electrode surfaces to catalyze the interconversion of oxidized and reduced substrates in solution, driven by the supply or removal of electrons by the electrode. The catalytic current is directly proportional to the rate of enzyme turnover, and its dependence on the electrode potential can be exploited to define both the kinetics and thermodynamics of the enzyme's catalytic cycle. However, observed electrocatalytic voltammograms are often complex because the identity of the rate limiting step changes with the electrode potential and under different experimental conditions. Consequently, extracting mechanistic information requires that accurate models be constructed to deconvolute and analyze the observed behavior. Here, a basic model for catalysis by an adsorbed enzyme is described. It incorporates substrate mass transport, enzyme kinetics, and interfacial electron transport, and it accurately reproduces experimentally recorded voltammograms from the oxidation of NADH by subcomplex Ilambda (the hydrophilic subcomplex of NADH:ubiquinone oxidoreductase), under a range of conditions. Mass transport is imposed by a rotating disk electrode and described by the Levich equation. Interfacial electron transport is controlled by the electrode potential and characterized by a dispersion of rate constants, according to the model of Léger and co-workers. Here, the Michaelis-Menten equation is used for the enzyme kinetics, but our methodology can also be readily applied to derive and apply analogous equations relating to alternative enzyme mechanisms. Therefore, our results are highly relevant to the interpretation of electrocatalytic voltammograms for adsorbed enzymes in general. PMID:16471690

Reda, Torsten; Hirst, Judy



Dependence of the direct electron transfer activity and adsorption kinetics of cytochrome c on interfacial charge properties.  


With the advantages of in situ analysis and high surface sensitivity, surface-enhanced infrared absorption spectroscopy in attenuated total reflection mode (ATR-SEIRAS) combined with electrochemical methods has been employed to examine the interfacial direct electron transfer activity and adsorption kinetics of cytochrome c (cyt c). This work presents data on cyt c adsorption onto negatively charged mercaptohexanoic acid (MHA) and positively charged 6-amino-1-hexanethiol (MHN) self-assembled monolayers (SAMs) on gold nanofilm surfaces. The adsorbed cyt c displays a higher apparent electron transfer rate constant (33.5 ± 2.4 s(-1)) and apparent binding rate constant (73.1 ± 5.2 M(-1) s(-1)) at the MHA SAMs surface than those on the MHN SAMs surface. The results demonstrate that the surface charge density determines the protein adsorption kinetics, while the surface charge character determines the conformation and orientation of proteins assembled which in turn affects the direct electron transfer activity. PMID:23912152

Wang, Gui-Xia; Wang, Min; Wu, Zeng-Qiang; Bao, Wen-Jing; Zhou, Yue; Xia, Xing-Hua



Mapping projected potential, interfacial roughness, and composition in general crystalline solids by quantitative transmission electron microscopy  

Microsoft Academic Search

We describe how general lattice images may be used to measure the variation of the potential in crystalline solids in any projection, with no knowledge of the imaging conditions. This approach is applicable to structurally perfect samples, in which interfacial topography or changes in composition are of interest. We present the first atomic-level topographic map of a Si\\/SiO2 interface in

P. Schwander; C. Kisielowski; M. Seibt; F. H. Baumann; Y. Kim; A. Ourmazd



Single molecule quantum-confined Stark effect measurements of semiconductor nanoparticles at room temperature  

NASA Astrophysics Data System (ADS)

We investigate the quantum confined Stark effect (QCSE) of various nanoparticles (NPs) on the single molecule level at room temperature. We tested 8 different NPs with different geometry, material composition and electronic structure, and measured their QCSE by single molecule spectroscopy. This study reveals that suppressing the Coulomb interaction force between electron and hole by asymmetric type-II interface is critical for an enhanced QCSE. For example, ZnSe-CdS and CdSe(Te)-CdS-CdZnSe asymmetric nanorods (type-II) display respectively twice and more than three times larger QCSE than that of simple type-I nanorods (CdSe). In addition, wavelength blue-shift of QCSE and roughly linear ??-F (emission wavelength shift vs. the applied electric field) relation are observed for the type-II nanorods. Experimental results (??-F or ?E-F) are successfully reproduced by self-consistent quantum mechanical calculation. Intensity reduction in blue-shifted spectrum is also accounted for. Both calculations and experiments suggest that the magnitude of the QCSE is predominantly determined by the degree of initial charge separation in these structures.

Park, Kyoung Won; Deutsch, Zvicka; Li, J. Jack; Oron, Dan; Weiss, Shimon



Spin transport and tunable Gilbert damping in a single-molecule magnet junction  

NASA Astrophysics Data System (ADS)

We study time-dependent electronic and spin transport through an electronic level connected to two leads and coupled with a single-molecule magnet via exchange interaction. The molecular spin is treated as a classical variable and precesses around an external magnetic field. We derive expressions for charge and spin currents by means of the Keldysh nonequilibrium Green's functions technique in linear order with respect to the time-dependent magnetic field created by this precession. The coupling between the electronic spins and the magnetization dynamics of the molecule creates inelastic tunneling processes which contribute to the spin currents. The inelastic spin currents, in turn, generate a spin-transfer torque acting on the molecular spin. This back-action includes a contribution to the Gilbert damping and a modification of the precession frequency. The Gilbert damping coefficient can be controlled by the bias and gate voltages or via the external magnetic field and has a nonmonotonic dependence on the tunneling rates.

Filipovi?, Milena; Holmqvist, Cecilia; Haupt, Federica; Belzig, Wolfgang



Simultaneous, Coincident Optical Trapping and Single-Molecule Fluorescence  

PubMed Central

We constructed a microscope-based instrument capable of simultaneous, spatially coincident optical trapping and single-molecule fluorescence. The capabilities of this apparatus were demonstrated by studying the force-induced strand separation of a dye-labeled, 15-basepair region of double stranded DNA, with force applied either parallel (“unzipping” mode) or perpendicular (“shearing” mode) to the long axis of the region. Mechanical transitions corresponding to DNA hybrid rupture occurred simultaneously with discontinuous changes in the fluorescence emission. The rupture force was strongly dependent on the direction of applied force, indicating the existence of distinct unbinding pathways for the two force-loading modes. From the rupture force histograms, we determined the distance to the thermodynamic transition state and the thermal off rates in the absence of load for both processes.

Lang, Matthew J.; Fordyce, Polly M.; Engh, Anita M.; Neuman, Keir C.; Block, Steven M.



Single-Molecule Detection in Micron-Sized Capillaries  

NASA Astrophysics Data System (ADS)

The detection of individual molecules in solution by laser-induced fluorescence is becoming an increasingly important tool for biophysics research and biotechnology applications. In a typical single-molecule detection (SMD) experiment, diffusion is the dominant mode of transport of fluorophores through the focused laser beam. In order to more rapidly process a large number of slowly diffusing bio-molecules for applications in pharmaceutical drug discovery, a flow can be introduced within a capillary. If the flow speed is sufficient, bio-molecules will be carried through the probe volume significantly faster than by diffusion alone. Here we discuss SMD near the tip of, and in, such micron-sized capillaries, with a high numerical-aperture microscope objective used for confocal-epi-illumination along the axis of the capillary. Problems such as molecular adsorption to the glass are also addressed.

Ball, David A.; Shen, Guoqing; Davis, Lloyd M.



Toward single-molecule microscopy on a smart phone.  


Thanks to fluorescence, single nano-objects down to individual fluorophores can now be imaged in optical microscopes. Fluorescence imaging is still restricted to laboratory facilities as it usually involves expensive and bulky instrumentation. A report by Wei et al. in this issue of ACS Nano, however, shows that a sensitive, cost-effective, and portable device can be developed to image individual nano-objects as small as large viruses. This work opens the fascinating prospects of single-molecule microscopy and spectroscopy on a smart phone. We speculate on the possible applications of such a portable imaging device and on the perspectives it may open in different fields of science and technology. PMID:24112048

Khatua, Saumyakanti; Orrit, Michel



Ultrasensitive nucleic acid sequence detection by single-molecule electrophoresis  

SciTech Connect

This is the final report of a one-year laboratory-directed research and development project at Los Alamos National Laboratory. There has been considerable interest in the development of very sensitive clinical diagnostic techniques over the last few years. Many pathogenic agents are often present in extremely small concentrations in clinical samples, especially at the initial stages of infection, making their detection very difficult. This project sought to develop a new technique for the detection and accurate quantification of specific bacterial and viral nucleic acid sequences in clinical samples. The scheme involved the use of novel hybridization probes for the detection of nucleic acids combined with our recently developed technique of single-molecule electrophoresis. This project is directly relevant to the DOE`s Defense Programs strategic directions in the area of biological warfare counter-proliferation.

Castro, A; Shera, E.B.



Robust spin crossover and memristance across a single molecule.  


A nanoscale molecular switch can be used to store information in a single molecule. Although the switching process can be detected electrically in the form of a change in the molecule's conductance, adding spin functionality to molecular switches is a key concept for realizing molecular spintronic devices. Here we show that iron-based spin-crossover molecules can be individually and reproducibly switched between a combined high-spin, high-conduction state and a low-spin, low-conduction state, provided the individual molecule is decoupled from a metallic substrate by a thin insulating layer. These results represent a step to achieving combined spin and conduction switching functionality on the level of individual molecules. PMID:22760637

Miyamachi, Toshio; Gruber, Manuel; Davesne, Vincent; Bowen, Martin; Boukari, Samy; Joly, Loïc; Scheurer, Fabrice; Rogez, Guillaume; Yamada, Toyo Kazu; Ohresser, Philippe; Beaurepaire, Eric; Wulfhekel, Wulf



Force spectroscopy of barnase-barstar single molecule interaction.  


Results of the single molecule force spectroscopy study of specific interactions between ribonuclease barnase and its inhibitor barstar are presented. Experimental data obtained for the force loading rate ranging 2-70 nN/s are well approximated by a single straight line, from which the dissociation barrier of the width of 0.12 nm and height of 0.75-0.85 × 10(-19)J can be inferred. The measured value of specific interaction does not depend on the NaCl concentration. This apparently contradicts the well-known dependence of the binding energy of this pair on the salt concentration, but such a "contradiction" is explained by the insensitivity of the force spectroscopy data to the relatively long-range electrostatic interaction. The latter essentially contributes to the value of barnase-barstar binding energy revealed by biochemical measurements, and it is exactly this electrostatic interaction which is influenced by the salt concentration. PMID:21038358

Sekatskii, S K; Favre, M; Dietler, G; Mikhailov, A G; Klinov, D V; Lukash, S V; Deyev, S M


Eukaryotic transcriptional dynamics: from single molecules to cell populations  

PubMed Central

Transcriptional regulation is achieved through combinatorial interactions between regulatory elements in the human genome and a vast range of factors that modulate the recruitment and activity of RNA polymerase. Experimental approaches for studying transcription in vivo now extend from single-molecule techniques to genome-wide measurements. Parallel to these developments is the need for testable quantitative and predictive models for understanding gene regulation. These conceptual models must also provide insight into the dynamics of transcription and the variability that is observed at the single-cell level. In this Review, we discuss recent results on transcriptional regulation and also the models those results engender. We show how a non-equilibrium description informs our view of transcription by explicitly considering time-and energy-dependence at the molecular level.

Coulon, Antoine; Chow, Carson C.; Singer, Robert H.; Larson, Daniel R.



Super-resolution fluorescence imaging with single molecules.  


The ability to detect, image and localize single molecules optically with high spatial precision by their fluorescence enables an emergent class of super-resolution microscopy methods which have overcome the longstanding diffraction barrier for far-field light-focusing optics. Achieving spatial resolutions of 20-40nm or better in both fixed and living cells, these methods are currently being established as powerful tools for minimally-invasive spatiotemporal analysis of structural details in cellular processes which benefit from enhanced resolution. Briefly covering the basic principles, this short review then summarizes key recent developments and application examples of two-dimensional and three-dimensional (3D) multi-color techniques and faster time-lapse schemes. The prospects for quantitative imaging - in terms of improved ability to correct for dipole-emission-induced systematic localization errors and to provide accurate counts of molecular copy numbers within nanoscale cellular domains - are discussed. PMID:23932284

Sahl, Steffen J; Moerner, We



Single Molecule Detection Using Surface-Enhanced Raman Scattering (SERS)  

SciTech Connect

By exploiting the extremely large effective cross sections (10{sup -17}{endash}10{sup -16}cm{sup 2}/molecule) available from surface-enhanced Raman scattering (SERS), we achieved the first observation of single molecule Raman scattering. Measured spectra of a single crystal violet molecule in aqueous colloidal silver solution using one second collection time and about 2{times}10{sup 5}W/cm{sup 2} nonresonant near-infrared excitation show a clear {open_quotes}fingerprint{close_quotes} of its Raman features between 700 and 1700cm{sup -1}. Spectra observed in a time sequence for an average of 0.6 dye molecule in the probed volume exhibited the expected Poisson distribution for actually measuring 0, 1, 2, or 3 molecules. {copyright} {ital 1997} {ital The American Physical Society}

Kneipp, K.; Wang, Y.; Kneipp, H.; Perelman, L.T.; Itzkan, I.; Dasari, R.R.; Feld, M.S. [George R. Harrison Spectroscopy Laboratory, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 (United States)]|[Department of Physics, Technical University of Berlin, D 10623 Berlin (Germany)



Single Molecule Probing of Exocytotic Protein Interactions Using Force Spectroscopy  

PubMed Central

Relatively recently, the Atomic Force Microscope (AFM) emerged as a powerful tool for single molecule nanomechanical investigations. Parameters that can be measured by force spectroscopy using AFM, such as the force and total mechanical extension required to break bonds between various proteins can yield valuable insights into the nature of the bond (zippering vs. highly localized binding site), the sequence of its interactions and the energy landscape along the length of the interaction. In this review we discuss the use of AFM in force spectroscopy mode to study intermolecular interactions between the exocytotic proteins of the core SNARE complex. Information gathered by force spectroscopy of protein-protein interactions of this complex supplement previous results acquired with other techniques, and allows a deeper understanding of SNARE protein interactions and their role in exocytosis.

Montana, Vedrana; Liu, Wei; Mohideen, Umar; Parpura, Vladimir



Single-Molecule Encoders for Tracking Motor Proteins on DNA  

NASA Astrophysics Data System (ADS)

Devices such as inkjet printers and disk drives track position and velocity using optical encoders, which produce periodic signals precisely synchronized with linear or rotational motion. We have implemented this technique at the nanometer scale by labeling DNA with regularly spaced fluorescent dyes. The resulting molecular encoders can be used in several ways for high-resolution continuous tracking of individual motor proteins. These measurements do not require mechanical coupling to macroscopic instrumentation, are automatically calibrated by the underlying structure of DNA, and depend on signal periodicity rather than absolute level. I will describe the synthesis of single-molecule encoders, data from and modeling of experiments on a helicase and a DNA polymerase, and some ideas for future work.

Lipman, Everett A.



Quantitative single-molecule imaging by confocal laser scanning microscopy  

PubMed Central

A new approach to quantitative single-molecule imaging by confocal laser scanning microscopy (CLSM) is presented. It relies on fluorescence intensity distribution to analyze the molecular occurrence statistics captured by digital imaging and enables direct determination of the number of fluorescent molecules and their diffusion rates without resorting to temporal or spatial autocorrelation analyses. Digital images of fluorescent molecules were recorded by using fast scanning and avalanche photodiode detectors. In this way the signal-to-background ratio was significantly improved, enabling direct quantitative imaging by CLSM. The potential of the proposed approach is demonstrated by using standard solutions of fluorescent dyes, fluorescently labeled DNA molecules, quantum dots, and the Enhanced Green Fluorescent Protein in solution and in live cells. The method was verified by using fluorescence correlation spectroscopy. The relevance for biological applications, in particular, for live cell imaging, is discussed.

Vukojevic, Vladana; Heidkamp, Marcus; Ming, Yu; Johansson, Bjorn; Terenius, Lars; Rigler, Rudolf



Freezing single molecule dynamics on interfaces and in polymers.  


Heterogeneous line broadening and spectral diffusion of the fluorescence emission spectra of perylene diimide molecules have been investigated by means of time dependent single molecule spectroscopy. The influence of temperature and environment has been studied and reveals strong correlation to spectral diffusion processes. We followed the freezing of the molecular mobility of quasi free molecules on the surface upon temperature lowering and by embedding into a poly(methyl methacrylate) (PMMA) polymer. Thereby changes of optical transition energies as a result of both intramolecular changes of conformation and external induced dynamics by the surrounding polymer matrix could be observed. Simulations of spectral fluctuations within a two-level system (TLS) model showed good agreement with the experimental findings. PMID:21152494

Krause, Stefan; Aramendia, Pedro F; Täuber, Daniela; von Borczyskowski, Christian



Analysis of DNA interactions using single-molecule force spectroscopy.  


Protein-DNA interactions are involved in many biochemical pathways and determine the fate of the corresponding cell. Qualitative and quantitative investigations on these recognition and binding processes are of key importance for an improved understanding of biochemical processes and also for systems biology. This review article focusses on atomic force microscopy (AFM)-based single-molecule force spectroscopy and its application to the quantification of forces and binding mechanisms that lead to the formation of protein-DNA complexes. AFM and dynamic force spectroscopy are exciting tools that allow for quantitative analysis of biomolecular interactions. Besides an overview on the method and the most important immobilization approaches, the physical basics of the data evaluation is described. Recent applications of AFM-based force spectroscopy to investigate DNA intercalation, complexes involving DNA aptamers and peptide- and protein-DNA interactions are given. PMID:23468137

Ritzefeld, Markus; Walhorn, Volker; Anselmetti, Dario; Sewald, Norbert



Tristability in a light-actuated single-molecule magnet.  


Molecules exhibiting bistability have been proposed as elementary binary units (bits) for information storage, potentially enabling fast and efficient computing. In particular, transition metal complexes can display magnetic bistability via either spin-crossover or single-molecule magnet behavior. We now show that the octahedral iron(II) complexes in the molecular salt [Fe(1-propyltetrazole)6](BF4)2, when placed in its high-symmetry form, can combine both types of behavior. Light irradiation under an applied magnetic field enables fully reversible switching between an S = 0 state and an S = 2 state with either up (MS = +2) or down (MS = -2) polarities. The resulting tristability suggests the possibility of using molecules for ternary information storage in direct analogy to current binary systems that employ magnetic switching and the magneto-optical Kerr effect as write and read mechanisms. PMID:24066720

Feng, Xiaowen; Mathonière, Corine; Jeon, Ie-Rang; Rouzières, Mathieu; Ozarowski, Andrew; Aubrey, Michael L; Gonzalez, Miguel I; Clérac, Rodolphe; Long, Jeffrey R



Enhancing Single Molecule Imaging in Optofluidics and Microfluidics  

PubMed Central

Microfluidics and optofluidics have revolutionized high-throughput analysis and chemical synthesis over the past decade. Single molecule imaging has witnessed similar growth, due to its capacity to reveal heterogeneities at high spatial and temporal resolutions. However, both resolution types are dependent on the signal to noise ratio (SNR) of the image. In this paper, we review how the SNR can be enhanced in optofluidics and microfluidics. Starting with optofluidics, we outline integrated photonic structures that increase the signal emitted by single chromophores and minimize the excitation volume. Turning then to microfluidics, we review the compatible functionalization strategies that reduce noise stemming from non-specific interactions and architectures that minimize bleaching and blinking.

Vasdekis, Andreas E.; Laporte, Gregoire P.J.



Progress towards DNA sequencing at the single molecule level  

SciTech Connect

We describe progress towards sequencing DNA at the single molecule level. Our technique involves incorporation of fluorescently tagged nucleotides into a targeted sequence, anchoring the labeled DNA strand in a flowing stream, sequential exonuclease digestion of the DNA strand, and efficient detection and identification of single tagged nucleotides. Experiments demonstrating strand specific exonuclease digestion of fluorescently labeled DNA anchored in flow as well as the detection of single cleaved fluorescently tagged nucleotides from a small number of anchored DNA fragments axe described. We find that the turnover rate of Esherichia coli exonuclease III on fluorescently labeled DNA in flow at 36{degree}C is {approximately}7 nucleotides per DNA strand per second, which is approximately the same as that measured for this enzyme on native DNA under static, saturated (excess enzyme) conditions. Experiments demonstrating the efficient detection of single fluorescent molecules delivered electrokinetically to a {approximately}3 pL probe volume are also described.

Goodwin, P.M.; Affleck, R.L.; Ambrose, W.P. [and others



Continuous base identification for single-molecule nanopore DNA sequencing  

NASA Astrophysics Data System (ADS)

A single-molecule method for sequencing DNA that does not require fluorescent labelling could reduce costs and increase sequencing speeds. An exonuclease enzyme might be used to cleave individual nucleotide molecules from the DNA, and when coupled to an appropriate detection system, these nucleotides could be identified in the correct order. Here, we show that a protein nanopore with a covalently attached adapter molecule can continuously identify unlabelled nucleoside 5'-monophosphate molecules with accuracies averaging 99.8%. Methylated cytosine can also be distinguished from the four standard DNA bases: guanine, adenine, thymine and cytosine. The operating conditions are compatible with the exonuclease, and the kinetic data show that the nucleotides have a high probability of translocation through the nanopore and, therefore, of not being registered twice. This highly accurate tool is suitable for integration into a system for sequencing nucleic acids and for analysing epigenetic modifications.

Clarke, James; Wu, Hai-Chen; Jayasinghe, Lakmal; Patel, Alpesh; Reid, Stuart; Bayley, Hagan



Single Molecule Views of Protein Movement on Single Stranded DNA  

PubMed Central

The advent of new technologies allowing the study of single biological molecules continues to have a major impact on studies of interacting systems as well as enzyme reactions. These approaches (fluorescence, optical and magnetic “tweezers”), in combination with ensemble methods, have been particularly useful for mechanistic studies of protein-nucleic acid interactions and enzymes that function on nucleic acids. We review progress in the use of single molecule methods to observe and perturb the activities of proteins and enzymes that function on flexible single stranded DNA. These include single stranded (ss)DNA binding (SSB) proteins, recombinases (RecA/Rad51) and helicases/translocases that operate as motor proteins and play central roles in genome maintenance. We emphasize methods that have been used to detect and study the movement of these proteins (both ATP-dependent directional and random movement) along the ssDNA and the mechanistic and functional information that can result from detailed analysis of such movement.

Ha, Taekjip; Kozlov, Alexander G.; Lohman, Timothy M.



Spin-coated polyethylene films probed by single molecules.  


We have studied ultrathin spin-coated high-density polyethylene films by means of single-molecule spectroscopy and microscopy at 1.8 K. The films have been doped with 2.3,8.9-dibenzanthanthrene (DBATT) molecules, which function as local reporters of their immediate environment. The orientation distributions of single DBATT probe molecules in 100-200 nm thin films of high-density polyethylene differ markedly from those in low-density films. We have found a preferential orientation of dopant molecules along two well-defined, mutually perpendicular directions. These directions are preserved over at least a 2 mm distance. The strong orientation preference of the probe molecules requires the presence of abundant lateral crystal faces and is therefore not consistent with a spherulitic morphology. Instead, a "shish-kebab" crystal structure is invoked to explain our results. PMID:17064117

Wirtz, A C; Hofmann, C; Groenen, E J J



Enhancing single molecule imaging in optofluidics and microfluidics.  


Microfluidics and optofluidics have revolutionized high-throughput analysis and chemical synthesis over the past decade. Single molecule imaging has witnessed similar growth, due to its capacity to reveal heterogeneities at high spatial and temporal resolutions. However, both resolution types are dependent on the signal to noise ratio (SNR) of the image. In this paper, we review how the SNR can be enhanced in optofluidics and microfluidics. Starting with optofluidics, we outline integrated photonic structures that increase the signal emitted by single chromophores and minimize the excitation volume. Turning then to microfluidics, we review the compatible functionalization strategies that reduce noise stemming from non-specific interactions and architectures that minimize bleaching and blinking. PMID:21954349

Vasdekis, Andreas E; Laporte, Gregoire P J



Photon Statistics for Single-Molecule Nonlinear Spectroscopy  

NASA Astrophysics Data System (ADS)

We develop the theory of nonlinear spectroscopy for a single molecule undergoing stochastic dynamics and interacting with a sequence of two laser pulses. We find general expressions for the photon counting statistics and the exact solution to the problem for the Kubo-Anderson process. In the limit of impulsive pulses the information on the photon statistics is contained in the molecule’s dipole correlation function. The selective limit, the semiclassical approximation, and the fast modulation limit exhibit rich general behaviors of this new type of spectroscopy. We show how the design of external fields leads to insights on ultrafast dynamics of individual molecules that are different from those found for an ensemble.

Shikerman, F.; Barkai, E.



Coherent control of single molecules at room temperature.  


The detection of individual molecules allows to unwrap the inhomogeneously broadened ensemble and reveal the spatial disorder and temporal dynamics of single entities. During 20 years of increasing sophistication this approach has provided valuable insights into biomolecular interactions, cellular processes, polymer dynamics, etc. Unfortunately the detection of fluorescence, i.e. incoherent spontaneous emission, has essentially kept the time resolution of the single molecule approach out of the range of ultrafast coherent processes. In parallel coherent control of quantum interferences has developed as a powerful method to study and actively steer ultrafast molecular interactions and energy conversion processes. However the degree of coherent control that can be reached in ensembles is restricted, due to the intrinsic inhomogeneity of the synchronized subset. Clearly the only way to overcome spatio-temporal disorder and achieve key control is by addressing individual units: coherent control of single molecules. Here we report the observation and manipulation of vibrational wave-packet interference in individual molecules at ambient conditions. We show that adapting the time and phase distribution of the optical excitation field to the dynamics of each molecule results in a superior degree of control compared to the ensemble approach. Phase reversal does invert the molecular response, confirming the control of quantum coherence. Time-phase maps show a rich diversity in excited state dynamics between different, yet chemically identical, molecules. The presented approach is promising for single-unit coherent control in multichromophoric systems. Especially the role of coherence in the energy transfer of single antenna complexes under physiological conditions is subject of great attention. Now the role of energy disorder and variation in coupling strength can be explored, beyond the inhomogeneously broadened ensemble. PMID:22452073

Brinks, Daan; Hildner, Richard; Stefani, Fernando D; van Hulst, Niek F



Heteroepitaxial metallo-phthalocyanine (MPc, M = cobalt, nickel, copper) thin films on gold: Atomic and interfacial electronic structures  

NASA Astrophysics Data System (ADS)

Organic semiconductors have become a hot topic for research within the past few years. This work describes research into a family of organic semiconductors known as metallo-phthalocyanine (MPc) in which the electronic and optical properties can be easily tuned by the systematic modification of the metal cations and ligands. More specifically, thin films of CoPc, NiPc, and CuPc have been evaporated onto a "5 x 20" reconstructed Au(001) substrate and have been investigated by employing low energy electron diffraction and ultraviolet photoelectron spectroscopy. Low energy electron diffraction reveals that thin films of CuPc and NiPc are highly ordered with a square unit cell aligned along the substrate <110> and <11¯0> axes. In addition, deposition of CuPc onto the Au(001) substrate when at room temperature and elevated temperatures reveal that the square unit cell is larger when the substrate is heated. On the other hand, CoPc thin films are not well ordered as evidenced by multiple rotationally equivalent square domains, which are separated by 16°. Even more interesting is that the contrast between NiPc and CoPc on Au(001) is further found in the interfacial electronic structure. Ultraviolet photoelectron spectroscopy studies of the interfacial layers of NiPc deposited on the reconstructed gold substrate indicate that NiPc physisorbs on the gold surface as verified by a uniform molecular orbital (MO) shift. However, similar studies of the interfacial layers of CoPc depict an interaction between the CoPc 13a1g MO and the Au surface suggesting a charge transfer between the two. In addition to the research into MPc thin films, this work also describes the development of a Low Energy Electron Diffraction Intensity versus Voltage (LEED-IV) system for the Laboratory for Surface Analysis and Modification. This development involved the merging of various hardware and software systems by means of LabVIEW environment. Characterization of the system performance was carried out through the use of a MoS2 sample with known LEED-IV characteristics. The results of the system characterization revealed that the fully operational and ready to be applied to other samples.

Ellis, Trinity S.



Connecting Rare DNA Conformations and Surface Dynamics using Single-Molecule Resonance Energy Transfer  

PubMed Central

A mechanistic understanding of single-stranded DNA (ssDNA) behavior in the near-surface environment is critical to advancing DNA-directed self-assembled nanomaterials. A new approach is described that uses total internal reflection fluorescence microscopy to measure resonance energy transfer at the single-molecule level, providing a mechanistic understanding of the connection between molecular conformation and interfacial dynamics near amine-modified surfaces. Large numbers (>105) of ssDNA trajectories were observed, permitting dynamic correlation of molecular conformation with desorption and surface mobility. On the basis of dynamic behavior, molecules could be designated as members of the more common coiled population or a rare, weakly bound conformation. Molecules in the coiled state generally exhibited slow diffusion and conformational fluctuations that decreased with increasing average end-to-end distance. Lattice simulations of adsorbed self-avoiding polymers successfully predicted these trends. In contrast, the weakly bound conformation, observed in about 5% of molecules, had a large end-to-end distance but demonstrated conformational fluctuations that were much higher than predicted by simulations for adsorbed flexible chains. This conformation correlated positively with desorption events and led to fast diffusion, indicating weak surface associations. Understanding the role of the weakly bound conformation in DNA hybridization, and how solution conditions and surface properties may favor it, could lead to improved self-assembled nanomaterials.

Kastantin, Mark; Schwartz, Daniel K.



Direct observation of molecular orbitals in an individual single-molecule magnet Mn12 on Bi(111).  


Single-molecule nanomagnets have unique quantum properties, and their potential applications require characterization and accessibility of individual single-molecule magnets on various substrates. We develop a gentle tip-deposition method to bring individual prototype single-molecule magnets, manganese-12-acetate (Mn12) molecules, onto the semimetallic Bi(111) surface without linker molecules, using low-temperature scanning tunneling microscopy. We are able to identify both the almost flat-lying and side-lying orientations of Mn12 molecules at 4.5 K. Energy-resolved spectroscopic mapping enables the first observation of several molecular orbitals of individual Mn12 molecules in real space, which is consistent with density functional theory calculations. Both experimental and theoretical results suggest that an energy gap between the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) of the almost flat-lying Mn12 is only 40% of such a gap for an isolated (free) Mn12 molecule, which is caused by charge transfer from the metallic surface states of Bi to the Mn12. Despite the reduction of this gap, STM images show that the local lattices of Bi(111) covered with Mn12 remain essentially intact, indicating that Mn12-Bi interactions are not strong. Our findings open an avenue to address directly the local structural and electronic properties of individual single-molecule magnets on solid substrates. PMID:23829481

Sun, Kai; Park, Kyungwha; Xie, Jiale; Luo, Jiyong; Yuan, Hongkuan; Xiong, Zuhong; Wang, Junzhong; Xue, Qikun



Modification and characterization of fluorescent conjugated polymer nanoparticles for single molecule detection  

NASA Astrophysics Data System (ADS)

Single molecule tracking using fluorescent dye or nanoparticle labels has emerged as a useful technique for probing biomolecular processes. Considerable interest arises in the development of nanoparticle labels with brighter fluorescence in order to improve the spatial and temporal resolution of single molecule detection and to facilitate the application of single molecule detection methods to a wider range of intracellular processes. The McNeill laboratory recently reported that conjugated polymer nanoparticles exhibit fluorescence cross-sections roughly 10--100 times higher than other luminescent nanoparticles of similar size, excellent photostability (2.2x108 photons emitted per nanoparticle prior to photobleaching), and saturated emission rates roughly 100 times higher than that of the molecular dyes and more than 1000 times higher than that of colloidal semiconductor quantum dots. One purpose of this graduate research is the development of highly fluorescent, bioconjugated nanoparticle labels based on conjugated polymers for demanding fluorescence applications such as single molecule tracking in live cells. Three surface modification methods (conjugated polymer nanoparticles encapsulated with lipid silica agents, conjugated polymer nanoparticles encapsulated with tetraethyl orthosilicate(TEOS) and hybrid nanoparticles with thiol pendant groups by the Stober Method (3-mercaptopropyl trimethoxysilane (MPS))) have been developed to protect the conjugated polymer, passivate the nanoparticle surface, and provide a chemical handle for bioconjugation such as nanoparticle encapsulation with alkoxysilanes and Stober method. After encapsulation, the fluorescence quantum yield of silica-encapsulated nanoparticles is improved by 20% as compared to bare conjugated polymer nanoparticles, while the photostability is improved by a factor of 2, indicating that some protection of the polymer is provided by the encapsulating layer. Another purpose of my research is the manipulation of the photophysics and photochemistry of conjugated polymer nanoparticles based on developing a more complete understanding of the various processes that control or limit nanoparticle brightness and photostability. The results indicate that a combination of photophysical processes including electron transfer to molecular oxygen, energy transfer, and exciton diffusion result in saturation and photobleaching phenomena that currently limit brightness. This study provides the potential methods and strategies aimed at manipulating such processes or limiting their effect on fluorescence brightness. Finally, efficient intra-particle energy transfer has been demonstrated in dye-doped CP nanoparticles, which provides a new strategy for improving nanoparticle fluorescence brightness and photostability, obtaining nanoparticles with red emission to avoid autofluorescence in mammalian tissue, and for designing novel sensitive biosensors based on energy transfer to sensor dyes.

Zheng, Yueli


Single Molecule Manipulation and Spectroscopy of Chlorophyll-a from Spinach  

NASA Astrophysics Data System (ADS)

Chlorophyll-a, a molecule produced from `Spinach', adsorbed on a Au(111) surface has been investigated by using an ultra-high-vacuum low-temperature scanning-tunneling-microscope (UHV-LT-STM) at liquid helium temperatures. Studies are carried out both on isolated single molecules and on self-assembled molecular layers. The tunneling I-V and dI-dV spectroscopy of chlorophyll-a elucidate electronic properties of single molecule, such as the HOMO-LOMO gap and molecular orbital states. Mechanical stability of the chlorophyll-a is examined by using STM lateral manipulation (1,2). Here, the STM tip is placed just a few angstrom separation from the molecule to increase the tip-molecule interaction. Then the tip is laterally scanned across the surface resulting in pulling of the molecule. The detailed molecule movement is directly monitored through the corresponding STM-tip height signals. Our results reveal that the spinach molecule is a promising candidate for environmental friendly nano-device applications. (1). S.-W. Hla, K.-H. Rieder, Ann. Rev. Phys. Chem. 54 (2003) 307-330. (2). S.-W. Hla, et al. Phys. Rev. Lett. 93 (2004), 208302. This work is financially supported by the US-DOE grant DE-FG02-02ER46012.

Benson, Jessica-Jones



Multiple conformations of PEVK proteins detected by single-molecule techniques  

PubMed Central

An important component of muscle elasticity is the PEVK region of titin, so named because of the preponderance of these amino acids. However, the PEVK region, similar to other elastomeric proteins, is thought to form a random coil and therefore its structure cannot be determined by standard techniques. Here we combine single-molecule electron microscopy and atomic force microscopy to examine the conformations of the human cardiac titin PEVK region. In contrast to a simple random coil, we have found that cardiac PEVK shows a wide range of elastic conformations with end-to-end distances ranging from 9 to 24 nm and persistence lengths from 0.4 to 2.5 nm. Individual PEVK molecules retained their distinctive elastic conformations through many stretch-relaxation cycles, consistent with the view that these PEVK conformers cannot be interconverted by force. The multiple elastic conformations of cardiac PEVK may result from varying degrees of proline isomerization. The single-molecule techniques demonstrated here may help elucidate the conformation of other proteins that lack a well-defined structure.

Li, Hongbin; Oberhauser, Andres F.; Redick, Sambra D.; Carrion-Vazquez, Mariano; Erickson, Harold P.; Fernandez, Julio M.



Anomalous gate dependence of the Kondo effect in single-molecule transistors  

NASA Astrophysics Data System (ADS)

In semiconductor quantum dots, the Kondo temperature has been observed to depend exponentially on the gate voltage. This dependence arises because in these structures the gate capacitively shifts the energy of the singly occupied Kondo-active level relative to the chemical potential of the conduction electrons in the source and drain. In single-molecule transistors incorporating transition metal complexes, we find that the expected gate dependence in the Kondo regime is not observed. While the data show that the gate does shift electronic levels, the Kondo temperature found from both the differential conductance temperature and bias voltage dependence is approximately independent of gate voltage. We discuss possible explanations for this surprising observation, including the possible effect of molecular vibrational modes.

Natelson, D.; Yu, L. H.; Keane, Z. K.; Ciszek, J. W.; Tour, J. M.



Solution, surface, and single molecule platforms for the study of DNA-mediated charge transport.  


The structural core of DNA, a continuous stack of aromatic heterocycles, the base pairs, which extends down the helical axis, gives rise to the fascinating electronic properties of this molecule that is so critical for life. Our laboratory and others have developed diverse experimental platforms to investigate the capacity of DNA to conduct charge, termed DNA-mediated charge transport (DNA CT). Here, we present an overview of DNA CT experiments in solution, on surfaces, and with single molecules that collectively provide a broad and consistent perspective on the essential characteristics of this chemistry. DNA CT can proceed over long molecular distances but is remarkably sensitive to perturbations in base pair stacking. We discuss how this foundation, built with data from diverse platforms, can be used both to inform a mechanistic description of DNA CT and to inspire the next platforms for its study: living organisms and molecular electronics. PMID:22850865

Muren, Natalie B; Olmon, Eric D; Barton, Jacqueline K



Fabrication and interfacial electronic structure studies on polypyrrole/TiO2 nano hybrid systems for photovoltaic aspects.  


The progress in studying the interfacial electronic structures of the developing new class of hybrid organic/inorganic material systems have envisaged a new dimension into the field of photovoltaics, which could be of great help in understanding the nature of charge transfer in them. In this regard, electropolymerization of pyrrole monomers have been carried out at room temperature on the surface of TiO2 working electrodes (assisted by UV radiations) and their interfacial electronic structure has been studied as a function of the applied photo anodic potentials. The formation of polypyrrole deposits has been ensured using FT-IR and Raman spectroscopy. Surface analysis of the hybrid matrix revealed the tendency of polymer molecules to cover up the spherical surface of TiO2 nanoparticles that could help in improving the light absorption rate. Signals (bands) corresponding to pyrrole molecules observed in the ultraviolet photoelectron spectroscopy measurements have been correlated with the polaronic states formed and identified to shift as a function of the applied photo anodic potentials, revealing the decrease in work function of the hybrid system to take place (confirmed using cyclic voltammetry measurements). The decreasing trend in the work function elucidates the adjustment in electronic structure of the system (hybrid materials possessing smaller work functions are generally preferred for photovoltaic studies). The aforementioned behavioural aspects have been reasoned with the increase in overpotential values for polarization, from the decrease in up-take rate of the anionic dopant, which increases the current density values, thereby modifying the conductivity of the systems. PMID:21780379

Kumar, Ganesan Mohan; Kawakita, Jin; Jayavel, Ramasamy



Compact quantum dots for single-molecule imaging.  


Single-molecule imaging is an important tool for understanding the mechanisms of biomolecular function and for visualizing the spatial and temporal heterogeneity of molecular behaviors that underlie cellular biology (1-4). To image an individual molecule of interest, it is typically conjugated to a fluorescent tag (dye, protein, bead, or quantum dot) and observed with epifluorescence or total internal reflection fluorescence (TIRF) microscopy. While dyes and fluorescent proteins have been the mainstay of fluorescence imaging for decades, their fluorescence is unstable under high photon fluxes necessary to observe individual molecules, yielding only a few seconds of observation before complete loss of signal. Latex beads and dye-labeled beads provide improved signal stability but at the expense of drastically larger hydrodynamic size, which can deleteriously alter the diffusion and behavior of the molecule under study. Quantum dots (QDs) offer a balance between these two problematic regimes. These nanoparticles are composed of semiconductor materials and can be engineered with a hydrodynamically compact size with exceptional resistance to photodegradation (5). Thus in recent years QDs have been instrumental in enabling long-term observation of complex macromolecular behavior on the single molecule level. However these particles have still been found to exhibit impaired diffusion in crowded molecular environments such as the cellular cytoplasm and the neuronal synaptic cleft, where their sizes are still too large (4,6,7). Recently we have engineered the cores and surface coatings of QDs for minimized hydrodynamic size, while balancing offsets to colloidal stability, photostability, brightness, and nonspecific binding that have hindered the utility of compact QDs in the past (8,9). The goal of this article is to demonstrate the synthesis, modification, and characterization of these optimized nanocrystals, composed of an alloyed HgxCd1-xSe core coated with an insulating CdyZn1-yS shell, further coated with a multidentate polymer ligand modified with short polyethylene glycol (PEG) chains (Figure 1). Compared with conventional CdSe nanocrystals, HgxCd1-xSe alloys offer greater quantum yields of fluorescence, fluorescence at red and near-infrared wavelengths for enhanced signal-to-noise in cells, and excitation at non-cytotoxic visible wavelengths. Multidentate polymer coatings bind to the nanocrystal surface in a closed and flat conformation to minimize hydrodynamic size, and PEG neutralizes the surface charge to minimize nonspecific binding to cells and biomolecules. The end result is a brightly fluorescent nanocrystal with emission between 550-800 nm and a total hydrodynamic size near 12 nm. This is in the same size range as many soluble globular proteins in cells, and substantially smaller than conventional PEGylated QDs (25-35 nm). PMID:23093375

Smith, Andrew M; Nie, Shuming



Single-molecule photochemical reactions of Auger-ionized quantum dots.  


Photoinduced electron transfer in donor-acceptor systems composed of quantum dots (QDs) and electron donors or acceptors is a subject of considerable recent research interest due to the potential applications of such systems in both solar energy harvesting and degradation of organic pollutants. Herein, we employed single-molecule imaging and spectroscopy techniques for the detection of photochemical reactions between 1,4-diaminobutane (DAB) and CdSe/ZnS single QDs. We investigated the reactions by analyzing photoluminescence (PL) intensity and lifetime of QDs at ensemble and single-molecule levels. While DAB was applied to single QDs tethered on a cover slip or QDs dispersed in a solution, PL intensity of QD continuously decreased with a concomitant increase in the PL lifetime. Interestingly, these changes in the PL properties of QD were predominant under high-intensity photoactivation. We hypothesize that the above changes in the PL properties surface due to the transfer of an electron from DAB to Auger-ionized QD followed by elimination of a proton from DAB and the formation of a QD-DAB adduct. Thus, a continuous decrease in the PL intensity of QDs under high-intensity photoactivation is attributed to continuous photochemical reactions of DAB with single QDs and the formation of QD-(DAB)(n) adducts. We believe that detection and analysis of such photochemical reactions of single QDs with amines will be of considerable broad interest due to the significant impact of photoinduced electron transfer reactions in energy management and environmental remediation. PMID:22132300

Hamada, Morihiko; Shibu, Edakkattuparambil Sidharth; Itoh, Tamitake; Kiran, Manikantan Syamala; Nakanishi, Shunsuke; Ishikawa, Mitsuru; Biju, Vasudevanpillai



Quantum tunneling of magnetization in lanthanide single-molecule magnets, bis(phthalocyaninato)terbium and bis(phthalocyaninato)-dysprosium anions  

Microsoft Academic Search

Magnetization versus field measurements were performed on single crystals of [(Pc)2TbIII0.02YIII0.98]^TBA+ and [(Pc)2DyIII0.02YIII0.98]^TBA+ (Pc: phthalocyaninato, TBA: tetrabutylammonium) at 0.04 K. The [(Pc)2TbIII] complex, the first lanthanide single-molecule magnet, exhibited clear staircase-like structures, which are assigned to resonant quantum tunneling between entangled states of the electron and nuclear spin systems.

Naoto Ishikawa; Miki Sugita; Wolfgang Wernsdorfer



Multiplexed single-molecule measurements with magnetic tweezers.  


We present a method for performing multiple single-molecule manipulation experiments in parallel with magnetic tweezers. We use a microscope with a low magnification, and thus a wide field of view, to visualize multiple DNA-tethered paramagnetic beads and apply an optimized image analysis routine to track the three-dimensional position of each bead simultaneously in real time. Force is applied to each bead using an externally applied magnetic field. Since variations in the field parameters are negligible across the field of view, nearly identical manipulation of all visible beads is possible. However, we find that the error in the position measurement is inversely proportional to the microscope's magnification. To mitigate the increased error caused by demagnification, we have developed a strategy based on tracking multiple fixed beads. Our system is capable of simultaneously manipulating and tracking up to 34 DNA-tethered beads at 60 Hz with approximately 1.5 nm resolution and with approximately 10% variation in applied force. PMID:19044437

Ribeck, Noah; Saleh, Omar A



Mechanical Properties of ?-Catenin Revealed by Single-Molecule Experiments  

PubMed Central

?-catenin is a central component of the adaptor complex that links cadherins to the actin cytoskeleton in adherens junctions and thus, it is a good candidate to sense and transmit mechanical forces to trigger specific changes inside the cell. To fully understand its molecular physiology, we must first investigate its mechanical role in mechanotransduction within the cadherin system. We have studied the mechanical response of ?-catenin to stretching using single-molecule force spectroscopy and molecular dynamics. Unlike most proteins analyzed to date, which have a fixed mechanical unfolding pathway, the ?-catenin armadillo repeat region (ARM) displays low mechanostability and multiple alternative unfolding pathways that seem to be modulated by its unstructured termini. These results are supported by steered molecular dynamics simulations, which also predict its mechanical stabilization and unfolding pathway restrictions when the contiguous ?-helix of the C-terminal unstructured region is included. Furthermore, simulations of the ARM/E-cadherin cytosolic tail complex emulating the most probable stress geometry occurring in vivo show a mechanical stabilization of the interaction whose magnitude correlates with the length of the stretch of the cadherin cytosolic tail that is in contact with the ARM region.

Valbuena, Alejandro; Vera, Andres Manuel; Oroz, Javier; Menendez, Margarita; Carrion-Vazquez, Mariano



Quantifying multiscale noise sources in single-molecule time series  

PubMed Central

When analyzing single-molecule data, a low-dimensional set of system observables typically serve as the observational data. We calibrate stochastic dynamical models from time series that record such observables (our focus throughout is on a molecule’s end-to-end distance). Numerical techniques for quantifying noise from multiple time scales in a single trajectory, including experimental instrument and inherent thermal noise, are demonstrated. The techniques are applied to study time series coming from both simulations and experiments associated with the nonequilibrium mechanical unfolding of titin’s I27 domain. The estimated models can be used for several purposes: (1) detect dynamical signatures of “rare events” by analyzing the effective diffusion and force as a function of the monitored observable; (2) quantify the influence that experimentally unobservable conformational degrees of freedom have on the dynamics of the monitored observable; (3) quantitatively compare the inherent thermal noise to other noise sources, e.g. instrument noise, variation induced by conformational heterogeneity, etc.; (4) simulate random quantities associated with repeated experiments; (5) apply pathwise (i.e. trajectory-wise) hypothesis tests to assess the goodness-of-fit of models and even detect conformational transitions in noisy signals. These items are all illustrated with several examples.

Calderon, Christopher P.; Harris, Nolan C.; Kiang, Ching-Hwa; Cox, Dennis D.



Single Molecule Epigenetic Analysis in a Nanofluidic Channel  

PubMed Central

Epigenetic states are governed by DNA methylation and a host of modifications to histones bound with DNA. These states are essential for proper developmentally regulated gene expression and are perturbed in many diseases. There is great interest in identifying epigenetic mark placement genome-wide and understanding how these marks vary among cell types, with changes in environment or according to health and disease status. Current epigenomic analyses employ bisulfite sequencing and chromatin immunoprecipitation, but query only one type of epigenetic mark at a time, DNA methylation or histone modifications, and often require substantial input material. To overcome these limitations, we established a method using nanofluidics and multi-color fluorescence microscopy to detect DNA and histones in individual chromatin fragments at about 10 Mbp/min. We demonstrated its utility for epigenetic analysis by identifying DNA methylation on individual molecules. This technique will provide the unprecedented opportunity for genome-wide, simultaneous analysis of multiple epigenetic states on single molecules using femtogram quantities of material.

Cipriany, Benjamin R.; Zhao, Ruqian; Murphy, Patrick J.; Levy, Stephen L.; Tan, Christine P.; Craighead, Harold G.; Soloway, Paul D.



Thermopower Measurements of Highly Conducting Single-Molecule Devices  

NASA Astrophysics Data System (ADS)

We measure the conductance (G) and thermopower (S) of highly conducting single-molecule junctions with Au electrodes. The junctions are formed and measured using a scanning tunneling microscope-based break-junction technique. The target molecules are synthesized with SnMe3 terminations that cleave off in situ, allowing for the formation of direct Au-C covalent bonds to the electrodes[1,2]. We compare the conductance and thermopower for two families of molecules: pi-conjugated polyphenyls, which have a high conductance and thermopower, and sigma-bonded alkyl systems, where we observe a significant thermopower despite the low conductance. For these measurements, we use the most probable thermopower to determine a power factor, GS^2, for each molecular junction studied. Our results show that the molecular thermopower increases systematically and non-linearly with molecular length and also that the power factor is exceptionally large for the case of the biphenyl. [1] Z. L. Cheng, R. Skouta, H. Vazquez et al., Nat. Nano. 6, 353 (2011). [2] W. Chen, J. R. Widawsky, H. Vázquez et al., J. Am. Chem. Soc. 133, 17160 (2011).

Widawsky, Jonathan R.; Chen, Wenbo; Vazquez, Hector; Kim, Taekyeong; Hybertsen, Mark S.; Breslow, Ronald; Venkataraman, Latha



Analysis and Interpretation of Single Molecule Protein Unfolding Kinetics  

NASA Astrophysics Data System (ADS)

The kinetics of protein unfolding under a stretching force has been extensively studied by atomic force microscopy (AFM) over the past decade [1]. Experimental artifacts at the single molecule level introduce uncertainties in the data analysis that have led to several competing physical models for the unfolding process. For example, the unfolding dynamics of the protein ubiquitin under constant force has been described by probability distributions as diverse as exponential [2,3], a sum of exponentials, log-normal [4], and more recently a function describing static disorder in the Arrhenius model [5]. A new method for data analysis is presented that utilizes maximum likelihood estimation (MLE) combined with other traditional statistical tests to unambiguously rank the consistency of these and other models with the experimental data. These techniques applied to the ubiquitin unfolding data shows that the probability of unfolding is best fit with a stretched exponential distribution, with important implications on the complexity of the mechanism of protein unfolding. [4pt] [1] Carrion-Vazquez, et. al. Springer Series in Biophys. 2006 [0pt] [2] Fernandez et. al. Science 2004 [0pt] [3] Brujic et. al. Nat. Phys 2006 [0pt] [4] Garcia-Manyes et. al. Biophys. J. 2007 [0pt] [5] Kuo et. al. PNAS 2010

Lannon, Herbert; Brujic, Jasna



Flexible single molecule simulation of reaction-diffusion processes  

SciTech Connect

An algorithm is developed for simulation of the motion and reactions of single molecules at a microscopic level. The molecules diffuse in a solvent and react with each other or a polymer and molecules can dissociate. Such simulations are of interest e.g. in molecular biology. The algorithm is similar to the Green's function reaction dynamics (GFRD) algorithm by van Zon and ten Wolde where longer time steps can be taken by computing the probability density functions (PDFs) and then sample from the distribution functions. Our computation of the PDFs is much less complicated than GFRD and more flexible. The solution of the partial differential equation for the PDF is split into two steps to simplify the calculations. The sampling is without splitting error in two of the coordinate directions for a pair of molecules and a molecule-polymer interaction and is approximate in the third direction. The PDF is obtained either from an analytical solution or a numerical discretization. The errors due to the operator splitting, the partitioning of the system, and the numerical approximations are analyzed. The method is applied to three different systems involving up to four reactions. Comparisons with other mesoscopic and macroscopic models show excellent agreement.

Hellander, Stefan, E-mail: stefan.hellander@it.uu.s [Division of Scientific Computing, Department of Information Technology, Uppsala University, P.O. Box 337, SE-75105 Uppsala (Sweden); Loetstedt, Per, E-mail: perl@it.uu.s [Division of Scientific Computing, Department of Information Technology, Uppsala University, P.O. Box 337, SE-75105 Uppsala (Sweden)



Single Molecule Analysis of Serotonin Transporter Regulation Using Quantum Dots  

NASA Astrophysics Data System (ADS)

For the first time, we implement a novel, single molecule approach to define the localization and mobility of the brain's major target of widely prescribed antidepressant medications, the serotonin transporter (SERT). SERT labeled with single quantum dot (Qdot) revealed unsuspected features of transporter mobility with cholesterol-enriched membrane microdomains (often referred to as "lipid rafts") and cytoskeleton network linked to transporter activation. We document two pools of surface SERT proteins defined by their lateral mobility, one that exhibits relatively free diffusion in the plasma membrane and a second that displays significantly restricted mobility and localizes to cholesterol-enriched microdomains. Diffusion model prediction and instantaneous velocity analysis indicated that stimuli that act through p38 MAPK-dependent signaling pathways to activate SERT trigger rapid SERT movements within membrane microdomains. Cytoskeleton disruption showed that SERT lateral mobility behaves a membrane raft-constrained, cytoskeleton-associated manner. Our results identify an unsuspected aspect of neurotransmitter transporter regulation that we propose reflects the dissociation of inhibitory, SERT-associated cytoskeletal anchors.

Chang, Jerry; Tomlinson, Ian; Warnement, Michael; Ustione, Alessandro; Carneiro, Ana; Piston, David; Blakely, Randy; Rosenthal, Sandra



Single-molecule orientation measurements with a quadrated pupil.  


This Letter presents a means of measuring the dipole orientation of a fluorescent, orientationally fixed single molecule, which uses a specially designed phase mask, termed a "quadrated pupil," conjugate to the back focal plane of a conventional wide-field microscope. The method leverages the spatial anisotropy of the far-field emission pattern of a dipole emitter and makes this anisotropy amenable to quantitative analysis at the image plane. In comparison to older image-fitting techniques that infer orientation by matching simulations to defocused or excessively magnified images, the quadrated pupil approach is more robust to minor modeling discrepancies and optical aberrations. Precision of 1°-5° is achieved in proof-of-concept experiments for both azimuthal (?) and polar (?) angles without defocusing. Since the phase mask is implemented on a liquid-crystal spatial light modulator that may be deactivated without any mechanical perturbation of the sample or imaging system, the technique may be readily integrated into clear aperture imaging studies. PMID:23632538

Backer, Adam S; Backlund, Mikael P; Lew, Matthew D; Moerner, W E



A Single-Molecule Hershey-Chase Experiment  

PubMed Central

Summary Ever since Hershey and Chase used phages to establish DNA as the carrier of genetic information in 1952, the precise mechanisms of phage DNA translocation have been a mystery [1]. Although bulk measurements have set a time-scale for in vivo DNA translocation during bacteriophage infection, measurements of DNA ejection by single bacteriophages have only been made in vitro. Here, we present direct visualization of single bacteriophages infecting individual Escherichia coli cells. For bacteriophage ?, we establish a mean ejection time of roughly 5 min with significant cell-to-cell variability, including pausing events. In contrast, corresponding in vitro single-molecule ejections are more uniform and finish within 10 s. Our data reveal that when plotted against the amount of DNA ejected, the velocity of ejection for two different genome lengths collapses onto a single curve. This suggests that in vivo ejections are controlled by the amount of DNA ejected. In contrast, in vitro DNA ejections are governed by the amount of DNA left inside the capsid. This analysis provides evidence against a purely intrastrand repulsion-based mechanism and suggests that cell-internal processes dominate. This provides a picture of the early stages of phage infection and sheds light on the problem of polymer translocation.

Van Valen, David; Wu, David; Chen, Yi-Ju; Tuson, Hannah; Wiggins, Paul; Phillips, Rob



Single-molecule DNA repair in live bacteria.  


Cellular DNA damage is reversed by balanced repair pathways that avoid accumulation of toxic intermediates. Despite their importance, the organization of DNA repair pathways and the function of repair enzymes in vivo have remained unclear because of the inability to directly observe individual reactions in living cells. Here, we used photoactivation, localization, and tracking in live Escherichia coli to directly visualize single fluorescent labeled DNA polymerase I (Pol) and ligase (Lig) molecules searching for DNA gaps and nicks, performing transient reactions, and releasing their products. Our general approach provides enzymatic rates and copy numbers, substrate-search times, diffusion characteristics, and the spatial distribution of reaction sites, at the single-cell level, all in one measurement. Single repair events last 2.1 s (Pol) and 2.5 s (Lig), respectively. Pol and Lig activities increased fivefold over the basal level within minutes of DNA methylation damage; their rates were limited by upstream base excision repair pathway steps. Pol and Lig spent >80% of their time searching for free substrates, thereby minimizing both the number and lifetime of toxic repair intermediates. We integrated these single-molecule observations to generate a quantitative, systems-level description of a model repair pathway in vivo. PMID:23630273

Uphoff, Stephan; Reyes-Lamothe, Rodrigo; Garza de Leon, Federico; Sherratt, David J; Kapanidis, Achillefs N



A delocalized arene-bridged diuranium single-molecule magnet.  


Single-molecule magnets (SMMs) are compounds that, below a blocking temperature, exhibit stable magnetization purely of molecular origin, and not caused by long-range ordering of magnetic moments in the bulk. They thus show promise for applications such as data storage of ultra-high density. The stability of the magnetization increases with increasing ground-state spin and magnetic anisotropy. Transition-metal SMMs typically possess high-spin ground states, but insufficient magnetic anisotropies. Lanthanide SMMs exhibit large magnetic anisotropies, but building high-spin ground states is difficult because they tend to form ionic bonds that limit magnetic exchange coupling. In contrast, the significant covalent bonding and large spin-orbit contributions associated with uranium are particularly attractive for the development of improved SMMs. Here we report a delocalized arene-bridged diuranium SMM. This study demonstrates that arene-bridged polyuranium clusters can exhibit SMM behaviour without relying on the superexchange coupling of spins. This approach may lead to increased blocking temperatures. PMID:21602860

Mills, David P; Moro, Fabrizio; McMaster, Jonathan; van Slageren, Joris; Lewis, William; Blake, Alexander J; Liddle, Stephen T



Mechanical fatigue in repetitively stretched single molecules of titin.  

PubMed Central

Relaxed striated muscle cells exhibit mechanical fatigue when exposed to repeated stretch and release cycles. To understand the molecular basis of such mechanical fatigue, single molecules of the giant filamentous protein titin, which is the main determinant of sarcomeric elasticity, were repetitively stretched and released while their force response was characterized with optical tweezers. During repeated stretch-release cycles titin becomes mechanically worn out in a process we call molecular fatigue. The process is characterized by a progressive shift of the stretch-force curve toward increasing end-to-end lengths, indicating that repeated mechanical cycles increase titin's effective contour length. Molecular fatigue occurs only in a restricted force range (0-25 pN) during the initial part of the stretch half-cycle, whereas the rest of the force response is repeated from one mechanical cycle to the other. Protein-folding models fail to explain molecular fatigue on the basis of an incomplete refolding of titin's globular domains. Rather, the process apparently derives from the formation of labile nonspecific bonds cross-linking various sites along a pre-unfolded titin segment. Because titin's molecular fatigue occurs in a physiologically relevant force range, the process may play an important role in dynamically adjusting muscle's response to the recent history of mechanical perturbations.

Kellermayer, M S; Smith, S B; Bustamante, C; Granzier, H L



Single-Molecule Manipulation Studies of a Mechanically Activated Protein  

NASA Astrophysics Data System (ADS)

Plasma von Willebrand factor (pVWF) is the largest multimeric adhesion ligand found in human blood and must be adhesively activated by exposure to shear stress, like at sites of vascular injury, to initiate blood clotting. Sheared pVWF (sVWF) will undergo a conformational change from a loose tangled coil to elongated strings forming adhesive fibers by binding with other sVWF. VWF's adhesion activity is also related to its length, with the ultra-large form of VWF (ULVWF) being hyper-actively adhesive without exposure to shear stress; it has also been shown to spontaneously form fibers. We used single molecule manipulation techniques with the AFM to stretch pVWF, sVWF and ULVWF and monitor the forces as a function of molecular extension. We showed a similar increase in resistance to unfolding for sVWF and ULVWF when compared to pVWF. This mechanical resistance to forced unfolding is reduced when other molecules known to disrupt their fibril formation are present. Our results show that sVWF and ULVWF domains unfold at higher forces than pVWF, which is consistent with the hypothesis that shear stress induces lateral association that alters adhesion activity of pVWF.

Botello, Eric; Harris, Nolan; Choi, Huiwan; Bergeron, Angela; Dong, Jing-Fei; Kiang, Ching-Hwa



Bayesian Inference for Improved Single Molecule Fluorescence Tracking  

PubMed Central

Single molecule tracking is widely used to monitor the change in position of lipids and proteins in living cells. In many experiments in which molecules are tagged with a single or small number of fluorophores, the signal/noise ratio may be limiting, the number of molecules is not known, and fluorophore blinking and photobleaching can occur. All these factors make accurate tracking over long trajectories difficult and hence there is still a pressing need to develop better algorithms to extract the maximum information from a sequence of fluorescence images. We describe here a Bayesian-based inference approach, based on a trans-dimensional sequential Monte Carlo method that utilizes both the spatial and temporal information present in the image sequences. We show, using model data, where the real trajectory of the molecule is known, that our method allows accurate tracking of molecules over long trajectories even with low signal/noise ratio and in the presence of fluorescence blinking and photobleaching. The method is then applied to real experimental data.

Yoon, Ji Won; Bruckbauer, Andreas; Fitzgerald, William J.; Klenerman, David



Competition between Supercoils and Toroids in Single Molecule DNA Condensation  

PubMed Central

The condensation of free DNA into toroidal structures in the presence of multivalent ions and polypeptides is well known. Recent single molecule experiments have shown that condensation into toroids occurs even when the DNA molecule is subjected to tensile forces. Here we show that the combined tension and torsion of DNA in the presence of condensing agents dramatically modifies this picture by introducing supercoiled DNA as a competing structure in addition to toroids. We combine a fluctuating elastic rod model of DNA with phenomenological models for DNA interaction in the presence of condensing agents to compute the minimum energy configuration for given tension and end-rotations. We show that for each tension there is a critical number of end-rotations above which the supercoiled solution is preferred and below which toroids are the preferred state. Our results closely match recent extension rotation experiments on DNA in the presence of spermine and other condensing agents. Motivated by this, we construct a phase diagram for the preferred DNA states as a function of tension and applied end-rotations and identify a region where new experiments or simulations are needed to determine the preferred state.

Argudo, David; Purohit, Prashant K.



Stretching polysaccharides on live cells using single molecule force spectroscopy.  


The knowledge of molecular mechanisms underlying the adhesive and mechanical properties of cell surface-associated molecules is a key to understanding their functions. In this context, single-molecule force spectroscopy (SMFS) has recently offered new opportunities for probing the adhesion and mechanics of polysaccharides and proteins on live cells. Here we present a protocol that we have used to analyze polysaccharide chains of different nature on the bacterium Lactobacillus rhamnosus GG. We describe procedures (i) for functionalizing atomic force microscopy (AFM) tips with Pseudomonas aeruginosa-I or concanavalin A lectins, (ii) for stretching specific polysaccharide molecules on live bacteria using SMFS with lectin tips and (iii) for mapping the localization, adhesion and extension of individual polysaccharide chains. We also discuss data treatment, emphasizing how to gain insight into the elasticity of the stretched macromolecules using the extended freely jointed chain model. Even though the presented protocol is for L. rhamnosus, it can be easily modified for other cell types. For users having expertise in the field, the entire protocol can be completed in about 5 d. PMID:19478809

Francius, Grégory; Alsteens, David; Dupres, Vincent; Lebeer, Sarah; De Keersmaecker, Sigrid; Vanderleyden, Jos; Gruber, Hermann J; Dufrêne, Yves F



Optical Microcavity: Sensing down to Single Molecules and Atoms  

PubMed Central

This review article discusses fundamentals of dielectric, low-loss, optical micro-resonator sensing, including figures of merit and a variety of microcavity designs, and future perspectives in microcavity-based optical sensing. Resonance frequency and quality (Q) factor are altered as a means of detecting a small system perturbation, resulting in realization of optical sensing of a small amount of sample materials, down to even single molecules. Sensitivity, Q factor, minimum detectable index change, noises (in sensor system components and microcavity system including environments), microcavity size, and mode volume are essential parameters to be considered for optical sensing applications. Whispering gallery mode, photonic crystal, and slot-type microcavities typically provide compact, high-quality optical resonance modes for optical sensing applications. Surface Bloch modes induced on photonic crystals are shown to be a promising candidate thanks to large field overlap with a sample and ultra-high-Q resonances. Quantum optics effects based on microcavity quantum electrodynamics (QED) would provide novel single-photo-level detection of even single atoms and molecules via detection of doublet vacuum Rabi splitting peaks in strong coupling.

Yoshie, Tomoyuki; Tang, Lingling; Su, Shu-Yu



Mapping Transcription Factors on Extended DNA: A Single Molecule Approach  

NASA Astrophysics Data System (ADS)

The ability to determine the precise loci and distribution of nucleic acid binding proteins is instrumental to our detailed understanding of cellular processes such as transcription, replication, and chromatin reorganization. Traditional molecular biology approaches and above all Chromatin immunoprecipitation (ChIP) based methods have provided a wealth of information regarding protein-DNA interactions. Nevertheless, existing techniques can only provide average properties of these interactions, since they are based on the accumulation of data from numerous protein-DNA complexes analyzed at the ensemble level. We propose a single molecule approach for direct visualization of DNA binding proteins bound specifically to their recognition sites along a long stretch of DNA such as genomic DNA. Fluorescent Quantum dots are used to tag proteins bound to DNA, and the complex is deposited on a glass substrate by extending the DNA to a linear form. The sample is then imaged optically to determine the precise location of the protein binding site. The method is demonstrated by detecting individual, Quantum dot tagged T7-RNA polymerase enzymes on the bacteriophage T7 genomic DNA and assessing the relative occupancy of the different promoters.

Ebenstein, Yuval; Gassman, Natalie; Weiss, Shimon


Multiplexed single-molecule measurements with magnetic tweezers  

SciTech Connect

We present a method for performing multiple single-molecule manipulation experiments in parallel with magnetic tweezers. We use a microscope with a low magnification, and thus a wide field of view, to visualize multiple DNA-tethered paramagnetic beads and apply an optimized image analysis routine to track the three-dimensional position of each bead simultaneously in real time. Force is applied to each bead using an externally applied magnetic field. Since variations in the field parameters are negligible across the field of view, nearly identical manipulation of all visible beads is possible. However, we find that the error in the position measurement is inversely proportional to the microscope's magnification. To mitigate the increased error caused by demagnification, we have developed a strategy based on tracking multiple fixed beads. Our system is capable of simultaneously manipulating and tracking up to 34 DNA-tethered beads at 60 Hz with {approx}1.5 nm resolution and with {approx}10% variation in applied force.

Ribeck, Noah [Physics Department, University of California, Santa Barbara, Santa Barbara, California 93106 (United States); Saleh, Omar A. [Materials Department and Biomolecular Science and Engineering Program, University of California, Santa Barbara, Santa Barbara, California 93106 (United States)



A Single Molecule Investigation of the Photostability of Quantum Dots  

PubMed Central

Quantum dots (QDs) are very attractive probes for multi-color fluorescence imaging in biological applications because of their immense brightness and reported extended photostability. We report here however that single QDs, suitable for biological applications, that are subject to continuous blue excitation from a conventional 100 W mercury arc lamp will undergo a continuous blue-switching of the emission wavelength eventually reaching a permanent dark, photobleached state. We further show that ?-mercaptoethanol has a dual stabilizing effect on the fluorescence emission of QDs: 1) by increasing the frequency of time that a QD is in its fluorescent state, and 2) by decreasing the photobleaching rate. The observed QD color spectral switching is especially detrimental for multi-color single molecule applications, as we regularly observe spectral blue-shifts of 50 nm, or more even after only ten seconds of illumination. However, of significant importance for biological applications, we find that even small, biologically compatible, concentrations (25 µM) of ?-mercaptoethanol has a significant stabilizing effect on the emission color of QDs, but that greater amounts are required to completely abolish the spectral blue shifting or to minimize the emission intermittency of QDs.

Lagerholm, B. Christoffer



Temperature softening of a protein in single-molecule experiments.  


Mechanical flexibility is crucial for the function of proteins. However, such material properties are not easily accessible experimentally. We used single-molecule force spectroscopy to study the stiffness of a single domain of Dictyostelium discoideum filamin (ddFLN4) in a temperature range from 5 degrees C to 37 degrees C. Analyzing the distributions of unfolding forces allowed us to extract transition barrier heights and positions of the underlying energy landscape. We found a marked narrowing of unfolding force distributions with increasing temperature. This narrowing reflects an increase in transition state position from 2.7 A to 7.8 A and thus a reduction of the molecular spring constant of the protein by a factor of 7. We suggest this temperature softening reflects a shift in the nature of the interactions responsible for mechanical stability from hydrogen bonds to hydrophobic interactions. This result has important consequences for all interpretations of protein mechanical studies if experimental results obtained at room temperature are to be transferred to physiological temperatures. PMID:16246362

Schlierf, Michael; Rief, Matthias



Single Molecule Visualization of DNA in Pure Shear Flow  

NASA Astrophysics Data System (ADS)

Polymers are ever-present in society from plastic bottles to DNA. The study of single molecule dynamics will provide the opportunity for advances in fields from synthetic polymer coatings to gene therapy. Many applications involve flow of dilute polymer solutions in viscous solvents. These long, flexible polymer chains (DNA) are coiled at rest in solution. The configuration of the molecules is altered by the applied flow which, in turn, affects the dynamics of the flow. Control of flow allows for manipulation of the DNA molecules. Our apparatus consists of a rectangular channel that has been plasma etched into a silicon wafer with pressure driven flow (pulse-free syringe pump). The dynamics of the DNA molecules in flow are monitored using fluorescence microscopy and digital imaging. The flow channel was designed to allow for visualization of the molecules in the plane defined by velocity and velocity gradient instead of the plane identified by the velocity and the vorticity (previously studied by Smith et al (1999) and LeDuc et al (1999)). Moreover, we can visualize the DNA in a flow where the velocity gradient is not uniform. The individual and average conformations (size and orientation) of the flowing DNA molecules are being studied as a function of the Weissenberg number (product of strain rate and DNA relaxation time) and distance from the channel walls.

Smith, Connie; Duggal, Rajat; Pasquali, Matteo



Single molecule detection using charge-coupled device array technology  

NASA Astrophysics Data System (ADS)

An ultra sensitive technique for the detection of fluorescent species in a flowing stream has been developed. The extension of this technique to the detection of fluorescently tagged nucleotides will be a significant benefit to one of the novel approaches for DNA sequencing being developed at Los Alamos National Laboratories. The detection scheme is based on a novel mode of operating a charge-coupled device (CCD) which greatly enhances the discrimination between fluorescence from the analyte and the background Raman scattering from the solvent. Register shifts between rows in the CCD are synchronized with the sample flow velocity so that fluorescence from a single species is collected in a single moving charge packet occupying an area approaching that of a single pixel while the background is spread evenly among a large number of pixels. This research has demonstrated that this technique is highly effective for the detection of fluorescently labelled latex microspheres. With additional development, the authors believe that this technique will achieve single molecule detection.

Denton, M. B.



A single molecule study of cellulase hydrolysis of crystalline cellulose  

NASA Astrophysics Data System (ADS)

Cellobiohydrolase-I (CBH I), a processive exoglucanase secreted by Trichoderma reesei, is one of the key enzyme components in a commercial cellulase mixture currently used for processing biomass to biofuels. CBH I contains a family 7 glycoside hydrolase catalytic module, a family 1 carbohydrate-binding module (CBM), and a highlyglycosylated linker peptide. It has been proposed that the CBH I cellulase initiates the hydrolysis from the reducing end of one cellulose chain and successively cleaves alternate ?-1,4-glycosidic bonds to release cellobiose as its principal end product. The role each module of CBH I plays in the processive hydrolysis of crystalline cellulose has yet to be convincingly elucidated. In this report, we use a single-molecule approach that combines optical (Total Internal Reflection Fluorescence microscopy, or TIRF-M) and non-optical (Atomic Force Microscopy, or AFM) imaging techniques to analyze the molecular motion of CBM tagged with green fluorescence protein (GFP), and to investigate the surface structure of crystalline cellulose and changes made in the structure by CBM and CBH I. The preliminary results have revealed a confined nanometer-scale movement of the TrCBM1-GFP bound to cellulose, and decreases in cellulose crystal size as well as increases in surface roughness during CBH I hydrolysis of crystalline cellulose.

Liu, Yu-San; Luo, Yonghua; Baker, John O.; Zeng, Yining; Himmel, Michael E.; Smith, Steve; Ding, Shi-You



Electrostatic and Steric Interactions Determine Bacteriorhodopsin Single-Molecule Biomechanics  

PubMed Central

Bacteriorhodopsin (bR) is a haloarchaeal membrane protein that converts the energy of single photons into large structural changes to directionally pump protons across purple membrane. This is achieved by a complex combination of local dynamic interactions controlling bR biomechanics at the submolecular level, producing efficient amplification of the retinal photoisomerization. Using single molecule force spectroscopy at different salt concentrations, we show that tryptophan (Trp) residues use steric specific interactions to create a rigid scaffold in bR extracellular region and are responsible for the main unfolding barriers. This scaffold, which encloses the retinal, controls bR local mechanical properties and anchors the protein into the membrane. Furthermore, the stable Trp-based network allows ion binding to two specific sites on the extracellular loops (BC and FG), which are involved in proton release and lateral transport. In contrast, the cytoplasmic side of bR is mainly governed by relatively weak nonspecific electrostatic interactions that provide the flexibility necessary for large cytoplasmic structural rearrangements during the photocycle. The presence of an extracellular Trp-based network tightly enclosing the retinal seems common to most haloarchaeal rhodopsins, and could be relevant to their exceptional efficiency.

Voitchovsky, Kislon; Contera, Sonia Antoranz; Ryan, J. F.



Single-Molecule Spectroscopic Investigations of RNA Structural Dynamics  

NASA Astrophysics Data System (ADS)

To function properly, catalytic RNAs (ribozymes) fold into specific three-dimensional shapes stabilized by multiple tertiary interactions. However, only limited information is available on the contributions of individual tertiary contacts to RNA conformational dynamics. The Tetrahymena ribozymes's P4--P6 domain forms a hinged, ``candy-cane'' structure with parallel helices clamped by two motifs, the GAAA tetraloop-tetraloop receptor and adenosine (A)-rich bulge--P4 helix interactions. Previously, we characterized RNA folding due to a tetraloop-receptor interaction. In this study, we employ time-resolved single-molecule FRET methods to probe A-rich bulge induced structural dynamics. Specifically, fluorescently labeled RNA constructs excited by a pulsed 532 nm laser are detected in the confocal region of an inverted microscope, with each photon sorted by arrival time, color and polarization. We resolve the kinetic dependence of A-rich bulge-P4 helix docking/undocking on cationic environment (e.g. Na^+ and Mg^2+ concentration.) At saturating [Mg^2+], the docked structure appears only weakly stabilized, while only 50% of the molecules exhibit efficient folding.

Fiore, Julie L.; Nesbitt, David J.



Single-Molecule Mechanical Identification and Sequencing: Proof of Principle  

PubMed Central

High-throughput low-cost DNA sequencing has emerged as one of the challenges of the post-genomic era. Here we present the proof of concept for a new single-molecule platform that allows for DNA identification and sequencing. In contrast with most present methods, our scheme is not based on the detection of the fluorescence of incorporated nucleotides, but rather on the measurement of a DNA hairpin length. By cyclically modulating the force pulling on small magnetic beads tethered by a hairpin to a surface, one can unzip and rezip the molecule. In the presence of complementary oligonucleotides in solution, reziping may be transiently interrupted by the hybrids they form with the hairpin. By measuring the extension of the blocked hairpin, one can determine the position of the hybrid along the molecule with nearly single base precision. Our approach, well adapted to a high-throughput scheme, can be used to identify a DNA fragment of known sequence among a sample of various fragments and to sequence an unknown DNA fragment by hybridization or ligation.

Ding, Fangyuan; Manosas, Maria; Spiering, Michelle M.; Benkovic, Stephen J.; Bensimon, David; Allemand, Jean-Francois; Croquette, Vincent



Bayesian inference for improved single molecule fluorescence tracking.  


Single molecule tracking is widely used to monitor the change in position of lipids and proteins in living cells. In many experiments in which molecules are tagged with a single or small number of fluorophores, the signal/noise ratio may be limiting, the number of molecules is not known, and fluorophore blinking and photobleaching can occur. All these factors make accurate tracking over long trajectories difficult and hence there is still a pressing need to develop better algorithms to extract the maximum information from a sequence of fluorescence images. We describe here a Bayesian-based inference approach, based on a trans-dimensional sequential Monte Carlo method that utilizes both the spatial and temporal information present in the image sequences. We show, using model data, where the real trajectory of the molecule is known, that our method allows accurate tracking of molecules over long trajectories even with low signal/noise ratio and in the presence of fluorescence blinking and photobleaching. The method is then applied to real experimental data. PMID:18339757

Yoon, Ji Won; Bruckbauer, Andreas; Fitzgerald, William J; Klenerman, David



High-Resolution Optical Tweezers for Single-Molecule Manipulation  

PubMed Central

Forces hold everything together and determine its structure and dynamics. In particular, tiny forces of 1-100 piconewtons govern the structures and dynamics of biomacromolecules. These forces enable folding, assembly, conformational fluctuations, or directional movements of biomacromolecules over sub-nanometer to micron distances. Optical tweezers have become a revolutionary tool to probe the forces, structures, and dynamics associated with biomacromolecules at a single-molecule level with unprecedented resolution. In this review, we introduce the basic principles of optical tweezers and their latest applications in studies of protein folding and molecular motors. We describe the folding dynamics of two strong coiled coil proteins, the GCN4-derived protein pIL and the SNARE complex. Both complexes show multiple folding intermediates and pathways. ATP-dependent chromatin remodeling complexes translocate DNA to remodel chromatin structures. The detailed DNA translocation properties of such molecular motors have recently been characterized by optical tweezers, which are reviewed here. Finally, several future developments and applications of optical tweezers are discussed. These past and future applications demonstrate the unique advantages of high-resolution optical tweezers in quantitatively characterizing complex multi-scale dynamics of biomacromolecules.

Zhang, Xinming; Ma, Lu; Zhang, Yongli



Spectroelectrochemical investigation of intramolecular and interfacial electron-transfer rates reveals differences between nitrite reductase at rest and during turnover.  


A combined fluorescence and electrochemical method is described that is used to simultaneously monitor the type-1 copper oxidation state and the nitrite turnover rate of a nitrite reductase (NiR) from Alcaligenes faecalis S-6. The catalytic activity of NiR is measured electrochemically by exploiting a direct electron transfer to fluorescently labeled enzyme molecules immobilized on modified gold electrodes, whereas the redox state of the type-1 copper site is determined from fluorescence intensity changes caused by Fo?rster resonance energy transfer (FRET) between a fluorophore attached to NiR and its type-1 copper site. The homotrimeric structure of the enzyme is reflected in heterogeneous interfacial electron-transfer kinetics with two monomers having a 25-fold slower kinetics than the third monomer. The intramolecular electron-transfer rate between the type-1 and type-2 copper site changes at high nitrite concentration (?520 ?M), resulting in an inhibition effect at low pH and a catalytic gain in enzyme activity at high pH. We propose that the intramolecular rate is significantly reduced in turnover conditions compared to the enzyme at rest, with an exception at low pH/nitrite conditions. This effect is attributed to slower reduction rate of type-2 copper center due to a rate-limiting protonation step of residues in the enzyme's active site, gating the intramolecular electron transfer. PMID:21863850

Krzemi?ski, ?ukasz; Ndamba, Lionel; Canters, Gerard W; Aartsma, Thijs J; Evans, Stephen D; Jeuken, Lars J C



Many-body theory of electric and thermal transport in single-molecule heterojunctions  

NASA Astrophysics Data System (ADS)

Electron transport in single-molecule junctions (SMJ) is a key example of a strongly-correlated system far from equilibrium, with myriad potential applications in nanotechnology. When macroscopic leads are attached to a single molecule, a SMJ is formed, transforming the ``few-body'' molecular problem into a true ``many-body'' problem. Until recently, a theory of transport that properly accounts for both the particle and wave character of the electron has been lacking, so that the Coulomb blockade and coherent transport regimes were considered ``complementary.'' We have developed a nonequilibrium many-body theoryfootnotetextJ. P. Bergfield and C. A. Stafford, Phys. Rev. B 79, 245125 (2009). that reproduces the key features of both the Coulomb blockade and coherent transport regimes simultaneously. Our approach is based on nonequilibrium Green's functions, enabling physically motivated approximations that sum terms to all orders. The junction Green's functions are calculated exactly in the sequential-tunneling limit, and the corrections to the electron self-energy due to finite tunneling width are included via Dyson-Keldysh equations. In this talk, I will present a brief overview of our many-body theory of SMJ and discuss the simulated linear and nonlinear response of a benzenedithiol-gold junction. I will also outline our derivation of an exact expression for the heat current in an interacting nanostructure, highlighting our predictionfootnotetextJ. P. Bergfield and C. A. Stafford, Nano Letters 9, 3072 (2009). of a dramatic quantum-induced enhancement of thermoelectric effects in the vicinity of a transmission node. Finally, I will provide several striking examples where the predictions of our many-body theory differ drastically from those of mean-field (density functional) theory.

Bergfield, Justin



Lab-on-a-chip technologies for single-molecule studies.  


Recent developments on various lab-on-a-chip techniques allow miniaturized and integrated devices to perform on-chip single-molecule studies. Fluidic-based platforms that utilize unique microscale fluidic behavior are capable of conducting single-molecule experiments with high sensitivities and throughputs, while biomolecular systems can be studied on-chip using techniques such as DNA curtains, magnetic tweezers, and solid-state nanopores. The advances of these on-chip single-molecule techniques lead to next-generation lab-on-a-chip devices, such as DNA transistors, and single-molecule real-time (SMRT) technology for rapid and low-cost whole genome DNA sequencing. In this Focus article, we will discuss some recent successes in the development of lab-on-a-chip techniques for single-molecule studies and expound our thoughts on the near future of on-chip single-molecule studies. PMID:23670195

Zhao, Yanhui; Chen, Danqi; Yue, Hongjun; French, Jarrod B; Rufo, Joseph; Benkovic, Stephen J; Huang, Tony Jun



Single-molecule fluorescence probes dynamics of barrier crossing.  


Kramers developed the theory on how chemical reaction rates are influenced by the viscosity of the medium. At the viscosity of water, the kinetics of unimolecular reactions are described by diffusion of a Brownian particle over a free-energy barrier separating reactants and products. For reactions in solution this famous theory extended Eyring's transition state theory, and is widely applied in physics, chemistry and biology, including to reactions as complex as protein folding. Because the diffusion coefficient of Kramers' theory is determined by the dynamics in the sparsely populated region of the barrier top, its properties have not been directly measured for any molecular system. Here we show that the Kramers diffusion coefficient and free-energy barrier can be characterized by measuring the temperature- and viscosity-dependence of the transition path time for protein folding. The transition path is the small fraction of an equilibrium trajectory for a single molecule when the free-energy barrier separating two states is actually crossed. Its duration, the transition path time, can now be determined from photon trajectories for single protein molecules undergoing folding/unfolding transitions. Our finding of a long transition path time with an unusually small solvent viscosity dependence suggests that internal friction as well as solvent friction determine the Kramers diffusion coefficient for ?-helical proteins, as opposed to a breakdown of his theory, which occurs for many small-molecule reactions. It is noteworthy that the new and fundamental information concerning Kramers' theory and the dynamics of barrier crossings obtained here come from experiments on a protein rather than a much simpler chemical or physical system. PMID:24153185

Chung, Hoi Sung; Eaton, William A



Single Molecule Force Spectroscopy using Optical Traps and AFMs  

NASA Astrophysics Data System (ADS)

Force spectroscopy is an important single-molecule technique to study the energetics and dynamics of biological systems. Both optical traps and atomic force microscopes (AFMs) can measure the dynamics of individual molecules. My talk will focus on two intellectually distinct ways to improve these experiments: passive force clamps and an optically stabilized AFM. To increase measurement precision, feedback is used to maintain a constant force on a molecule - often called a force clamp. Precise yet rapid active feedback is limited by Brownian motion. This limited bandwidth leads to significant fluctuations in force that are particularly pronounced for the rapid, large changes in extension seen in nucleic acid structures (e.g. DNA hairpins, ribozymes, riboswitches). Here, we show that the dynamics determined in active force clamps are five-to-seven fold different than in a passive force clamp, which has a (˜30-fold faster control of force. Thus, the dynamics of biological molecules can be significantly altered by the mechanism of force feedback. In AFM-based force spectroscopy experiments, force versus extension curves are generated by retracting the tip using a PZT stage while measuring force via cantilever deflection. Extension is not stable over the long times due to drift in the AFM assembly (˜10 nm/min). We developed an ultrastable AFM by measuring and thereby stabilizing the tip in 3D using a laser scattered off the apex of a commercial AFM tip, not its back side. A second laser detected and thereby stabilized the sample. We next demonstrated simultaneous and independent measurement of extension and force. Preliminary studies of bacteriorhodopsin, a model membrane protein, highlight this instruments unique force- and position-clamp modes.

Perkins, Tom



Single Molecule Force Spectroscopy using Optical Traps and AFMs  

NASA Astrophysics Data System (ADS)

Force spectroscopy is an important single-molecule technique to study the energetics and dynamics of biological systems. Both optical traps and atomic force microscopes (AFMs) can measure the dynamics of individual molecules. My talk will focus on two intellectually distinct ways to improve these experiments: passive force clamps and an optically stabilized AFM. To increase measurement precision, feedback is used to maintain a constant force on a molecule -- often called a force clamp. Precise yet rapid active feedback is limited by Brownian motion. This limited bandwidth leads to significant fluctuations in force that are particularly pronounced for the rapid, large changes in extension seen in nucleic acid structures (e.g. DNA hairpins, ribozymes, riboswitches). Here, we show that the dynamics determined in active force clamps are five-to-seven fold different than in a passive force clamp, which has a ˜30-fold faster control of force. Thus, the dynamics of biological molecules can be significantly altered by the mechanism of force feedback. In AFM-based force spectroscopy experiments, force versus extension curves are generated by retracting the tip using a PZT stage while measuring force via cantilever deflection. Extension is inferred, not measured, and therefore convolved with drift in the AFM assembly (˜10 nm/min). We developed an ultrastable AFM by scattering a laser off the apex of a commercial AFM tip to measure and thereby stabilize the tip in 3D. A second laser stabilized the sample, leading to a 100-fold improvement in tip-sample stability compared to the previous state-of-the-art at ambient conditions (in air at room temperature). We next demonstrated simultaneous and independent measurement of extension and force in liquid. Preliminary studies of bacteriorhodopsin, a model membrane protein, highlight this instrument's unique force- and position-clamp modes.

Perkins, Thomas



Nucleosome disassembly intermediates characterized by single-molecule FRET.  


The nucleosome has a central role in the compaction of genomic DNA and the control of DNA accessibility for transcription and replication. To help understanding the mechanism of nucleosome opening and closing in these processes, we studied the disassembly of mononucleosomes by quantitative single-molecule FRET with high spatial resolution, using the SELEX-generated "Widom 601" positioning sequence labeled with donor and acceptor fluorophores. Reversible dissociation was induced by increasing NaCl concentration. At least 3 species with different FRET were identified and assigned to structures: (i) the most stable high-FRET species corresponding to the intact nucleosome, (ii) a less stable mid-FRET species that we attribute to a first intermediate with a partially unwrapped DNA and less histones, and (iii) a low-FRET species characterized by a very broad FRET distribution, representing highly unwrapped structures and free DNA formed at the expense of the other 2 species. Selective FCS analysis indicates that even in the low-FRET state, some histones are still bound to the DNA. The interdye distance of 54.0 A measured for the high-FRET species corresponds to a compact conformation close to the known crystallographic structure. The coexistence and interconversion of these species is first demonstrated under non-invasive conditions. A geometric model of the DNA unwinding predicts the presence of the observed FRET species. The different structures of these species in the disassembly pathway map the energy landscape indicating major barriers for 10-bp and minor ones for 5-bp DNA unwinding steps. PMID:19706432

Gansen, Alexander; Valeri, Alessandro; Hauger, Florian; Felekyan, Suren; Kalinin, Stanislav; Tóth, Katalin; Langowski, Jörg; Seidel, Claus A M



Novel polymer linkers for single molecule AFM force spectroscopy.  


Flexible polymer linkers play an important role in various imaging and probing techniques that require surface immobilization, including atomic force microscopy (AFM). In AFM force spectroscopy, polymer linkers are necessary for the covalent attachment of molecules of interest to the AFM tip and the surface. The polymer linkers tether the molecules and provide their proper orientation in probing experiments. Additionally, the linkers separate specific interactions from nonspecific short-range adhesion and serve as a reference point for the quantitative analysis of single molecule probing events. In this report, we present our results on the synthesis and testing of a novel polymer linker and the identification of a number of potential applications for its use in AFM force spectroscopy experiments. The synthesis of the linker is based on the well-developed phosphoramidate (PA) chemistry that allows the routine synthesis of linkers with predetermined lengths and PA composition. These linkers are homogeneous in length and can be terminated with various functional groups. PA linkers with different functional groups were synthesized and tested in experimental systems utilizing different immobilization chemistries. We probed interactions between complementary DNA oligonucleotides; DNA and protein complexes formed by the site-specific binding protein SfiI; and interactions between amyloid peptide (A?42). The results of the AFM force spectroscopy experiments validated the feasibility of the proposed approach for the linker design and synthesis. Furthermore, the properties of the tether (length, functional groups) can be adjusted to meet the specific requirements for different force spectroscopy experiments and system characteristics, suggesting that it could be used for a large number of various applications. PMID:23624104

Tong, Zenghan; Mikheikin, Andrey; Krasnoslobodtsev, Alexey; Lv, Zhengjian; Lyubchenko, Yuri L



Single-molecule enzyme kinetics in the presence of inhibitors  

NASA Astrophysics Data System (ADS)

Recent studies in single-molecule enzyme kinetics reveal that the turnover statistics of a single enzyme is governed by the waiting time distribution that decays as mono-exponential at low substrate concentration and multi-exponential at high substrate concentration. The multi-exponentiality arises due to protein conformational fluctuations, which act on the time scale longer than or comparable to the catalytic reaction step, thereby inducing temporal fluctuations in the catalytic rate resulting in dynamic disorder. In this work, we study the turnover statistics of a single enzyme in the presence of inhibitors to show that the multi-exponentiality in the waiting time distribution can arise even when protein conformational fluctuations do not influence the catalytic rate. From the Michaelis-Menten mechanism of inhibited enzymes, we derive exact expressions for the waiting time distribution for competitive, uncompetitive, and mixed inhibitions to quantitatively show that the presence of inhibitors can induce dynamic disorder in all three modes of inhibitions resulting in temporal fluctuations in the reaction rate. In the presence of inhibitors, dynamic disorder arises due to transitions between active and inhibited states of enzymes, which occur on time scale longer than or comparable to the catalytic step. In this limit, the randomness parameter (dimensionless variance) is greater than unity indicating the presence of dynamic disorder in all three modes of inhibitions. In the opposite limit, when the time scale of the catalytic step is longer than the time scale of transitions between active and inhibited enzymatic states, the randomness parameter is unity, implying no dynamic disorder in the reaction pathway.

Saha, Soma; Sinha, Antara; Dua, Arti



Efficient unfolding pattern recognition in single molecule force spectroscopy data  

PubMed Central

Background Single-molecule force spectroscopy (SMFS) is a technique that measures the force necessary to unfold a protein. SMFS experiments generate Force-Distance (F-D) curves. A statistical analysis of a set of F-D curves reveals different unfolding pathways. Information on protein structure, conformation, functional states, and inter- and intra-molecular interactions can be derived. Results In the present work, we propose a pattern recognition algorithm and apply our algorithm to datasets from SMFS experiments on the membrane protein bacterioRhodopsin (bR). We discuss the unfolding pathways found in bR, which are characterised by main peaks and side peaks. A main peak is the result of the pairwise unfolding of the transmembrane helices. In contrast, a side peak is an unfolding event in the alpha-helix or other secondary structural element. The algorithm is capable of detecting side peaks along with main peaks. Therefore, we can detect the individual unfolding pathway as the sequence of events labeled with their occurrences and co-occurrences special to bR's unfolding pathway. We find that side peaks do not co-occur with one another in curves as frequently as main peaks do, which may imply a synergistic effect occurring between helices. While main peaks co-occur as pairs in at least 50% of curves, the side peaks co-occur with one another in less than 10% of curves. Moreover, the algorithm runtime scales well as the dataset size increases. Conclusions Our algorithm satisfies the requirements of an automated methodology that combines high accuracy with efficiency in analyzing SMFS datasets. The algorithm tackles the force spectroscopy analysis bottleneck leading to more consistent and reproducible results.



Photonic Methods to Enhance Fluorescence Correlation Spectroscopy and Single Molecule Fluorescence Detection  

PubMed Central

Recent advances in nanophotonics open the way for promising applications towards efficient single molecule fluorescence analysis. In this review, we discuss how photonic methods bring innovative solutions for two essential questions: how to detect a single molecule in a highly concentrated solution, and how to enhance the faint optical signal emitted per molecule? The focus is set primarily on the widely used technique of fluorescence correlation spectroscopy (FCS), yet the discussion can be extended to other single molecule detection methods.

Wenger, Jerome; Rigneault, Herve




Microsoft Academic Search

The 2010 Gordon Conference on Single-Molecule Approaches to Biology focuses on cutting-edge research in single-molecule science. Tremendous technical developments have made it possible to detect, identify, track, and manipulate single biomolecules in an ambient environment or even in a live cell. Single-molecule approaches have changed the way many biological problems are addressed, and new knowledge derived from these approaches continues

William Moerner



A CMOS enhanced solid-state nanopore based single molecule detection platform.  


Solid-state nanopores have emerged as a single molecule label-free electronic detection platform. Existing transimpedance stages used to measure ionic current nanopores suffer from dynamic range limitations resulting from steady-state baseline currents. We propose a digitally-assisted baseline cancellation CMOS platform that circumvents this issue. Since baseline cancellation is a form of auto-zeroing, the 1/f noise of the system is also reduced. Our proposed design can tolerate a steady state baseline current of 10µA and has a usable bandwidth of 750kHz. Quantitative DNA translocation experiments on 5kbp DNA was performed using a 5nm silicon nitride pore using both the CMOS platform and a commercial system. Comparison of event-count histograms show that the CMOS platform clearly outperforms the commercial system, allowing for unambiguous interpretation of the data. PMID:24109650

Chen, Chinhsuan; Yemenicioglu, Sukru; Uddin, Ashfaque; Corgliano, Ellie; Theogarajan, Luke



Spectroscopy and single-molecule emission of a fluorene-terthiophene oligomer.  


We study the thiophene-based oligomer poly[2,7-(9,9-bis(2'-ethylhexyl)fluorene)-alt-2,5-terthiophene] (PF3T) in solution and when dispersed at low concentration into a polynorbornene matrix. We find that at high concentration in solution the 0-0 electronic transition observed in fluorescence is suppressed, a result indicative of the formation of weakly coupled H-aggregates. At low concentration in a polymer matrix, emission from both single molecules and molecular aggregates is observed. We find that the fluorescence spectra of most PF3T emitters are composed of a number of relatively narrow emission features, indicating that the emission usually occurs from multiple chromophores. A small number of PF3T molecules are however characterized by single chromophore emission, spectral blinking, and narrowed emission peaks. PMID:21916445

Khalil, G E; Adawi, A M; Robinson, B; Cadby, A J; Tsoi, W C; Kim, J-S; Charas, A; Morgado, J; Lidzey, D G



Frequency domain Fourier transform THz-EPR on single molecule magnets using coherent synchrotron radiation.  


Frequency domain Fourier transform THz electron paramagnetic resonance (FD-FT THz-EPR) based on coherent synchrotron radiation (CSR) is presented as a novel tool to ascertain very large zero field splittings in transition metal ion complexes. A description of the FD-FT THz-EPR at the BESSY II storage ring providing CSR in a frequency range from 5 cm(-1) up to 40 cm(-1) at external magnetic fields from -10 T to +10 T is given together with first measurements on the single molecule magnet Mn(12)Ac where we studied DeltaM(S) = +/-1 spin transition energies as a function of the external magnetic field and temperature. PMID:19639156

Schnegg, Alexander; Behrends, Jan; Lips, Klaus; Bittl, Robert; Holldack, Karsten



Single-Molecule Tracking in Living Cells Using Single Quantum Dot Applications  

PubMed Central

Revealing the behavior of single molecules in living cells is very useful for understanding cellular events. Quantum dot probes are particularly promising tools for revealing how biological events occur at the single molecule level both in vitro and in vivo. In this review, we will introduce how single quantum dot applications are used for single molecule tracking. We will discuss how single quantum dot tracking has been used in several examples of complex biological processes, including membrane dynamics, neuronal function, selective transport mechanisms of the nuclear pore complex, and in vivo real-time observation. We also briefly discuss the prospects for single molecule tracking using advanced probes.

Baba, Koichi; Nishida, Kohji



Photophysical Properties of Acene DCDHF Fluorophores: Long-Wavelength Single-Molecule Emitters Designed for Cellular Imaging  

PubMed Central

We report the solvatochromic, viscosity-sensitive, and single-molecule photophysics of the fluorophores DCDHF-N-6 and DCDHF-A-6. These molecules are members of the dicyanomethylenedihydrofuran (DCDHF) class of single-molecule emitters that contain an amine electron donor and a DCDHF acceptor linked by a conjugated unit; DCDHF-N-6 and DCDHF-A-6 have naphthalene- and anthracene-conjugated linkers, respectively. These molecules maintain the beneficial photophysics of the phenylene-linked DCDHF (i.e., photostability, emission wavelength dependence on solvent polarity, and quantum yield sensitivity to solvent viscosity), yet offer absorption and emission at longer wavelengths that are more appropriate for cellular imaging. We demonstrate that these new fluorophores are less photolabile in an aqueous environment than several other commonly used dyes (rhodamine 6G, Texas Red, and fluorescein). Finally, we image single copies of the acene DCDHFs diffusing in the plasma membrane of living cells.

Lord, Samuel J.; Lu, Zhikuan; Wang, Hui; Willets, Katherine A.; Schuck, P. James; Lee, Hsiao-lu D.; Nishimura, Stefanie Y.; Twieg, Robert J.; Moerner, W. E.



Characterization of interfacial thermal resistance for packaging high-power electronics modules  

Microsoft Academic Search

In high-power electronics modules, the heat generated by the power devices is transferred to the ambient environment by attaching a heat spreader to the semiconductor package. Once the heat spreader is selected, it has to be attached optimally to the semiconductor package to ensure efficient thermal management through the thermal interface. In most power electronics modules, solder or thermally conductive

Shatil Haque; Guo-Quan Lu



Improved fabrication of zero-mode waveguides for single-molecule detection  

NASA Astrophysics Data System (ADS)

Metallic subwavelength apertures can be used in epi-illumination fluorescence to achieve focal volume confinement. Because of the near field components inherent to small metallic structures, observation volumes are formed that are much smaller than the conventional diffraction limited volume attainable by high numerical aperture far field optics (circa a femtoliter). Observation volumes in the range of 10-4 fl have been reported previously. Such apertures can be used for single-molecule detection at relatively high concentrations (up to 20 ?M) of fluorophores. Here, we present a novel fabrication of metallic subwavelength apertures in the visible range. Using a new electron beam lithography process, uniform arrays of such apertures can be manufactured efficiently in large numbers with diameters in the range of 60-100 nm. The apertures were characterized by scanning electron microscopy, optical microscopy, focused ion beam cross sections/transmission electron microscopy, and fluorescence correlation spectroscopy measurements, which confirmed their geometry and optical confinement. Process throughput can be further increased using deep ultraviolet photolithography to replace electron beam lithography. This enables the production of aperture arrays in a high volume manufacturing environment.

Foquet, Mathieu; Samiee, Kevan T.; Kong, Xiangxu; Chauduri, Bidhan P.; Lundquist, Paul M.; Turner, Stephen W.; Freudenthal, Jake; Roitman, Daniel B.



Single molecule kinetics of ENTH binding to lipid membranes.  


Transient recruitment of proteins to membranes is a fundamental mechanism by which the cell exerts spatial and temporal control over proteins' localization and interactions. Thus, the specificity and the kinetics of peripheral proteins' membrane residence are an attribute of their function. Here, we describe the membrane interactions of the interfacial epsin N-terminal homology (ENTH) domain with its target lipid phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P(2)). The direct visualization and quantification of interactions of single ENTH molecules with supported lipid bilayers is achieved using total internal reflection fluorescence microscopy (TIRFM) with a time resolution of 13 ms. This enables the recording of the kinetic behavior of ENTH interacting with membranes with physiologically relevant concentrations of PtdIns(4,5)P(2) despite the low effective binding affinity. Subsequent single fluorophore tracking permits us to build up distributions of residence times and to measure ENTH dissociation rates as a function of membrane composition. Furthermore, due to the high time resolution, we are able to resolve details of the motion of ENTH associated with a simple, homogeneous membrane. In this case ENTH's diffusive transport appears to be the result of at least three different diffusion processes. PMID:22471245

Rozovsky, Sharon; Forstner, Martin B; Sondermann, Holger; Groves, Jay T



Moving into the cell: single-molecule studies of molecular motors in complex environments  

Microsoft Academic Search

Much has been learned in the past decades about molecular force generation. Single-molecule techniques, such as atomic force microscopy, single-molecule fluorescence microscopy and optical tweezers, have been key in resolving the mechanisms behind the power strokes, 'processive' steps and forces of cytoskeletal motors. However, it remains unclear how single force generators are integrated into composite mechanical machines in cells to

Claudia Veigel; Christoph F. Schmidt



Excitation of a single molecule on the surface of a spherical microcavity  

Microsoft Academic Search

We use the optical resonance of a spherical microcavity (quality factor ?106) to excite a single molecule. By attaching a p-terphenyl crystal doped with pentacene and terrylene molecules to a dielectric sphere, we detect individual molecules excited by the near field of the cavity. A low-temperature optical microscope is utilized to image the emission from the single molecule, determine its

D. J. Norris; M. Kuwata-Gonokami; W. E. Moerner



SingleParticle Tracking Photoactivated Localization Microscopy for Mapping Single-Molecule Dynamics  

Microsoft Academic Search

Recent developments in single-molecule localization techniques using photoactivatable fluorescent proteins have allowed the probing of single-molecule motion in a living cell with high specificity, millisecond time resolution, and nanometer spatial resolution. Analyzing the dynamics of individual molecules at high densities in this manner promises to provide new insights into the mechanisms of many biological processes, including protein heterogeneity in the

Suliana Manley; Jennifer M. Gillette; Jennifer Lippincott-Schwartz



PET-FCS: Probing Rapid Structural Fluctuations of Proteins and Nucleic Acids by Single-Molecule Fluorescence Quenching.  


Quenching of organic fluorophores by aromatic amino acids and DNA nucleotides with expelled electron donating properties allows the study of conformational dynamics of biomolecules. Efficient fluorescence quenching via photoinduced electron transfer (PET) requires van der Waals contact and can be used as reporter for structural fluctuations at the 1-nm scale in proteins, peptides, and nucleic acids. The combination of PET with fluorescence correlation spectroscopy (FCS) establishes a powerful method (PET-FCS) to study equilibrium dynamics at the single-molecule level on time scales from nano- to milliseconds. We delineate the fundamentals of PET-based fluorescence quenching, reporter engineering, instrumental and experimental design, and provide examples. PMID:24108646

Sauer, Markus; Neuweiler, Hannes



Integration of biological ion channels onto optically addressable micro-fluidic electrode arrays for single molecule characterization.  

SciTech Connect

The challenge of modeling the organization and function of biological membranes on a solid support has received considerable attention in recent years, primarily driven by potential applications in biosensor design. Affinity-based biosensors show great promise for extremely sensitive detection of BW agents and toxins. Receptor molecules have been successfully incorporated into phospholipid bilayers supported on sensing platforms. However, a collective body of data detailing a mechanistic understanding of membrane processes involved in receptor-substrate interactions and the competition between localized perturbations and delocalized responses resulting in reorganization of transmembrane protein structure, has yet to be produced. This report describes a systematic procedure to develop detailed correlation between (recognition-induced) protein restructuring and function of a ligand gated ion channel by combining single molecule fluorescence spectroscopy and single channel current recordings. This document is divided into three sections: (1) reported are the thermodynamics and diffusion properties of gramicidin using single molecule fluorescence imaging and (2) preliminary work on the 5HT{sub 3} serotonin receptor. Thirdly, we describe the design and fabrication of a miniaturized platform using the concepts of these two technologies (spectroscopic and single channel electrochemical techniques) for single molecule analysis, with a longer term goal of using the physical and electronic changes caused by a specific molecular recognition event as a transduction pathway in affinity based biosensors for biotoxin detection.

Brozik, Susan Marie; Frink, Laura J. Douglas; Bachand, George David; Keller, David J. (University of New Mexico, Albuquerque, NM); Patrick, Elizabeth L.; Marshall, Jason A. (University of New Mexico, Albuquerque, NM); Ortiz, Theodore P. (University of New Mexico, Albuquerque, NM); Meyer, Lauren A. (University of New Mexico, Albuquerque, NM); Davis, Ryan W. (University of New Mexico, Albuquerque, NM); Brozik, James A. (University of New Mexico, Albuquerque, NM); Flemming, Jeb Hunter



Manipulating Protein Conformations by Single-Molecule AFM-FRET Nanoscopy  

PubMed Central

Combining atomic force microscopy and fluorescence resonance energy transfer spectroscopy (AFM-FRET), we have developed a single-molecule AFM-FRET nanoscopy approach capable of effectively pinpointing and mechanically manipulating a targeted dye-labeled single protein in a large sampling area, and simultaneously monitoring the conformational changes of the targeted protein by recording single-molecule FRET time trajectories. We have further demonstrated an application of using this nanoscopy on manipulation of single-molecule protein conformation and simultaneous single-molecule FRET measurement of a Cy3–Cy5 labeled kinase enzyme, HPPK (6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase). By analyzing time-resolved FRET trajectories and correlated AFM force pulling curves of the targeted single-molecule enzyme, we are able to observe the protein conformational changes of a specific coordination by AFM mechanic force pulling.

He, Yufan; Lu, Maolin; Cao, Jin; Lu, H. Peter



TOPICAL REVIEW: Surfing on a new wave of single-molecule fluorescence methods  

NASA Astrophysics Data System (ADS)

Single-molecule fluorescence microscopy is currently one of the most popular methods in the single-molecule toolbox. In this review, we discuss recent advances in fluorescence instrumentation and assays: these methods are characterized by a substantial increase in complexity of the instrumentation or biological samples involved. Specifically, we describe new multi-laser and multi-colour fluorescence spectroscopy and imaging techniques, super-resolution microscopy imaging and the development of instruments that combine fluorescence detection with other single-molecule methods such as force spectroscopy. We also highlight two pivotal developments in basic and applied biosciences: the new information available from detection of single molecules in single biological cells and exciting developments in fluorescence-based single-molecule DNA sequencing.

Hohlbein, Johannes; Gryte, Kristofer; Heilemann, Mike; Kapanidis, Achillefs N.



Directional Raman scattering from single molecules in the feed gaps of optical antennas.  


Controlling light from single emitters is an overarching theme of nano-optics. Antennas are routinely used to modify the angular emission patterns of radio wave sources. "Optical antennas" translate these principles to visible and infrared wavelengths and have been recently used to modify fluorescence from single quantum dots and single molecules. Understanding the properties of single molecules, however, would be advanced were one able to observe their vibrational spectra through Raman scattering in a very reproducible manner but it is a hugely challenging task, as Raman scattering cross sections are very weak. Here we measure for the first time the highly directional emission patterns of Raman scattering from single molecules in the feed gaps of optical antennas fabricated on a chip. More than a thousand single molecule events are observed, revealing that an unprecedented near-unity fraction of optical antennas have single molecule sensitivity. PMID:23550513

Wang, Dongxing; Zhu, Wenqi; Best, Michael D; Camden, Jon P; Crozier, Kenneth B



Kinetics of interfacial electron transfer at single-layer graphene electrodes in aqueous and nonaqueous solutions.  


We present a catalog of electron transfer mediators for investigating the heterogeneous electron transfer kinetics of large-area, single-layer graphene electrodes. Scanning electrochemical microscopy (SECM) was used to probe the apparent standard electron transfer rate constant of mediators in aqueous solutions and in acetonitrile and dimethylformamide, allowing for studies of graphene electroactivity at different potentials and in both aqueous and nonaqueous media. In aqueous solution, iron(III) ethylenediaminetetraacetic acid, hexacyanoruthenate(II), hexacyanoferrate(II), hexacyanoferrate(III), octacyanomalybdate(IV), cobalt(III) sepulchrate, and hydroxymethylferrocene exhibited quasi-reversible electron transfer behavior. The electron transfer kinetics of hexaammineruthenium(III), methyl viologen, and tris(2,2'-bipyridyl)ruthenium(II) were found to be reversible in these studies. The electron transfer rate constant of hydroxymethylferrocene and ferrocene, in organic media, was similar to that for hydroxymethylferrocene in water, which, although fast, shows clear kinetic complications that we believe are inherent to graphene. A series of viologens, known to be reversible at metal electrodes, exhibited quasi-reversible electron transfer. For [Co(dapa)(2)](2+), where dapa is 2,6-bis[1-(phenylimino)ethyl]pyridine, in dimethylformamide, the oxidation state of the redox pair investigated affected the observed kinetics. Under similar experimental conditions, the Co(I/II) couple exhibited nearly reversible behavior whereas Co(II/III) had finite kinetics. This behavior was ascribed to the large difference in self-exchange rates for these two processes. To demonstrate the utility of using these mediators for examining graphene electrodes, the kinetics of two mediators with quasi-reversible electron transfer behavior, iron ethylenediaminetetraacetic acid and hexacyanoruthenate, were measured in the presence of a redox-active species [Os(bpy)(2)(dipy)Cl]PF(6), where bpy is 2,2'-bipyridine and dipy is 4,4'-trimethylenedipyridine, adsorbed onto the graphene surface. The kinetics of both mediators were enhanced in the presence of one-hundredth of a monolayer of the osmium complex, showing that even small amounts of impurities on the graphene surface are capable of enhancing the observed kinetics. PMID:23305445

Ritzert, Nicole L; Rodríguez-López, Joaquín; Tan, Cen; Abruña, Héctor D



Low Temperature STM Manipulation and Spectroscopy of Chlorophyll-a Single Molecules from Spinach  

NASA Astrophysics Data System (ADS)

We interrogate single chlorophyll-a, a molecule produced from Spinach, on Cu(111) surface to check its mechanical stability and electronic properties using an ultra-high-vacuum low-temperature scanning-tunneling-microscope (UHV-LT-STM) at liquid helium temperatures. The measured results of isolated single chlorophyll-a molecules are then compared with that of self-assembled molecular layer. The tunneling I/V and dI/dV spectroscopy techniques are used to probe the electronic properties of the chlorophyll-a molecule with atomic precision (1). These spectroscopic investigations elucidate properties of the single molecule such as the band gap and additional molecular orbital states. Mechanical stability of the chlorophyll-a molecule is examined using lateral manipulation techniques with the STM tip (2). In this procedure, the STM tip is placed in close proximity to the molecule (just a few angstrom separation) to increase the tip-molecule interaction. Then the tip is laterally moved across the surface, which results in pulling of the chlorophyll-a molecule to relocate to the specific surface sites. The detailed molecule movement during this manipulation is directly monitored through the corresponding STM-tip height signals. Our results highlight that the Spinach molecule is a promising candidate for environmental friendly nano-electronic device applications. (1) F. Moresco et al, Phy. Rev. Lett. 86, 672-675, (2001) (2) S-W. Hla, K-H. Rieder, Ann. Phy. Chem. 54, 307-330, (2003)

Benson, Jessica J.; Iancu, Violeta; Deshpande, Aparna; Hla, Saw-Wai



CRADA Final Report for CRADA No. ORNL99-0544, Interfacial Properties of Electron Beam Cured Composites  

SciTech Connect

Electron beam (EB) curing is a technology that promises, in certain applications, to deliver lower cost and higher performance polymer matrix composite (PMC) structures compared to conventional thermal curing processes. PMCs enhance performance by making products lighter, stronger, more durable, and less energy demanding. They are essential in weight- and performance-dominated applications. Affordable PMCs can enhance US economic prosperity and national security. US industry expects rapid implementation of electron beam cured composites in aircraft and aerospace applications as satisfactory properties are demonstrated, and implementation in lower performance applications will likely follow thereafter. In fact, at this time and partly because of discoveries made in this project, field demonstrations are underway that may result in the first fielded applications of electron beam cured composites. Serious obstacles preventing the widespread use of electron beam cured PMCs in many applications are their relatively poor interfacial properties and resin toughness. The composite shear strength and resin toughness of electron beam cured carbon fiber reinforced epoxy composites were about 25% and 50% lower, respectively, than those of thermally cured composites of similar formulations. The essential purpose of this project was to improve the mechanical properties of electron beam cured, carbon fiber reinforced epoxy composites, with a specific focus on composite shear properties for high performance aerospace applications. Many partners, sponsors, and subcontractors participated in this project. There were four government sponsors from three federal agencies, with the US Department of Energy (DOE) being the principal sponsor. The project was executed by Oak Ridge National Laboratory (ORNL), NASA and Department of Defense (DOD) participants, eleven private CRADA partners, and two subcontractors. A list of key project contacts is provided in Appendix A. In order to properly manage the large project team and properly address the various technical tasks, the CRADA team was organized into integrated project teams (IPT's) with each team focused on specific research areas. Early in the project, the end user partners developed ''exit criteria'', recorded in Appendix B, against which the project's success was to be judged. The project team made several important discoveries. A number of fiber coatings or treatments were developed that improved fiber-matrix adhesion by 40% or more, according to microdebond testing. The effects of dose-time and temperature-time profiles during the cure were investigated, and it was determined that fiber-matrix adhesion is relatively insensitive to the irradiation procedure, but can be elevated appreciably by thermal postcuring. Electron beam curable resin properties were improved substantially, with 80% increase in electron beam 798 resin toughness, and {approx}25% and 50% improvement, respectively, in ultimate tensile strength and ultimate tensile strain vs. earlier generation electron beam curable resins. Additionally, a new resin electron beam 800E was developed with generally good properties, and a very notable 120% improvement in transverse composite tensile strength vs. earlier generation electron beam cured carbon fiber reinforced epoxies. Chemical kinetics studies showed that reaction pathways can be affected by the irradiation parameters, although no consequential effects on material properties have been noted to date. Preliminary thermal kinetics models were developed to predict degree of cure vs. irradiation and thermal parameters. These models are continually being refined and validated. Despite the aforementioned impressive accomplishments, the project team did not fully realize the project objectives. The best methods for improving adhesion were combined with the improved electron beam 3K resin to make prepreg and uni-directional test laminates from which composite properties could be determined. Nevertheless, only minor improvements in the composite shear strength, and moderate improvements i

Janke, C.J.



Preparation of zinc sulfide nanocrystallites from single-molecule precursors  

Microsoft Academic Search

Zinc sulfide nanocrystallites were prepared using Zinc(II) thiosemicarbazone complexes of the types Zn(L)2 and ZnCl2(LH)2 (where, LH=thiosemicarbazones of cinnamaldehyde, 4-chlorobenzaldehyde, indol-3-carboxaldehyde and thiophene-2-carboxaldehyde) as single source precursors by solvothermal decomposition in ethylene glycol and ethylene diamine in few cases. The materials were characterized by powder X-ray diffraction (XRD), transmission electron microscopy (TEM), selected area electron diffraction, energy dispersive X-ray analysis

Anil M. Palve; Shivram S. Garje



Interfacial thermal resistance and temperature dependence of three adhesives for electronic packaging  

Microsoft Academic Search

The thermal resistance and its temperature dependence was measured for three industrial adhesives used for electronic packaging. Measurements were made by the laser-flash method from room temperature to 300°C. The samples were in the form of sandwiches consisting of two platelets of silicon carbide-reinforced aluminum (AlSiC) bonded together with the adhesives. The total thermal resistance of the bond (the sum

D. P. H. Hasselman; Kimberly Y. Donaldson; Fred D. Barlow; Aicha A. Elshabini; Gerhard H. Schiroky; Josh P. Yaskoff; Raymond L. Dietz



Single-molecule photon stamping FRET spectroscopy study of enzymatic conformational dynamics.  


The fluorescence resonant energy transfer (FRET) from a donor to an acceptor via transition dipole-dipole interactions decreases the donor's fluorescent lifetime. The donor's fluorescent lifetime decreases as the FRET efficiency increases, following the equation: E(FRET) = 1 - ?(DA)/?(D), where ?(D) and ?(DA) are the donor fluorescence lifetime without FRET and with FRET. Accordingly, the FRET time trajectories associated with single-molecule conformational dynamics can be recorded by measuring the donor's lifetime fluctuations. In this article, we report our work on the use of a Cy3/Cy5-labeled enzyme, HPPK to demonstrate probing single-molecule conformational dynamics in an enzymatic reaction by measuring single-molecule FRET donor lifetime time trajectories. Compared with single-molecule fluorescence intensity-based FRET measurements, single-molecule lifetime-based FRET measurements are independent of fluorescence intensity. The latter has an advantage in terms of eliminating the analysis background noise from the acceptor fluorescence detection leak through noise, excitation light intensity noise, or light scattering noise due to local environmental factors, for example, in a AFM-tip correlated single-molecule FRET measurements. Furthermore, lifetime-based FRET also supports simultaneous single-molecule fluorescence anisotropy. PMID:23085845

He, Yufan; Lu, Maolin; Lu, H Peter



A quantum digital half adder inside a single molecule  

NASA Astrophysics Data System (ADS)

A dinitro[1,3]anthracene molecule is shown to perform an half adder logic function with no resemblance to the topology of an electronic circuit performing the same function. The logic function is obtained by the control of the molecule quantum trajectory in its molecular orbitals electronic quantum state space by changing the conformation of the NO 2 groups, starting from an initial non-stationary state independent of the input configuration. Time dependent quantum interference effects controlled by the relative position and degeneracy of the molecule eigenstates is at the origin of this half adder logic gate.

Duchemin, I.; Joachim, C.



Do-it-yourself guide: How to use the modern single molecule toolkit  

PubMed Central

Single molecule microscopy has evolved into the ultimate-sensitivity toolkit to study systems from small molecules to living cells, with the prospect of revolutionizing the modern biosciences. Here we survey the current state-of-the-art in single molecule tools including fluorescence spectroscopy, tethered particle microscopy, optical and magnetic tweezers, and atomic force microscopy. Our review seeks to guide the biological scientist in choosing the right approach from the available single molecule toolkit for applications ranging as far as structural biology, enzymology, nanotechnology, and systems biology.

Walter, Nils G.; Huang, Cheng-Yen; Manzo, Anthony J.; Sobhy, Mohamed A.



Experimental approaches for addressing fundamental biological questions in living, functioning cells with single molecule precision  

PubMed Central

In recent years, single molecule experimentation has allowed researchers to observe biological processes at the sensitivity level of single molecules in actual functioning, living cells, thereby allowing us to observe the molecular basis of the key mechanistic processes in question in a very direct way, rather than inferring these from ensemble average data gained from traditional molecular and biochemical techniques. In this short review, we demonstrate the impact that the application of single molecule bioscience experimentation has had on our understanding of various cellular systems and processes, and the potential that this approach has for the future to really address very challenging and fundamental questions in the life sciences.

Lenn, Tchern; Leake, Mark C.



Central dogma at the single-molecule level in living cells  

PubMed Central

Gene expression originates from individual DNA molecules within living cells. Like many single-molecule processes, gene expression and regulation are stochastic, that is, sporadic in time. This leads to heterogeneity in the messenger RNA and protein copy numbers in a population of cells with identical genomes. With advanced single-cell fluorescence microscopy, it is now possible to quantify transcriptomes and proteomes with single-molecule sensitivity. Dynamic processes such as transcription factor binding, transcription and translation can be monitored in real time, providing quantitative descriptions of gene expression and regulation, and the demonstration that a single-molecule event can determine the phenotype of a cell.

Li, Gene-Wei; Xie, X. Sunney



Development of new microscope unit for single molecule spectroscopy under various ambient conditions  

NASA Astrophysics Data System (ADS)

This paper introduces techniques we previously developed for single molecule spectroscopy and continues on to describe our studies on dipole orientation imaging of single molecules under various ambient conditions. In these studies, we successfully obtained defocused images of single perylene diimide (PDI) molecules under air, high-vacuum, and pure N2 gas conditions by utilizing the advantages of our new microscope unit. The studies are positioned as one of the important applications of our microscope unit for single molecule spectroscopy. We expect a wide range of applications for this unit for various microscope measurements for many types of materials.

Yamada, T.; Kaji, T.; Ueda, R.; Otomo, A.



Fabrication of metallized nanopores in silicon nitride membranes for single-molecule sensing.  


The fabrication and characterization of a metallized nanopore structure for the sensing of single molecules is described. Pores of varying diameters (>10 nm) are patterned into free-standing silicon nitride membranes by electron-beam lithography and reactive ion etching. Structural characterization by transmission electron microscopy (TEM) and tomography reveals a conical pore shape with a 40 degrees aperture. Metal films of Ti/Au are vapor deposited and the pore shape and shrinking are studied as a function of evaporated film thickness. TEM tomography analysis confirms metalization of the inner pore walls as well as conservation of the conical pore shape. In electrical measurements of the transpore current in aqueous electrolyte solution, the pores feature very low noise. The applicability of the metallized pores for stochastic sensing is demonstrated in real-time translocation experiments of single lambda-DNA molecules. We observe exceptionally long-lasting current blockades with a fine structure of distinct current levels, suggesting an attractive interaction between the DNA and the PEGylated metallic pore walls. PMID:20564484

Wei, Ruoshan; Pedone, Daniel; Zürner, Andreas; Döblinger, Markus; Rant, Ulrich



Effects of magnetic field and transverse anisotropy on full counting statistics in single-molecule magnet  

NASA Astrophysics Data System (ADS)

We have theoretically studied the full counting statistics of electron transport through a single-molecule magnet (SMM) with an arbitrary angle between the applied magnetic field and the SMM's easy axis above the sequential tunneling threshold, since the angle ? cannot be controlled in present-day SMM experiments. In the absence of the small transverse anisotropy, when the coupling of the SMM with the incident-electrode is stronger than that with the outgoing-electrode, i.e., ?L/?R>>1, the maximum peak of shot noise first increases and then decreases with increasing ? from 0 to 0.5?. In particular, the shot noise can reach up to a super-Poissonian value from a sub-Poissonian value when considering the small transverse anisotropy. For ?L/?R<<1, the maximum peaks of the shot noise and skewness can be reduced from a super-Poissonian to a sub-Poissonian value with increasing ? from 0 to 0.5? the super-Poissonian behavior of the skewness is more sensitive to the small ? than shot noise, which is suppressed when taking into account the small transverse anisotropy. These characteristics of shot noise can be qualitatively attributed to the competition between the fast and slow transport channels. The predictions regarding the ?- dependence of high order current cumulants are very interesting for a better understanding of electron transport through SMM, and will allow for experimental tests in the near future.

Xue, Hai-Bin; Nie, Y.-H.; Li, Z.-J.; Liang, J.-Q.



Molecular Quantum Spintronics: Supramolecular Spin Valves Based on Single-Molecule Magnets and Carbon Nanotubes  

PubMed Central

We built new hybrid devices consisting of chemical vapor deposition (CVD) grown carbon nanotube (CNT) transistors, decorated with TbPc2 (Pc = phthalocyanine) rare-earth based single-molecule magnets (SMMs). The drafting was achieved by tailoring supramolecular ?-? interactions between CNTs and SMMs. The magnetoresistance hysteresis loop measurements revealed steep steps, which we can relate to the magnetization reversal of individual SMMs. Indeed, we established that the electronic transport properties of these devices depend strongly on the relative magnetization orientations of the grafted SMMs. The SMMs are playing the role of localized spin polarizer and analyzer on the CNT electronic conducting channel. As a result, we measured magneto-resistance ratios up to several hundred percent. We used this spin valve effect to confirm the strong uniaxial anisotropy and the superparamagnetic blocking temperature (TB ~ 1 K) of isolated TbPc2 SMMs. For the first time, the strength of exchange interaction between the different SMMs of the molecular spin valve geometry could be determined. Our results introduce a new design for operable molecular spintronic devices using the quantum effects of individual SMMs.

Urdampilleta, Matias; Nguyen, Ngoc-Viet; Cleuziou, Jean-Pierre; Klyatskaya, Svetlana; Ruben, Mario; Wernsdorfer, Wolfgang