Simultaneous monitoring of the two coupled motors of a single FoF1-ATP synthase by three-color FRET using duty cycle-optimized triple-ALEX

Science.gov (United States)

Zarrabi, N.; Ernst, S.; Düser, M. G.; Golovina-Leiker, A.; Becker, W.; Erdmann, R.; Dunn, S. D.; Börsch, M.

2009-02-01

FoF1-ATP synthase is the enzyme that provides the 'chemical energy currency' adenosine triphosphate, ATP, for living cells. The formation of ATP is accomplished by a stepwise internal rotation of subunits within the enzyme. Briefly, proton translocation through the membrane-bound Fo part of ATP synthase drives a 10-step rotary motion of the ring of c subunits with respect to the non-rotating subunits a and b. This rotation is transmitted to the γ and ɛ subunits of the F1 sector resulting in 120° steps. In order to unravel this symmetry mismatch we monitor subunit rotation by a single-molecule fluorescence resonance energy transfer (FRET) approach using three fluorophores specifically attached to the enzyme: one attached to the F1 motor, another one to the Fo motor, and the third one to a non-rotating subunit. To reduce photophysical artifacts due to spectral fluctuations of the single fluorophores, a duty cycle-optimized alternating three-laser scheme (DCO-ALEX) has been developed. Simultaneous observation of the stepsizes for both motors allows the detection of reversible elastic deformations between the rotor parts of Fo and F1.

Parallel multispot smFRET analysis using an 8-pixel SPAD array

Science.gov (United States)

Ingargiola, A.; Colyer, R. A.; Kim, D.; Panzeri, F.; Lin, R.; Gulinatti, A.; Rech, I.; Ghioni, M.; Weiss, S.; Michalet, X.

2012-02-01

Single-molecule Förster resonance energy transfer (smFRET) is a powerful tool for extracting distance information between two fluorophores (a donor and acceptor dye) on a nanometer scale. This method is commonly used to monitor binding interactions or intra- and intermolecular conformations in biomolecules freely diffusing through a focal volume or immobilized on a surface. The diffusing geometry has the advantage to not interfere with the molecules and to give access to fast time scales. However, separating photon bursts from individual molecules requires low sample concentrations. This results in long acquisition time (several minutes to an hour) to obtain sufficient statistics. It also prevents studying dynamic phenomena happening on time scales larger than the burst duration and smaller than the acquisition time. Parallelization of acquisition overcomes this limit by increasing the acquisition rate using the same low concentrations required for individual molecule burst identification. In this work we present a new two-color smFRET approach using multispot excitation and detection. The donor excitation pattern is composed of 4 spots arranged in a linear pattern. The fluorescent emission of donor and acceptor dyes is then collected and refocused on two separate areas of a custom 8-pixel SPAD array. We report smFRET measurements performed on various DNA samples synthesized with various distances between the donor and acceptor fluorophores. We demonstrate that our approach provides identical FRET efficiency values to a conventional single-spot acquisition approach, but with a reduced acquisition time. Our work thus opens the way to high-throughput smFRET analysis on freely diffusing molecules.

Multispot single-molecule FRET: High-throughput analysis of freely diffusing molecules.

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Antonino Ingargiola

Full Text Available We describe an 8-spot confocal setup for high-throughput smFRET assays and illustrate its performance with two characteristic experiments. First, measurements on a series of freely diffusing doubly-labeled dsDNA samples allow us to demonstrate that data acquired in multiple spots in parallel can be properly corrected and result in measured sample characteristics consistent with those obtained with a standard single-spot setup. We then take advantage of the higher throughput provided by parallel acquisition to address an outstanding question about the kinetics of the initial steps of bacterial RNA transcription. Our real-time kinetic analysis of promoter escape by bacterial RNA polymerase confirms results obtained by a more indirect route, shedding additional light on the initial steps of transcription. Finally, we discuss the advantages of our multispot setup, while pointing potential limitations of the current single laser excitation design, as well as analysis challenges and their solutions.

Click strategies for single-molecule protein fluorescence.

Science.gov (United States)

Milles, Sigrid; Tyagi, Swati; Banterle, Niccolò; Koehler, Christine; VanDelinder, Virginia; Plass, Tilman; Neal, Adrian P; Lemke, Edward A

2012-03-21

Single-molecule methods have matured into central tools for studies in biology. Foerster resonance energy transfer (FRET) techniques, in particular, have been widely applied to study biomolecular structure and dynamics. The major bottleneck for a facile and general application of these studies arises from the need to label biological samples site-specifically with suitable fluorescent dyes. In this work, we present an optimized strategy combining click chemistry and the genetic encoding of unnatural amino acids (UAAs) to overcome this limitation for proteins. We performed a systematic study with a variety of clickable UAAs and explored their potential for high-resolution single-molecule FRET (smFRET). We determined all parameters that are essential for successful single-molecule studies, such as accessibility of the probes, expression yield of proteins, and quantitative labeling. Our multiparameter fluorescence analysis allowed us to gain new insights into the effects and photophysical properties of fluorescent dyes linked to various UAAs for smFRET measurements. This led us to determine that, from the extended tool set that we now present, genetically encoding propargyllysine has major advantages for state-of-the-art measurements compared to other UAAs. Using this optimized system, we present a biocompatible one-step dual-labeling strategy of the regulatory protein RanBP3 with full labeling position freedom. Our technique allowed us then to determine that the region encompassing two FxFG repeat sequences adopts a disordered but collapsed state. RanBP3 serves here as a prototypical protein that, due to its multiple cysteines, size, and partially disordered structure, is not readily accessible to any of the typical structure determination techniques such as smFRET, NMR, and X-ray crystallography.

Graphical models for inferring single molecule dynamics

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Gonzalez Ruben L

2010-10-01

Full Text Available Abstract Background The recent explosion of experimental techniques in single molecule biophysics has generated a variety of novel time series data requiring equally novel computational tools for analysis and inference. This article describes in general terms how graphical modeling may be used to learn from biophysical time series data using the variational Bayesian expectation maximization algorithm (VBEM. The discussion is illustrated by the example of single-molecule fluorescence resonance energy transfer (smFRET versus time data, where the smFRET time series is modeled as a hidden Markov model (HMM with Gaussian observables. A detailed description of smFRET is provided as well. Results The VBEM algorithm returns the model’s evidence and an approximating posterior parameter distribution given the data. The former provides a metric for model selection via maximum evidence (ME, and the latter a description of the model’s parameters learned from the data. ME/VBEM provide several advantages over the more commonly used approach of maximum likelihood (ML optimized by the expectation maximization (EM algorithm, the most important being a natural form of model selection and a well-posed (non-divergent optimization problem. Conclusions The results demonstrate the utility of graphical modeling for inference of dynamic processes in single molecule biophysics.

Versatile single-molecule multi-color excitation and detection fluorescence setup for studying biomolecular dynamics

KAUST Repository

Sobhy, M. A.; Elshenawy, M. M.; Takahashi, Masateru; Whitman, B. H.; Walter, N. G.; Hamdan, S. M.

2011-01-01

Single-molecule fluorescence imaging is at the forefront of tools applied to study biomolecular dynamics both in vitro and in vivo. The ability of the single-molecule fluorescence microscope to conduct simultaneous multi-color excitation

A New Theoretical Approach to Single-Molecule Fluorescence Optical Studies of RNA Dynamics

International Nuclear Information System (INIS)

Zhao Xinghai; Shan Guangcun; Bao Shuying

2011-01-01

Single-molecule fluorescence spectroscopy in condensed phases has many important chemical and biological applications. The single-molecule fluorescence measurements contain information about conformational dynamics on a vast range of time scales. Based on the data analysis protocols methodology proposed by X. Sunney Xie, the theoretical study here mainly focuses on the single-molecule studies of single RNA with interconversions among different conformational states, to with a single FRET pair attached. We obtain analytical expressions for fluorescence lifetime correlation functions that relate changes in fluorescence lifetime to the distance-dependent FRET mechanism within the context of the Smoluchowski diffusion model. The present work establishes useful guideline for the single-molecule studies of biomolecules to reveal the complicated folding dynamics of single RNA molecules at nanometer scale.

A rapid, ratiometric, enzyme-free, and sensitive single-step miRNA detection using three-way junction based FRET probes

Science.gov (United States)

Luo, Qingying; Liu, Lin; Yang, Cai; Yuan, Jing; Feng, Hongtao; Chen, Yan; Zhao, Peng; Yu, Zhiqiang; Jin, Zongwen

2018-03-01

MicroRNAs (miRNAs) are single stranded endogenous molecules composed of only 18-24 nucleotides which are critical for gene expression regulating the translation of messenger RNAs. Conventional methods based on enzyme-assisted nucleic acid amplification techniques have many problems, such as easy contamination, high cost, susceptibility to false amplification, and tendency to have sequence mismatches. Here we report a rapid, ratiometric, enzyme-free, sensitive, and highly selective single-step miRNA detection using three-way junction assembled (or self-assembled) FRET probes. The developed strategy can be operated within the linear range from subnanomolar to hundred nanomolar concentrations of miRNAs. In comparison with the traditional approaches, our method showed high sensitivity for the miRNA detection and extreme selectivity for the efficient discrimination of single-base mismatches. The results reveal that the strategy paved a new avenue for the design of novel highly specific probes applicable in diagnostics and potentially in microscopic imaging of miRNAs in real biological environments.

Inter-Dye Distance Distributions Studied by a Combination of Single-Molecule FRET-Filtered Lifetime Measurements and a Weighted Accessible Volume (wAV Algorithm

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Henning Höfig

2014-11-01

Full Text Available Förster resonance energy transfer (FRET is an important tool for studying the structural and dynamical properties of biomolecules. The fact that both the internal dynamics of the biomolecule and the movements of the biomolecule-attached dyes can occur on similar timescales of nanoseconds is an inherent problem in FRET studies. By performing single-molecule FRET-filtered lifetime measurements, we are able to characterize the amplitude of the motions of fluorescent probes attached to double-stranded DNA standards by means of flexible linkers. With respect to previously proposed experimental approaches, we improved the precision and the accuracy of the inter-dye distance distribution parameters by filtering out the donor-only population with pulsed interleaved excitation. A coarse-grained model is employed to reproduce the experimentally determined inter-dye distance distributions. This approach can easily be extended to intrinsically flexible proteins allowing, under certain conditions, to decouple the macromolecule amplitude of motions from the contribution of the dye linkers.

The role of FRET in solar concentrator efficiency and color tunability

Energy Technology Data Exchange (ETDEWEB)

Balaban, Benjamin, E-mail: bbalaban@ucsc.edu; Doshay, Sage; Osborn, Melissa; Rodriguez, Yvonne; Carter, Sue A., E-mail: sacarter@ucsc.edu

2014-02-15

We demonstrate concentration-dependent Förster-type energy transfer in a luminescent solar concentrator (LSC) material containing two high quantum yield laser dyes in a PMMA matrix. FRET heterotransfer is shown to be approximately 50% efficient in the regime of 2×10{sup −3}molal acceptor dye by weight in the host polymer. The two dyes used have been well studied for solar concentrator applications: BASF's Lumogen Red 305, and Exciton Chemical Company's DCM both demonstrate desirable stability, quantum yield, and complementary absorption spectra. We demonstrate how multiple-dye LSC devices employing FRET increase the absorption of air mass 1.5 solar irradiance without affecting the self-absorption properties of the film. Color tunability may be achieved through the addition of additional absorbers while minimizing the impact on waveguide efficiency. -- Highlights: • Förster Resonance Energy Transfer is demonstrated in a two-dye luminescent solar concentrator. • Donor-acceptor pair distance is related to the dye concentration in PMMA. • FRET's benefit to waveguide transport losses and color tunability is discussed.

Fast-NPS-A Markov Chain Monte Carlo-based analysis tool to obtain structural information from single-molecule FRET measurements

Science.gov (United States)

Eilert, Tobias; Beckers, Maximilian; Drechsler, Florian; Michaelis, Jens

2017-10-01

The analysis tool and software package Fast-NPS can be used to analyse smFRET data to obtain quantitative structural information about macromolecules in their natural environment. In the algorithm a Bayesian model gives rise to a multivariate probability distribution describing the uncertainty of the structure determination. Since Fast-NPS aims to be an easy-to-use general-purpose analysis tool for a large variety of smFRET networks, we established an MCMC based sampling engine that approximates the target distribution and requires no parameter specification by the user at all. For an efficient local exploration we automatically adapt the multivariate proposal kernel according to the shape of the target distribution. In order to handle multimodality, the sampler is equipped with a parallel tempering scheme that is fully adaptive with respect to temperature spacing and number of chains. Since the molecular surrounding of a dye molecule affects its spatial mobility and thus the smFRET efficiency, we introduce dye models which can be selected for every dye molecule individually. These models allow the user to represent the smFRET network in great detail leading to an increased localisation precision. Finally, a tool to validate the chosen model combination is provided. Programme Files doi:http://dx.doi.org/10.17632/7ztzj63r68.1 Licencing provisions: Apache-2.0 Programming language: GUI in MATLAB (The MathWorks) and the core sampling engine in C++ Nature of problem: Sampling of highly diverse multivariate probability distributions in order to solve for macromolecular structures from smFRET data. Solution method: MCMC algorithm with fully adaptive proposal kernel and parallel tempering scheme.

Spectroscopic characterization of Venus at the single molecule level.

Science.gov (United States)

David, Charlotte C; Dedecker, Peter; De Cremer, Gert; Verstraeten, Natalie; Kint, Cyrielle; Michiels, Jan; Hofkens, Johan

2012-02-01

Venus is a recently developed, fast maturating, yellow fluorescent protein that has been used as a probe for in vivo applications. In the present work the photophysical characteristics of Venus were analyzed spectroscopically at the bulk and single molecule level. Through time-resolved single molecule measurements we found that single molecules of Venus display pronounced fluctuations in fluorescence emission, with clear fluorescence on- and off-times. These fluorescence intermittencies were found to occupy a broad range of time scales, ranging from milliseconds to several seconds. Such long off-times can complicate the analysis of single molecule counting experiments or single-molecule FRET experiments. This journal is © The Royal Society of Chemistry and Owner Societies 2012

Expectation-maximization of the potential of mean force and diffusion coefficient in Langevin dynamics from single molecule FRET data photon by photon.

Science.gov (United States)

Haas, Kevin R; Yang, Haw; Chu, Jhih-Wei

2013-12-12

The dynamics of a protein along a well-defined coordinate can be formally projected onto the form of an overdamped Lagevin equation. Here, we present a comprehensive statistical-learning framework for simultaneously quantifying the deterministic force (the potential of mean force, PMF) and the stochastic force (characterized by the diffusion coefficient, D) from single-molecule Förster-type resonance energy transfer (smFRET) experiments. The likelihood functional of the Langevin parameters, PMF and D, is expressed by a path integral of the latent smFRET distance that follows Langevin dynamics and realized by the donor and the acceptor photon emissions. The solution is made possible by an eigen decomposition of the time-symmetrized form of the corresponding Fokker-Planck equation coupled with photon statistics. To extract the Langevin parameters from photon arrival time data, we advance the expectation-maximization algorithm in statistical learning, originally developed for and mostly used in discrete-state systems, to a general form in the continuous space that allows for a variational calculus on the continuous PMF function. We also introduce the regularization of the solution space in this Bayesian inference based on a maximum trajectory-entropy principle. We use a highly nontrivial example with realistically simulated smFRET data to illustrate the application of this new method.

Single particle tracking and single molecule energy transfer

CERN Document Server

Bräuchle, Christoph; Michaelis, Jens

2009-01-01

Closing a gap in the literature, this handbook gathers all the information on single particle tracking and single molecule energy transfer. It covers all aspects of this hot and modern topic, from detecting virus entry to membrane diffusion, and from protein folding using spFRET to coupled dye systems, as well recent achievements in the field. Throughout, the first-class editors and top international authors present content of the highest quality, making this a must-have for physical chemists, spectroscopists, molecular physicists and biochemists.

Single cell FRET analysis for the identification of optimal FRET-pairs in Bacillus subtilis using a prototype MEM-FLIM system.

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Ruud G J Detert Oude Weme

Full Text Available Protein-protein interactions can be studied in vitro, e.g. with bacterial or yeast two-hybrid systems or surface plasmon resonance. In contrast to in vitro techniques, in vivo studies of protein-protein interactions allow examination of spatial and temporal behavior of such interactions in their native environment. One approach to study protein-protein interactions in vivo is via Förster Resonance Energy Transfer (FRET. Here, FRET efficiency of selected FRET-pairs was studied at the single cell level using sensitized emission and Frequency Domain-Fluorescence Lifetime Imaging Microscopy (FD-FLIM. For FRET-FLIM, a prototype Modulated Electron-Multiplied FLIM system was used, which is, to the best of our knowledge, the first account of Frequency Domain FLIM to analyze FRET in single bacterial cells. To perform FRET-FLIM, we first determined and benchmarked the best fluorescent protein-pair for FRET in Bacillus subtilis using a novel BglBrick-compatible integration vector. We show that GFP-tagRFP is an excellent donor-acceptor pair for B. subtilis in vivo FRET studies. As a proof of concept, selected donor and acceptor fluorescent proteins were fused using a linker that contained a tobacco etch virus (TEV-protease recognition sequence. Induction of TEV-protease results in loss of FRET efficiency and increase in fluorescence lifetime. The loss of FRET efficiency after TEV induction can be followed in time in single cells via time-lapse microscopy. This work will facilitate future studies of in vivo dynamics of protein complexes in single B. subtilis cells.

Probing Protein Multidimensional Conformational Fluctuations by Single-Molecule Multiparameter Photon Stamping Spectroscopy

Science.gov (United States)

2015-01-01

Conformational motions of proteins are highly dynamic and intrinsically complex. To capture the temporal and spatial complexity of conformational motions and further to understand their roles in protein functions, an attempt is made to probe multidimensional conformational dynamics of proteins besides the typical one-dimensional FRET coordinate or the projected conformational motions on the one-dimensional FRET coordinate. T4 lysozyme hinge-bending motions between two domains along α-helix have been probed by single-molecule FRET. Nevertheless, the domain motions of T4 lysozyme are rather complex involving multiple coupled nuclear coordinates and most likely contain motions besides hinge-bending. It is highly likely that the multiple dimensional protein conformational motions beyond the typical enzymatic hinged-bending motions have profound impact on overall enzymatic functions. In this report, we have developed a single-molecule multiparameter photon stamping spectroscopy integrating fluorescence anisotropy, FRET, and fluorescence lifetime. This spectroscopic approach enables simultaneous observations of both FRET-related site-to-site conformational dynamics and molecular rotational (or orientational) motions of individual Cy3-Cy5 labeled T4 lysozyme molecules. We have further observed wide-distributed rotational flexibility along orientation coordinates by recording fluorescence anisotropy and simultaneously identified multiple intermediate conformational states along FRET coordinate by monitoring time-dependent donor lifetime, presenting a whole picture of multidimensional conformational dynamics in the process of T4 lysozyme open-close hinge-bending enzymatic turnover motions under enzymatic reaction conditions. By analyzing the autocorrelation functions of both lifetime and anisotropy trajectories, we have also observed the dynamic and static inhomogeneity of T4 lysozyme multidimensional conformational fluctuation dynamics, providing a fundamental

Assembly and dynamics of the U4/U6 di-snRNP by single-molecule FRET

Science.gov (United States)

Hardin, John W.; Warnasooriya, Chandani; Kondo, Yasushi; Nagai, Kiyoshi; Rueda, David

2015-01-01

In large ribonucleoprotein machines, such as ribosomes and spliceosomes, RNA functions as an assembly scaffold as well as a critical catalytic component. Protein binding to the RNA scaffold can induce structural changes, which in turn modulate subsequent binding of other components. The spliceosomal U4/U6 di-snRNP contains extensively base paired U4 and U6 snRNAs, Snu13, Prp31, Prp3 and Prp4, seven Sm and seven LSm proteins. We have studied successive binding of all protein components to the snRNA duplex during di-snRNP assembly by electrophoretic mobility shift assay and accompanying conformational changes in the U4/U6 RNA 3-way junction by single-molecule FRET. Stems I and II of the duplex were found to co-axially stack in free RNA and function as a rigid scaffold during the entire assembly, but the U4 snRNA 5′ stem-loop adopts alternative orientations each stabilized by Prp31 and Prp3/4 binding accounting for altered Prp3/4 binding affinities in presence of Prp31. PMID:26503251

The multi-state energy landscape of the SAM-I riboswitch: A single-molecule Förster resonance energy transfer spectroscopy study

Science.gov (United States)

Manz, Christoph; Kobitski, Andrei Yu.; Samanta, Ayan; Jäschke, Andres; Nienhaus, G. Ulrich

2018-03-01

RNA (ribonucleic acid) molecules are highly flexible biopolymers fluctuating at physiological temperatures among many different conformations that are represented by minima in a hierarchical conformational free energy landscape. Here we have employed single-molecule FRET (smFRET) to explore the energy landscape of the B. subtilis yitJ SAM-I riboswitch (RS). In this small RNA molecule, specific binding of an S-adenosyl-L-methionine (SAM) ligand in the aptamer domain regulates gene expression by inducing structural changes in another domain, the expression platform, causing transcription termination by the RNA polymerase. We have measured smFRET histograms over wide ranges of Mg2+ concentration for three RS variants that were specifically labeled with fluorescent dyes on different sites. In the analysis, different conformations are associated with discrete Gaussian model distributions, which are typically fairly broad on the FRET efficiency scale and thus can be extremely challenging to unravel due to their mutual overlap. Our earlier work on two SAM-I RS variants revealed four major conformations. By introducing a global fitting procedure which models both the Mg2+ concentration dependencies of the fractional populations and the average FRET efficiencies of the individual FRET distributions according to Mg2+ binding isotherms, we were able to consistently describe the histogram data of both variants at all studied Mg2+ concentrations. With the third FRET-labeled variant, however, we found significant deviations when applying the four-state model to the data. This can arise because the different FRET labeling of the new variant allows two states to be distinguished that were previously not separable due to overlap. Indeed, the resulting five-state model presented here consistently describes the smFRET histograms of all three variants as well as their variations with Mg2+ concentration. We also performed a triangulation of the donor position for two of the constructs

Subunit rotation in a single FoF1-ATP synthase in a living bacterium monitored by FRET

Science.gov (United States)

Seyfert, K.; Oosaka, T.; Yaginuma, H.; Ernst, S.; Noji, H.; Iino, R.; Börsch, M.

2011-03-01

FoF1-ATP synthase is the ubiquitous membrane-bound enzyme in mitochondria, chloroplasts and bacteria which provides the 'chemical energy currency' adenosine triphosphate (ATP) for cellular processes. In Escherichia coli ATP synthesis is driven by a proton motive force (PMF) comprising a proton concentration difference ΔpH plus an electric potential ΔΨ across the lipid membrane. Single-molecule in vitro experiments have confirmed that proton-driven subunit rotation within FoF1-ATP synthase is associated with ATP synthesis. Based on intramolecular distance measurements by single-molecule fluorescence resonance energy transfer (FRET) the kinetics of subunit rotation and the step sizes of the different rotor parts have been unraveled. However, these experiments were accomplished in the presence of a PMF consisting of a maximum ΔpH ~ 4 and an unknown ΔΨ. In contrast, in living bacteria the maximum ΔpH across the plasma membrane is likely 0.75, and ΔΨ has been measured between -80 and -140 mV. Thus the problem of in vivo catalytic turnover rates, or the in vivo rotational speed in single FoF1-ATP synthases, respectively, has to be solved. In addition, the absolute number of functional enzymes in a single bacterium required to maintain the high ATP levels has to be determined. We report our progress of measuring subunit rotation in single FoF1-ATP synthases in vitro and in vivo, which was enabled by a new labeling approach for single-molecule FRET measurements.

Extraction of information on macromolecular interactions from fluorescence micro-spectroscopy measurements in the presence and absence of FRET

Science.gov (United States)

Raicu, Valerică

2018-06-01

Investigations of static or dynamic interactions between proteins or other biological macromolecules in living cells often rely on the use of fluorescent tags with two different colors in conjunction with adequate theoretical descriptions of Förster Resonance Energy Transfer (FRET) and molecular-level micro-spectroscopic technology. One such method based on these general principles is FRET spectrometry, which allows determination of the quaternary structure of biomolecules from cell-level images of the distributions, or spectra of occurrence frequency of FRET efficiencies. Subsequent refinements allowed combining FRET frequency spectra with molecular concentration information, thereby providing the proportion of molecular complexes with various quaternary structures as well as their binding/dissociation energies. In this paper, we build on the mathematical principles underlying FRET spectrometry to propose two new spectrometric methods, which have distinct advantages compared to other methods. One of these methods relies on statistical analysis of color mixing in subpopulations of fluorescently tagged molecules to probe molecular association stoichiometry, while the other exploits the color shift induced by FRET to also derive geometric information in addition to stoichiometry. The appeal of the first method stems from its sheer simplicity, while the strength of the second consists in its ability to provide structural information.

N-way FRET microscopy of multiple protein-protein interactions in live cells.

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Adam D Hoppe

Full Text Available Fluorescence Resonance Energy Transfer (FRET microscopy has emerged as a powerful tool to visualize nanoscale protein-protein interactions while capturing their microscale organization and millisecond dynamics. Recently, FRET microscopy was extended to imaging of multiple donor-acceptor pairs, thereby enabling visualization of multiple biochemical events within a single living cell. These methods require numerous equations that must be defined on a case-by-case basis. Here, we present a universal multispectral microscopy method (N-Way FRET to enable quantitative imaging for any number of interacting and non-interacting FRET pairs. This approach redefines linear unmixing to incorporate the excitation and emission couplings created by FRET, which cannot be accounted for in conventional linear unmixing. Experiments on a three-fluorophore system using blue, yellow and red fluorescent proteins validate the method in living cells. In addition, we propose a simple linear algebra scheme for error propagation from input data to estimate the uncertainty in the computed FRET images. We demonstrate the strength of this approach by monitoring the oligomerization of three FP-tagged HIV Gag proteins whose tight association in the viral capsid is readily observed. Replacement of one FP-Gag molecule with a lipid raft-targeted FP allowed direct observation of Gag oligomerization with no association between FP-Gag and raft-targeted FP. The N-Way FRET method provides a new toolbox for capturing multiple molecular processes with high spatial and temporal resolution in living cells.

Solid-phase single molecule biosensing using dual-color colocalization of fluorescent quantum dot nanoprobes

Science.gov (United States)

Liu, Jianbo; Yang, Xiaohai; Wang, Kemin; Wang, Qing; Liu, Wei; Wang, Dong

2013-10-01

The development of solid-phase surface-based single molecule imaging technology has attracted significant interest during the past decades. Here we demonstrate a sandwich hybridization method for highly sensitive detection of a single thrombin protein at a solid-phase surface based on the use of dual-color colocalization of fluorescent quantum dot (QD) nanoprobes. Green QD560-modified thrombin binding aptamer I (QD560-TBA I) were deposited on a positive poly(l-lysine) assembled layer, followed by bovine serum albumin blocking. It allowed the thrombin protein to mediate the binding of the easily detectable red QD650-modified thrombin binding aptamer II (QD650-TBA II) to the QD560-TBA I substrate. Thus, the presence of the target thrombin can be determined based on fluorescent colocalization measurements of the nanoassemblies, without target amplification or probe separation. The detection limit of this assay reached 0.8 pM. This fluorescent colocalization assay has enabled single molecule recognition in a separation-free detection format, and can serve as a sensitive biosensing platform that greatly suppresses the nonspecific adsorption false-positive signal. This method can be extended to other areas such as multiplexed immunoassay, single cell analysis, and real time biomolecule interaction studies.The development of solid-phase surface-based single molecule imaging technology has attracted significant interest during the past decades. Here we demonstrate a sandwich hybridization method for highly sensitive detection of a single thrombin protein at a solid-phase surface based on the use of dual-color colocalization of fluorescent quantum dot (QD) nanoprobes. Green QD560-modified thrombin binding aptamer I (QD560-TBA I) were deposited on a positive poly(l-lysine) assembled layer, followed by bovine serum albumin blocking. It allowed the thrombin protein to mediate the binding of the easily detectable red QD650-modified thrombin binding aptamer II (QD650-TBA II) to

Application of FRET probes in the analysis of neuronal plasticity

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Yoshibumi eUeda

2013-10-01

Full Text Available Breakthroughs in imaging techniques and optical probes in recent years have revolutionized the field of life sciences in ways that traditional methods could never match. The spatial and temporal regulation of molecular events can now be studied with great precision. There have been several key discoveries that have made this possible. Since GFP was cloned in 1992, it has become the dominant tracer of proteins in living cells. Then the evolution of color variants of GFP opened the door to the application of Förster resonance energy transfer (FRET, which is now widely recognized as a powerful tool to study complicated signal transduction events and interactions between molecules. Employment of fluorescent lifetime imaging microscopy (FLIM allows the precise detection of FRET in small subcellular structures such as dendritic spines. In this review, we provide an overview of the basic and practical aspects of FRET imaging and discuss how different FRET probes have revealed insights into the molecular mechanisms of synaptic plasticity and enabled visualization of neuronal network activity both in vitro and in vivo.

Multi step FRET among three laser dyes Pyrene, Acriflavine and Rhodamine B

International Nuclear Information System (INIS)

Saha, Jaba; Dey, Dibyendu; Roy, Arpan Datta; Bhattacharjee, D.; Hussain, Syed Arshad

2016-01-01

Fluorescence Resonance Energy Transfer (FRET) system using three dyes has been demonstrated. It has been observed that multi step energy transfer occurred from Pyrene to Rhodamine B via Acriflavine. Here Acriflavine acts as an antenna to receive energy from Pyrene and transfer the same to Rhodamine B. This multi step FRET system is advantageous compared to the conventional FRET as this can be used to study molecular level interaction beyond conventional FRET distance (1–10 nm) as well as studying multi-branched macromolecules. The introduction of clay enhances the FRET efficiencies among the dye pair, which is an advantage to make the multi step system more useful. Similar approach can be used for increasing FRET efficiencies by using other dyes. - Highlights: • Multi-step FRET occurred from Pyrene (Py) to Rhodamine B (RhB) via Acriflavine (Acf). • Acf acts as an antenna to receive energy from Py and to transfer energy to RhB. • Multi-step FRET can be used to study molecular level interaction beyond 1–10 nm. • Incorporation of nanoclay laponite enhances the energy transfer efficiency.

A Starting Point for Fluorescence-Based Single-Molecule Measurements in Biomolecular Research

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Alexander Gust

2014-09-01

Full Text Available Single-molecule fluorescence techniques are ideally suited to provide information about the structure-function-dynamics relationship of a biomolecule as static and dynamic heterogeneity can be easily detected. However, what type of single-molecule fluorescence technique is suited for which kind of biological question and what are the obstacles on the way to a successful single-molecule microscopy experiment? In this review, we provide practical insights into fluorescence-based single-molecule experiments aiming for scientists who wish to take their experiments to the single-molecule level. We especially focus on fluorescence resonance energy transfer (FRET experiments as these are a widely employed tool for the investigation of biomolecular mechanisms. We will guide the reader through the most critical steps that determine the success and quality of diffusion-based confocal and immobilization-based total internal reflection fluorescence microscopy. We discuss the specific chemical and photophysical requirements that make fluorescent dyes suitable for single-molecule fluorescence experiments. Most importantly, we review recently emerged photoprotection systems as well as passivation and immobilization strategies that enable the observation of fluorescently labeled molecules under biocompatible conditions. Moreover, we discuss how the optical single-molecule toolkit has been extended in recent years to capture the physiological complexity of a cell making it even more relevant for biological research.

Single-molecule analysis reveals the kinetics and physiological relevance of MutL-ssDNA binding.

Directory of Open Access Journals (Sweden)

Jonghyun Park

2010-11-01

Full Text Available DNA binding by MutL homologs (MLH/PMS during mismatch repair (MMR has been considered based on biochemical and genetic studies. Bulk studies with MutL and its yeast homologs Mlh1-Pms1 have suggested an integral role for a single-stranded DNA (ssDNA binding activity during MMR. We have developed single-molecule Förster resonance energy transfer (smFRET and a single-molecule DNA flow-extension assays to examine MutL interaction with ssDNA in real time. The smFRET assay allowed us to observe MutL-ssDNA association and dissociation. We determined that MutL-ssDNA binding required ATP and was the greatest at ionic strength below 25 mM (K(D = 29 nM while it dramatically decreases above 100 mM (K(D>2 µM. Single-molecule DNA flow-extension analysis suggests that multiple MutL proteins may bind ssDNA at low ionic strength but this activity does not enhance stability at elevated ionic strengths. These studies are consistent with the conclusion that a stable MutL-ssDNA interaction is unlikely to occur at physiological salt eliminating a number of MMR models. However, the activity may infer some related dynamic DNA transaction process during MMR.

A communication theoretical analysis of FRET-based mobile ad hoc molecular nanonetworks.

Science.gov (United States)

Kuscu, Murat; Akan, Ozgur B

2014-09-01

Nanonetworks refer to a group of nanosized machines with very basic operational capabilities communicating to each other in order to accomplish more complex tasks such as in-body drug delivery, or chemical defense. Realizing reliable and high-rate communication between these nanomachines is a fundamental problem for the practicality of these nanonetworks. Recently, we have proposed a molecular communication method based on Förster Resonance Energy Transfer (FRET) which is a nonradiative excited state energy transfer phenomenon observed among fluorescent molecules, i.e., fluorophores. We have modeled the FRET-based communication channel considering the fluorophores as single-molecular immobile nanomachines, and shown its reliability at high rates, and practicality at the current stage of nanotechnology. In this study, for the first time in the literature, we investigate the network of mobile nanomachines communicating through FRET. We introduce two novel mobile molecular nanonetworks: FRET-based mobile molecular sensor/actor nanonetwork (FRET-MSAN) which is a distributed system of mobile fluorophores acting as sensor or actor node; and FRET-based mobile ad hoc molecular nanonetwork (FRET-MAMNET) which consists of fluorophore-based nanotransmitter, nanoreceivers and nanorelays. We model the single message propagation based on birth-death processes with continuous time Markov chains. We evaluate the performance of FRET-MSAN and FRET-MAMNET in terms of successful transmission probability and mean extinction time of the messages, system throughput, channel capacity and achievable communication rates.

Step size of the rotary proton motor in single FoF1-ATP synthase from a thermoalkaliphilic bacterium by DCO-ALEX FRET

Science.gov (United States)

Hammann, Eva; Zappe, Andrea; Keis, Stefanie; Ernst, Stefan; Matthies, Doreen; Meier, Thomas; Cook, Gregory M.; Börsch, Michael

2012-02-01

Thermophilic enzymes operate at high temperatures but show reduced activities at room temperature. They are in general more stable during preparation and, accordingly, are considered to be more rigid in structure. Crystallization is often easier compared to proteins from bacteria growing at ambient temperatures, especially for membrane proteins. The ATP-producing enzyme FoF1-ATP synthase from thermoalkaliphilic Caldalkalibacillus thermarum strain TA2.A1 is driven by a Fo motor consisting of a ring of 13 c-subunits. We applied a single-molecule Förster resonance energy transfer (FRET) approach using duty cycle-optimized alternating laser excitation (DCO-ALEX) to monitor the expected 13-stepped rotary Fo motor at work. New FRET transition histograms were developed to identify the smaller step sizes compared to the 10-stepped Fo motor of the Escherichia coli enzyme. Dwell time analysis revealed the temperature and the LDAO dependence of the Fo motor activity on the single molecule level. Back-and-forth stepping of the Fo motor occurs fast indicating a high flexibility in the membrane part of this thermophilic enzyme.

FRET structure with non-radiative acceptor provided by dye-linker-glass surface complex and single-molecule photodynamics by TIRFM-polarized imaging

International Nuclear Information System (INIS)

Tani, Toshiro; Mashimo, Kei; Suzuki, Tetsu; Horiuchi, Hiromi; Oda, Masaru

2008-01-01

We present our recent study of microscopic single-molecule imaging on the artificial complex of tetramethylrhodamine linked with a propyl chain onto silica glass surface, i.e. an asymmetric fluorescence resonance energy transfer (FRET) structure with non-radiative acceptor. In the synthesis of the complex, we used a mixture of two kinds of isomers to introduce rather small photodynamic difference among them. This isomeric structure change will provide more or less a distinctive photophysical change in e.g. non-radiative relaxation rate. Our recent observation at room temperatures, so far, shows that such contributions can be discriminated in the histograms of the fluorescent spot intensities; broad but distinctive multi-components appear. To identify the isomeric difference as a cause of structures, some configurational assumptions are necessary. One such basic prerequisite is that the transition dipoles of the chromophores should be oriented almost parallel to the glass surface. In order to make clear the modeling, we also provide preliminary experiments on the polarization dependence of the imaging under rotating polarization in epi-illumination

FRET two-hybrid assay by linearly fitting FRET efficiency to concentration ratio between acceptor and donor

Science.gov (United States)

Du, Mengyan; Yang, Fangfang; Mai, Zihao; Qu, Wenfeng; Lin, Fangrui; Wei, Lichun; Chen, Tongsheng

2018-04-01

We here introduce a fluorescence resonance energy transfer (FRET) two-hybrid assay method to measure the maximal donor(D)- and acceptor(A)-centric FRET efficiency (ED,max and EA,max) of the D-A complex and its stoichiometry by linearly fitting the donor-centric FRET efficiency (ED) to the acceptor-to-donor concentration ratio (RC) and acceptor-centric FRET efficiency (EA) to 1/RC, respectively. We performed this method on a wide-field fluorescence microscope for living HepG2 cells co-expressing FRET tandem constructs and free donor/acceptor and obtained correct ED, EA, and stoichiometry values of those tandem constructs. Evaluation on the binding of Bad with Bcl-XL in Hela cells showed that Bad interacted strongly with Bcl-XL to form a Bad-Bcl-XL complex on mitochondria, and one Bad interacted mainly with one Bcl-XL molecule in healthy cells, while with multiple (maybe 2) Bcl-XL molecules in apoptotic cells.

Two states or not two states: Single-molecule folding studies of protein L

Science.gov (United States)

Aviram, Haim Yuval; Pirchi, Menahem; Barak, Yoav; Riven, Inbal; Haran, Gilad

2018-03-01

Experimental tools of increasing sophistication have been employed in recent years to study protein folding and misfolding. Folding is considered a complex process, and one way to address it is by studying small proteins, which seemingly possess a simple energy landscape with essentially only two stable states, either folded or unfolded. The B1-IgG binding domain of protein L (PL) is considered a model two-state folder, based on measurements using a wide range of experimental techniques. We applied single-molecule fluorescence resonance energy transfer (FRET) spectroscopy in conjunction with a hidden Markov model analysis to fully characterize the energy landscape of PL and to extract the kinetic properties of individual molecules of the protein. Surprisingly, our studies revealed the existence of a third state, hidden under the two-state behavior of PL due to its small population, ˜7%. We propose that this minority intermediate involves partial unfolding of the two C-terminal β strands of PL. Our work demonstrates that single-molecule FRET spectroscopy can be a powerful tool for a comprehensive description of the folding dynamics of proteins, capable of detecting and characterizing relatively rare metastable states that are difficult to observe in ensemble studies.

Inferring properties of disordered chains from FRET transfer efficiencies

Science.gov (United States)

Zheng, Wenwei; Zerze, Gül H.; Borgia, Alessandro; Mittal, Jeetain; Schuler, Benjamin; Best, Robert B.

2018-03-01

Förster resonance energy transfer (FRET) is a powerful tool for elucidating both structural and dynamic properties of unfolded or disordered biomolecules, especially in single-molecule experiments. However, the key observables, namely, the mean transfer efficiency and fluorescence lifetimes of the donor and acceptor chromophores, are averaged over a broad distribution of donor-acceptor distances. The inferred average properties of the ensemble therefore depend on the form of the model distribution chosen to describe the distance, as has been widely recognized. In addition, while the distribution for one type of polymer model may be appropriate for a chain under a given set of physico-chemical conditions, it may not be suitable for the same chain in a different environment so that even an apparently consistent application of the same model over all conditions may distort the apparent changes in chain dimensions with variation of temperature or solution composition. Here, we present an alternative and straightforward approach to determining ensemble properties from FRET data, in which the polymer scaling exponent is allowed to vary with solution conditions. In its simplest form, it requires either the mean FRET efficiency or fluorescence lifetime information. In order to test the accuracy of the method, we have utilized both synthetic FRET data from implicit and explicit solvent simulations for 30 different protein sequences, and experimental single-molecule FRET data for an intrinsically disordered and a denatured protein. In all cases, we find that the inferred radii of gyration are within 10% of the true values, thus providing higher accuracy than simpler polymer models. In addition, the scaling exponents obtained by our procedure are in good agreement with those determined directly from the molecular ensemble. Our approach can in principle be generalized to treating other ensemble-averaged functions of intramolecular distances from experimental data.

A Quantitative Theoretical Framework For Protein-Induced Fluorescence Enhancement-Förster-Type Resonance Energy Transfer (PIFE-FRET).

Science.gov (United States)

Lerner, Eitan; Ploetz, Evelyn; Hohlbein, Johannes; Cordes, Thorben; Weiss, Shimon

2016-07-07

Single-molecule, protein-induced fluorescence enhancement (PIFE) serves as a molecular ruler at molecular distances inaccessible to other spectroscopic rulers such as Förster-type resonance energy transfer (FRET) or photoinduced electron transfer. In order to provide two simultaneous measurements of two distances on different molecular length scales for the analysis of macromolecular complexes, we and others recently combined measurements of PIFE and FRET (PIFE-FRET) on the single molecule level. PIFE relies on steric hindrance of the fluorophore Cy3, which is covalently attached to a biomolecule of interest, to rotate out of an excited-state trans isomer to the cis isomer through a 90° intermediate. In this work, we provide a theoretical framework that accounts for relevant photophysical and kinetic parameters of PIFE-FRET, show how this framework allows the extraction of the fold-decrease in isomerization mobility from experimental data, and show how these results provide information on changes in the accessible volume of Cy3. The utility of this model is then demonstrated for experimental results on PIFE-FRET measurement of different protein-DNA interactions. The proposed model and extracted parameters could serve as a benchmark to allow quantitative comparison of PIFE effects in different biological systems.

Towards characterization of DNA structure under physiological conditions in vivo at the single-molecule level using single-pair FRET

Czech Academy of Sciences Publication Activity Database

Fessl, Tomáš; Adamec, František; Polívka, Tomáš; Foldynová-Trantírková, Silvie; Vácha, František; Trantírek, L.

2012-01-01

Roč. 40, č. 16 (2012), s. 10 ISSN 0305-1048 Institutional research plan: CEZ:AV0Z50510513; CEZ:AV0Z60220518 Keywords : in-cell FRET * fluorescence * DNA * nucleic acid * ATTO * in vivo Subject RIV: BO - Biophysics Impact factor: 8.278, year: 2012

Dual color single particle tracking via nanobodies

International Nuclear Information System (INIS)

Albrecht, David; Winterflood, Christian M; Ewers, Helge

2015-01-01

Single particle tracking is a powerful tool to investigate the function of biological molecules by following their motion in space. However, the simultaneous tracking of two different species of molecules is still difficult to realize without compromising the length or density of trajectories, the localization accuracy or the simplicity of the assay. Here, we demonstrate a simple dual color single particle tracking assay using small, bright, high-affinity labeling via nanobodies of accessible targets with widely available instrumentation. We furthermore apply a ratiometric step-size analysis method to visualize differences in apparent membrane viscosity. (paper)

A New Generation of FRET Sensors for Robust Measurement of Gαi1, Gαi2 and Gαi3 Activation Kinetics in Single Cells.

Directory of Open Access Journals (Sweden)

Jakobus van Unen

Full Text Available G-protein coupled receptors (GPCRs can activate a heterotrimeric G-protein complex with subsecond kinetics. Genetically encoded biosensors based on Förster resonance energy transfer (FRET are ideally suited for the study of such fast signaling events in single living cells. Here we report on the construction and characterization of three FRET biosensors for the measurement of Gαi1, Gαi2 and Gαi3 activation. To enable quantitative long-term imaging of FRET biosensors with high dynamic range, fluorescent proteins with enhanced photophysical properties are required. Therefore, we use the currently brightest and most photostable CFP variant, mTurquoise2, as donor fused to Gαi subunit, and cp173Venus fused to the Gγ2 subunit as acceptor. The Gαi FRET biosensors constructs are expressed together with Gβ1 from a single plasmid, providing preferred relative expression levels with reduced variation in mammalian cells. The Gαi FRET sensors showed a robust response to activation of endogenous or over-expressed alpha-2A-adrenergic receptors, which was inhibited by pertussis toxin. Moreover, we observed activation of the Gαi FRET sensor in single cells upon stimulation of several GPCRs, including the LPA2, M3 and BK2 receptor. Furthermore, we show that the sensors are well suited to extract kinetic parameters from fast measurements in the millisecond time range. This new generation of FRET biosensors for Gαi1, Gαi2 and Gαi3 activation will be valuable for live-cell measurements that probe Gαi activation.

Applications of a single-molecule detection in early disease diagnosis and enzymatic reaction study

Energy Technology Data Exchange (ETDEWEB)

Li, Jiangwei [Iowa State Univ., Ames, IA (United States)

2008-01-01

Various single-molecule techniques were utilized for ultra-sensitive early diagnosis of viral DNA and antigen and basic mechanism study of enzymatic reactions. DNA of human papilloma virus (HPV) served as the screening target in a flow system. Alexa Fluor 532 (AF532) labeled single-stranded DNA probes were hybridized to the target HPV-16 DNA in solution. The individual hybridized molecules were imaged with an intensified charge-coupled device (ICCD) in two ways. In the single-color mode, target molecules were detected via fluorescence from hybridized probes only. This system could detect HPV-16 DNA in the presence of human genomic DNA down to 0.7 copy/cell and had a linear dynamic range of over 6 orders of magnitude. In the dual-color mode, fluorescence resonance energy transfer (FRET) was employed to achieve zero false-positive count. We also showed that DNA extracts from Pap test specimens did not interfere with the system. A surface-based method was used to improve the throughput of the flow system. HPV-16 DNA was hybridized to probes on a glass surface and detected with a total internal reflection fluorescence (TIRF) microscope. In the single-probe mode, the whole genome and target DNA were fluorescently labeled before hybridization, and the detection limit is similar to the flow system. In the dual-probe mode, a second probe was introduced. The linear dynamic range covers 1.44-7000 copies/cell, which is typical of early infection to near-cancer stages. The dual-probe method was tested with a crudely prepared sample. Even with reduced hybridization efficiency caused by the interference of cellular materials, we were still able to differentiate infected cells from healthy cells. Detection and quantification of viral antigen with a novel single-molecule immunosorbent assay (SMISA) was achieved. Antigen from human immunodeficiency virus type 1(HIV-1) was chosen to be the target in this study. The target was sandwiched between a monoclonal capture antibody and a

Continuous throughput and long-term observation of single-molecule FRET without immobilization.

Science.gov (United States)

Tyagi, Swati; VanDelinder, Virginia; Banterle, Niccolò; Fuertes, Gustavo; Milles, Sigrid; Agez, Morgane; Lemke, Edward A

2014-03-01

We present an automated microfluidic platform that performs multisecond observation of single molecules with millisecond time resolution while bypassing the need for immobilization procedures. With this system, we confine biomolecules to a thin excitation field by reversibly collapsing microchannels to nanochannels. We demonstrate the power of our method by studying a variety of complex nucleic acid and protein systems, including DNA Holliday junctions, nucleosomes and human transglutaminase 2.

Three-color confocal Förster (or fluorescence) resonance energy transfer microscopy: Quantitative analysis of protein interactions in the nucleation of actin filaments in live cells.

Science.gov (United States)

Wallrabe, Horst; Sun, Yuansheng; Fang, Xiaolan; Periasamy, Ammasi; Bloom, George S

2015-06-01

Experiments using live cell 3-color Förster (or fluorescence) resonance energy transfer (FRET) microscopy and corresponding in vitro biochemical reconstitution of the same proteins were conducted to evaluate actin filament nucleation. A novel application of 3-color FRET data is demonstrated, extending the analysis beyond the customary energy-transfer efficiency (E%) calculations. MDCK cells were transfected for coexpression of Teal-N-WASP/Venus-IQGAP1/mRFP1-Rac1, Teal-N-WASP/Venus-IQGAP1/mRFP1-Cdc42, CFP-Rac1/Venus-IQGAP1/mCherry-actin, or CFP-Cdc42/Venus-IQGAP1/mCherry-actin, and with single-label equivalents for spectral bleedthrough correction. Using confirmed E% as an entry point, fluorescence levels and related ratios were correlated at discrete accumulating levels at cell peripheries. Rising ratios of CFP-Rac1:Venus-IQGAP1 were correlated with lower overall actin fluorescence, whereas the CFP-Cdc42:Venus-IQGAP1 ratio correlated with increased actin fluorescence at low ratios, but was neutral at higher ratios. The new FRET analyses also indicated that rising levels of mRFP1-Cdc42 or mRFP1-Rac1, respectively, promoted or suppressed the association of Teal-N-WASP with Venus-IQGAP1. These 3-color FRET assays further support our in vitro results about the role of IQGAP1, Rac1, and Cdc42 in actin nucleation, and the differential impact of Rac1 and Cdc42 on the association of N-WASP with IQGAP1. In addition, this study emphasizes the power of 3-color FRET as a systems biology strategy for simultaneous evaluation of multiple interacting proteins in individual live cells. © 2015 International Society for Advancement of Cytometry.

Experimental and Numerical Investigations of Fretting Fatigue Behavior for Steel Q235 Single-Lap Bolted Joints

Directory of Open Access Journals (Sweden)

Yazhou Xu

2016-01-01

Full Text Available This work aims to investigate the fretting fatigue life and failure mode of steel Q235B plates in single-lap bolted joints. Ten specimens were prepared and tested to fit the S-N curve. SEM (scanning electron microscope was then employed to observe fatigue crack surfaces and identify crack initiation, crack propagation, and transient fracture zones. Moreover, a FEM model was established to simulate the stress and displacement fields. The normal contact stress, tangential contact stress, and relative slipping displacement at the critical fretting zone were used to calculate FFD values and assess fretting fatigue crack initiation sites, which were in good agreement with SEM observations. Experimental results confirmed the fretting fatigue failure mode for these specimens. It was found that the crack initiation resulted from wear regions at the contact surfaces between plates, and fretting fatigue cracks occurred at a certain distance away from hole edges. The proposed FFD-N relationship is an alternative approach to evaluate fretting fatigue life of steel plates in bolted joints.

An in vitro tag-and-modify protein sample generation method for single-molecule fluorescence resonance energy transfer.

Science.gov (United States)

Hamadani, Kambiz M; Howe, Jesse; Jensen, Madeleine K; Wu, Peng; Cate, Jamie H D; Marqusee, Susan

2017-09-22

Biomolecular systems exhibit many dynamic and biologically relevant properties, such as conformational fluctuations, multistep catalysis, transient interactions, folding, and allosteric structural transitions. These properties are challenging to detect and engineer using standard ensemble-based techniques. To address this drawback, single-molecule methods offer a way to access conformational distributions, transient states, and asynchronous dynamics inaccessible to these standard techniques. Fluorescence-based single-molecule approaches are parallelizable and compatible with multiplexed detection; to date, however, they have remained limited to serial screens of small protein libraries. This stems from the current absence of methods for generating either individual dual-labeled protein samples at high throughputs or protein libraries compatible with multiplexed screening platforms. Here, we demonstrate that by combining purified and reconstituted in vitro translation, quantitative unnatural amino acid incorporation via AUG codon reassignment, and copper-catalyzed azide-alkyne cycloaddition, we can overcome these challenges for target proteins that are, or can be, methionine-depleted. We present an in vitro parallelizable approach that does not require laborious target-specific purification to generate dual-labeled proteins and ribosome-nascent chain libraries suitable for single-molecule FRET-based conformational phenotyping. We demonstrate the power of this approach by tracking the effects of mutations, C-terminal extensions, and ribosomal tethering on the structure and stability of three protein model systems: barnase, spectrin, and T4 lysozyme. Importantly, dual-labeled ribosome-nascent chain libraries enable single-molecule co-localization of genotypes with phenotypes, are well suited for multiplexed single-molecule screening of protein libraries, and should enable the in vitro directed evolution of proteins with designer single-molecule conformational

Multi-Color Single Particle Tracking with Quantum Dots

DEFF Research Database (Denmark)

Christensen, Eva Arnspang; Brewer, J. R.; Lagerholm, B. C.

2012-01-01

. multiplex single molecule sensitivity applications such as single particle tracking (SPT). In order to fully optimize single molecule multiplex application with QDs, we have in this work performed a comprehensive quantitative investigation of the fluorescence intensities, fluorescence intensity fluctuations......Quantum dots (QDs) have long promised to revolutionize fluorescence detection to include even applications requiring simultaneous multi-species detection at single molecule sensitivity. Despite the early promise, the unique optical properties of QDs have not yet been fully exploited in e. g...... further show that there is only a small size advantage in using blue-shifted QDs in biological applications because of the additional size of the water-stabilizing surface coat. Extending previous work, we finally also show that parallel four color multicolor (MC)-SPT with QDs is possible at an image...

Double-labeled donor probe can enhance the signal of fluorescence resonance energy transfer (FRET) in detection of nucleic acid hybridization

Science.gov (United States)

Okamura, Yukio; Kondo, Satoshi; Sase, Ichiro; Suga, Takayuki; Mise, Kazuyuki; Furusawa, Iwao; Kawakami, Shigeki; Watanabe, Yuichiro

2000-01-01

A set of fluorescently-labeled DNA probes that hybridize with the target RNA and produce fluorescence resonance energy transfer (FRET) signals can be utilized for the detection of specific RNA. We have developed probe sets to detect and discriminate single-strand RNA molecules of plant viral genome, and sought a method to improve the FRET signals to handle in vivo applications. Consequently, we found that a double-labeled donor probe labeled with Bodipy dye yielded a remarkable increase in fluorescence intensity compared to a single-labeled donor probe used in an ordinary FRET. This double-labeled donor system can be easily applied to improve various FRET probes since the dependence upon sequence and label position in enhancement is not as strict. Furthermore this method could be applied to other nucleic acid substances, such as oligo RNA and phosphorothioate oligonucleotides (S-oligos) to enhance FRET signal. Although the double-labeled donor probes labeled with a variety of fluorophores had unexpected properties (strange UV-visible absorption spectra, decrease of intensity and decay of donor fluorescence) compared with single-labeled ones, they had no relation to FRET enhancement. This signal amplification mechanism cannot be explained simply based on our current results and knowledge of FRET. Yet it is possible to utilize this double-labeled donor system in various applications of FRET as a simple signal-enhancement method. PMID:11121494

Photon-counting single-molecule spectroscopy for studying conformational dynamics and macromolecular interactions

Energy Technology Data Exchange (ETDEWEB)

Laurence, Ted Alfred [Univ. of California, Berkeley, CA (United States)

2002-01-01

Single-molecule methods have the potential to provide information about conformational dynamics and molecular interactions that cannot be obtained by other methods. Removal of ensemble averaging provides several benefits, including the ability to detect heterogeneous populations and the ability to observe asynchronous reactions. Single-molecule diffusion methodologies using fluorescence resonance energy transfer (FRET) are developed to monitor conformational dynamics while minimizing perturbations introduced by interactions between molecules and surfaces. These methods are used to perform studies of the folding of Chymotrypsin Inhibitor 2, a small, single-domain protein, and of single-stranded DNA (ssDNA) homopolymers. Confocal microscopy is used in combination with sensitive detectors to detect bursts of photons from fluorescently labeled biomolecules as they diffuse through the focal volume. These bursts are analyzed to extract fluorescence resonance energy transfer (FRET) efficiency. Advances in data acquisition and analysis techniques that are providing a more complete picture of the accessible molecular information are discussed. Photon Arrival-time Interval Distribution (PAID) analysis is a new method for monitoring macromolecular interactions by fluorescence detection with simultaneous determination of coincidence, brightness, diffusion time, and occupancy (proportional to concentration) of fluorescently-labeled molecules undergoing diffusion in a confocal detection volume. This method is based on recording the time of arrival of all detected photons, and then plotting the two-dimensional histogram of photon pairs, where one axis is the time interval between each pair of photons 1 and 2, and the second axis is the number of other photons detected in the time interval between photons 1 and 2. PAID is related to Fluorescence Correlation Spectroscopy (FCS) by a collapse of this histogram onto the time interval axis. PAID extends auto- and cross-correlation FCS

Photon-counting single-molecule spectroscopy for studying conformational dynamics and macromolecular interactions

International Nuclear Information System (INIS)

Laurence, Ted Alfred

2002-01-01

Single-molecule methods have the potential to provide information about conformational dynamics and molecular interactions that cannot be obtained by other methods. Removal of ensemble averaging provides several benefits, including the ability to detect heterogeneous populations and the ability to observe asynchronous reactions. Single-molecule diffusion methodologies using fluorescence resonance energy transfer (FRET) are developed to monitor conformational dynamics while minimizing perturbations introduced by interactions between molecules and surfaces. These methods are used to perform studies of the folding of Chymotrypsin Inhibitor 2, a small, single-domain protein, and of single-stranded DNA (ssDNA) homopolymers. Confocal microscopy is used in combination with sensitive detectors to detect bursts of photons from fluorescently labeled biomolecules as they diffuse through the focal volume. These bursts are analyzed to extract fluorescence resonance energy transfer (FRET) efficiency. Advances in data acquisition and analysis techniques that are providing a more complete picture of the accessible molecular information are discussed. Photon Arrival-time Interval Distribution (PAID) analysis is a new method for monitoring macromolecular interactions by fluorescence detection with simultaneous determination of coincidence, brightness, diffusion time, and occupancy (proportional to concentration) of fluorescently-labeled molecules undergoing diffusion in a confocal detection volume. This method is based on recording the time of arrival of all detected photons, and then plotting the two-dimensional histogram of photon pairs, where one axis is the time interval between each pair of photons 1 and 2, and the second axis is the number of other photons detected in the time interval between photons 1 and 2. PAID is related to Fluorescence Correlation Spectroscopy (FCS) by a collapse of this histogram onto the time interval axis. PAID extends auto- and cross-correlation FCS

A Single Molecule Investigation of the Photostability of Quantum Dots

DEFF Research Database (Denmark)

Christensen, Eva Arnspang; Kulatunga, Pasad; Lagerholm, B. Christoffer

2012-01-01

Quantum dots (QDs) are very attractive probes for multi-color fluorescence applications. We report here however that single QDs that are subject to continuous blue excitation from a 100W mercury arc lamp will undergo a continuous blue-switching of the emission wavelength eventually reaching a per...... 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....

EXPERIMENTAL INVESTIGTION OF THE FRETTING PHENOMENON

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Ştefan GHIMISI

2015-12-01

Full Text Available Fretting is now fully identified as a small amplitude oscilatory motion which induces a harmonic tangential force between two surfaces in contact.It is related to three main loadings, i.e. fretting-wear, fretting-fatigue and fretting corrosion.Fretting regimes were first mapped by Vingsbo. In a similar way, three fretting regimes will be considered: stick regime,slip regime and mixed regime. The mixed regime was made up of initial gross slip followed by partial slip condition after a few hundred cycles. Obviously the partial slip transition develops the highest stress levels which can induce fatigue crack nucleation depending on the fatigue properties of the two contacting first bodies. Therefore prediction of the frontier between partial slip and gross slip is required.

A Method for Extracting the Free Energy Surface and Conformational Dynamics of Fast-Folding Proteins from Single Molecule Photon Trajectories

Science.gov (United States)

2015-01-01

Single molecule fluorescence spectroscopy holds the promise of providing direct measurements of protein folding free energy landscapes and conformational motions. However, fulfilling this promise has been prevented by technical limitations, most notably, the difficulty in analyzing the small packets of photons per millisecond that are typically recorded from individual biomolecules. Such limitation impairs the ability to accurately determine conformational distributions and resolve sub-millisecond processes. Here we develop an analytical procedure for extracting the conformational distribution and dynamics of fast-folding proteins directly from time-stamped photon arrival trajectories produced by single molecule FRET experiments. Our procedure combines the maximum likelihood analysis originally developed by Gopich and Szabo with a statistical mechanical model that describes protein folding as diffusion on a one-dimensional free energy surface. Using stochastic kinetic simulations, we thoroughly tested the performance of the method in identifying diverse fast-folding scenarios, ranging from two-state to one-state downhill folding, as a function of relevant experimental variables such as photon count rate, amount of input data, and background noise. The tests demonstrate that the analysis can accurately retrieve the original one-dimensional free energy surface and microsecond folding dynamics in spite of the sub-megahertz photon count rates and significant background noise levels of current single molecule fluorescence experiments. Therefore, our approach provides a powerful tool for the quantitative analysis of single molecule FRET experiments of fast protein folding that is also potentially extensible to the analysis of any other biomolecular process governed by sub-millisecond conformational dynamics. PMID:25988351

QD-Based FRET Probes at a Glance

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Armen Shamirian

2015-06-01

Full Text Available The unique optoelectronic properties of quantum dots (QDs give them significant advantages over traditional organic dyes, not only as fluorescent labels for bioimaging, but also as emissive sensing probes. QD sensors that function via manipulation of fluorescent resonance energy transfer (FRET are of special interest due to the multiple response mechanisms that may be utilized, which in turn imparts enhanced flexibility in their design. They may also function as ratiometric, or “color-changing” probes. In this review, we describe the fundamentals of FRET and provide examples of QD-FRET sensors as grouped by their response mechanisms such as link cleavage and structural rearrangement. An overview of early works, recent advances, and various models of QD-FRET sensors for the measurement of pH and oxygen, as well as the presence of metal ions and proteins such as enzymes, are also provided.

FRET enhancement close to gold nanoparticles positioned in DNA origami constructs.

Science.gov (United States)

Aissaoui, Nesrine; Moth-Poulsen, Kasper; Käll, Mikael; Johansson, Peter; Wilhelmsson, L Marcus; Albinsson, Bo

2017-01-05

Here we investigate the energy transfer rates of a Förster resonance energy transfer (FRET) pair positioned in close proximity to a 5 nm gold nanoparticle (AuNP) on a DNA origami construct. We study the distance dependence of the FRET rate by varying the location of the donor molecule, D, relative to the AuNP while maintaining a fixed location of the acceptor molecule, A. The presence of the AuNP induces an alteration in the spontaneous emission of the donor (including radiative and non-radiative rates) which is strongly dependent on the distance between the donor and AuNP surface. Simultaneously, the energy transfer rates are enhanced at shorter D-A (and D-AuNP) distances. Overall, in addition to the direct influence of the acceptor and AuNP on the donor decay there is also a significant increase in decay rate not explained by the sum of the two interactions. This leads to enhanced energy transfer between donor and acceptor in the presence of a 5 nm AuNP. We also demonstrate that the transfer rate in the three "particle" geometry (D + A + AuNP) depends approximately linearly on the transfer rate in the donor-AuNP system, suggesting the possibility to control FRET process with electric field induced by 5 nm AuNPs close to the donor fluorophore. It is concluded that DNA origami is a very versatile platform for studying interactions between molecules and plasmonic nanoparticles in general and FRET enhancement in particular.

Activation-induced deoxycytidine deaminase (AID) co-transcriptional scanning at single-molecule resolution

Science.gov (United States)

Senavirathne, Gayan; Bertram, Jeffrey G.; Jaszczur, Malgorzata; Chaurasiya, Kathy R.; Pham, Phuong; Mak, Chi H.; Goodman, Myron F.; Rueda, David

2015-12-01

Activation-induced deoxycytidine deaminase (AID) generates antibody diversity in B cells by initiating somatic hypermutation (SHM) and class-switch recombination (CSR) during transcription of immunoglobulin variable (IgV) and switch region (IgS) DNA. Using single-molecule FRET, we show that AID binds to transcribed dsDNA and translocates unidirectionally in concert with RNA polymerase (RNAP) on moving transcription bubbles, while increasing the fraction of stalled bubbles. AID scans randomly when constrained in an 8 nt model bubble. When unconstrained on single-stranded (ss) DNA, AID moves in random bidirectional short slides/hops over the entire molecule while remaining bound for ~5 min. Our analysis distinguishes dynamic scanning from static ssDNA creasing. That AID alone can track along with RNAP during transcription and scan within stalled transcription bubbles suggests a mechanism by which AID can initiate SHM and CSR when properly regulated, yet when unregulated can access non-Ig genes and cause cancer.

Spatial and temporal superresolution concepts to study plasma membrane organization by single molecule fluorescence techniques

International Nuclear Information System (INIS)

Ruprecht, V.

2010-01-01

Fluorescence microscopy techniques are currently among the most important experimental tools to study cellular processes. Ultra-sensitive detection devices nowadays allow for measuring even individual farnesylacetate labeled target molecules with nanometer spatial accuracy and millisecond time resolution. The emergence of single molecule fluorescence techniques especially contributed to the field of membrane biology and provided basic knowledge on structural and dynamic features of the cellular plasma membrane. However, we are still confronted with a rather fragmentary understanding of the complex architecture and functional interrelations of membrane constituents. In this thesis new concepts in one- and dual-color single molecule fluorescence techniques are presented that allow for addressing organization principles and interaction dynamics in the live cell plasma membrane. Two complementary experimental strategies are described which differ in their detection principle: single molecule fluorescence imaging and fluorescence correlation spectroscopy. The presented methods are discussed in terms of their implementation, accuracy, quantitative and statistical data analysis, as well as live cell applications. State-of-the-art dual color single molecule imaging is introduced as the most direct experimental approach to study interaction dynamics between differently labeled target molecules. New analytical estimates for robust data analysis are presented that facilitate quantitative recording and identification of co localizations in dual color single molecule images. A novel dual color illumination scheme is further described that profoundly extends the current range and sensitivity of conventional dual color single molecule experiments. The method enables working at high surface densities of fluorescent molecules - a feature typically incommensurable with single molecule imaging - and is especially suited for the detection of rare interactions by tracking co localized

Multi-color single particle tracking with quantum dots.

Directory of Open Access Journals (Sweden)

Eva C Arnspang

Full Text Available Quantum dots (QDs have long promised to revolutionize fluorescence detection to include even applications requiring simultaneous multi-species detection at single molecule sensitivity. Despite the early promise, the unique optical properties of QDs have not yet been fully exploited in e. g. multiplex single molecule sensitivity applications such as single particle tracking (SPT. In order to fully optimize single molecule multiplex application with QDs, we have in this work performed a comprehensive quantitative investigation of the fluorescence intensities, fluorescence intensity fluctuations, and hydrodynamic radii of eight types of commercially available water soluble QDs. In this study, we show that the fluorescence intensity of CdSe core QDs increases as the emission of the QDs shifts towards the red but that hybrid CdSe/CdTe core QDs are less bright than the furthest red-shifted CdSe QDs. We further show that there is only a small size advantage in using blue-shifted QDs in biological applications because of the additional size of the water-stabilizing surface coat. Extending previous work, we finally also show that parallel four color multicolor (MC-SPT with QDs is possible at an image acquisition rate of at least 25 Hz. We demonstrate the technique by measuring the lateral dynamics of a lipid, biotin-cap-DPPE, in the cellular plasma membrane of live cells using four different colors of QDs; QD565, QD605, QD655, and QD705 as labels.

Intramolecular three-colour single pair FRET of intrinsically disordered proteins with increased dynamic range.

Science.gov (United States)

Milles, Sigrid; Koehler, Christine; Gambin, Yann; Deniz, Ashok A; Lemke, Edward A

2012-10-01

Single molecule observation of fluorescence resonance energy transfer can be used to provide insight into the structure and dynamics of proteins. Using a straightforward triple-colour labelling strategy, we present a measurement and analysis scheme that can simultaneously study multiple regions within single intrinsically disordered proteins.

A versatile optical microscope for time-dependent single-molecule and single-particle spectroscopy

Science.gov (United States)

Li, Hao; Yang, Haw

2018-03-01

This work reports the design and implementation of a multi-function optical microscope for time-dependent spectroscopy on single molecules and single nanoparticles. It integrates the now-routine single-object measurements into one standalone platform so that no reconfiguration is needed when switching between different types of sample or spectroscopy modes. The illumination modes include evanescent field through total internal reflection, dark-field illumination, and epi-excitation onto a diffraction-limited spot suitable for confocal detection. The detection modes include spectrally resolved line imaging, wide-field imaging with dual-color capability, and two-color single-element photon-counting detection. The switch between different spectroscopy and data acquisition modes is fully automated and executed through computer programming. The capability of this microscope is demonstrated through selected proof-of-principle experiments.

A versatile optical microscope for time-dependent single-molecule and single-particle spectroscopy.

Science.gov (United States)

Li, Hao; Yang, Haw

2018-03-28

This work reports the design and implementation of a multi-function optical microscope for time-dependent spectroscopy on single molecules and single nanoparticles. It integrates the now-routine single-object measurements into one standalone platform so that no reconfiguration is needed when switching between different types of sample or spectroscopy modes. The illumination modes include evanescent field through total internal reflection, dark-field illumination, and epi-excitation onto a diffraction-limited spot suitable for confocal detection. The detection modes include spectrally resolved line imaging, wide-field imaging with dual-color capability, and two-color single-element photon-counting detection. The switch between different spectroscopy and data acquisition modes is fully automated and executed through computer programming. The capability of this microscope is demonstrated through selected proof-of-principle experiments.

Monitoring transient elastic energy storage within the rotary motors of single FoF1-ATP synthase by DCO-ALEX FRET

Science.gov (United States)

Ernst, Stefan; Düser, Monika G.; Zarrabi, Nawid; Börsch, Michael

2012-03-01

The enzyme FoF1-ATP synthase provides the 'chemical energy currency' adenosine triphosphate (ATP) for living cells. Catalysis is driven by mechanochemical coupling of subunit rotation within the enzyme with conformational changes in the three ATP binding sites. Proton translocation through the membrane-bound Fo part of ATP synthase powers a 10-step rotary motion of the ring of c subunits. This rotation is transmitted to the γ and ɛ subunits of the F1 part. Because γ and ɛ subunits rotate in 120° steps, we aim to unravel this symmetry mismatch by real time monitoring subunit rotation using single-molecule Förster resonance energy transfer (FRET). One fluorophore is attached specifically to the F1 motor, another one to the Fo motor of the liposome-reconstituted enzyme. Photophysical artifacts due to spectral fluctuations of the single fluorophores are minimized by a previously developed duty cycle-optimized alternating laser excitation scheme (DCO-ALEX). We report the detection of reversible elastic deformations between the rotor parts of Fo and F1 and estimate the maximum angular displacement during the load-free rotation using Monte Carlo simulations.

Hyperspectral imaging for simultaneous measurements of two FRET biosensors in pancreatic β-cells.

Science.gov (United States)

Elliott, Amicia D; Bedard, Noah; Ustione, Alessandro; Baird, Michelle A; Davidson, Michael W; Tkaczyk, Tomasz; Piston, David W

2017-01-01

Fluorescent protein (FP) biosensors based on Förster resonance energy transfer (FRET) are commonly used to study molecular processes in living cells. There are FP-FRET biosensors for many cellular molecules, but it remains difficult to perform simultaneous measurements of multiple biosensors. The overlapping emission spectra of the commonly used FPs, including CFP/YFP and GFP/RFP make dual FRET measurements challenging. In addition, a snapshot imaging modality is required for simultaneous imaging. The Image Mapping Spectrometer (IMS) is a snapshot hyperspectral imaging system that collects high resolution spectral data and can be used to overcome these challenges. We have previously demonstrated the IMS's capabilities for simultaneously imaging GFP and CFP/YFP-based biosensors in pancreatic β-cells. Here, we demonstrate a further capability of the IMS to image simultaneously two FRET biosensors with a single excitation band, one for cAMP and the other for Caspase-3. We use these measurements to measure simultaneously cAMP signaling and Caspase-3 activation in pancreatic β-cells during oxidative stress and hyperglycemia, which are essential components in the pathology of diabetes.

A new trend to determine biochemical parameters by quantitative FRET assays.

Science.gov (United States)

Liao, Jia-yu; Song, Yang; Liu, Yan

2015-12-01

Förster resonance energy transfer (FRET) has been widely used in biological and biomedical research because it can determine molecule or particle interactions within a range of 1-10 nm. The sensitivity and efficiency of FRET strongly depend on the distance between the FRET donor and acceptor. Historically, FRET assays have been used to quantitatively deduce molecular distances. However, another major potential application of the FRET assay has not been fully exploited, that is, the use of FRET signals to quantitatively describe molecular interactive events. In this review, we discuss the use of quantitative FRET assays for the determination of biochemical parameters, such as the protein interaction dissociation constant (K(d)), enzymatic velocity (k(cat)) and K(m). We also describe fluorescent microscopy-based quantitative FRET assays for protein interaction affinity determination in cells as well as fluorimeter-based quantitative FRET assays for protein interaction and enzymatic parameter determination in solution.

Medical diagnosis and remote sensing at fiber-tip: picosecond resolved FRET sensor

Science.gov (United States)

Polley, Nabarun; Pal, Samir Kumar

2016-03-01

Förster Resonance Energy Transfer (FRET) strategy in popular in fiber-optic sensing. However, the steady state emission quenching of the donor is inadequate to conclude FRET. The resonance type energy transfer from one molecule (donor) to other (acceptor) should meet few key properties including donor to acceptor energy migration in non-radiative way. In the present study, we have coupled the evanescent field of an optical fiber to the covalently attached donor (dansyl) molecules at the fiber tip. By using picosecond resolved time correlated single photon counting (TCSPC) we have demonstrated that dansyl at the fiber tip transfers energy to a well known DNA-intercalating dye ethidium. Our ultrafast detection scheme selectively distinguishes the probe (dansyl) emission from the intrinsic emission of the fiber. We have also used the setup for the remote sensing of the dielectric constant (polarity) of an environment. We have finally implemented the detection mechanism to detect an industrial synthetic dye methylene blue (MB) in water.

Electroporation and microinjection successfully deliver single-stranded and duplex DNA into live cells as detected by FRET measurements.

Directory of Open Access Journals (Sweden)

Rosemary A Bamford

Full Text Available Förster resonance energy transfer (FRET technology relies on the close proximity of two compatible fluorophores for energy transfer. Tagged (Cy3 and Cy5 complementary DNA strands forming a stable duplex and a doubly-tagged single strand were shown to demonstrate FRET outside of a cellular environment. FRET was also observed after transfecting these DNA strands into fixed and live cells using methods such as microinjection and electroporation, but not when using lipid based transfection reagents, unless in the presence of the endosomal acidification inhibitor bafilomycin. Avoiding the endocytosis pathway is essential for efficient delivery of intact DNA probes into cells.

Revealing time bunching effect in single-molecule enzyme conformational dynamics.

Science.gov (United States)

Lu, H Peter

2011-04-21

In this perspective, we focus our discussion on how the single-molecule spectroscopy and statistical analysis are able to reveal enzyme hidden properties, taking the study of T4 lysozyme as an example. Protein conformational fluctuations and dynamics play a crucial role in biomolecular functions, such as in enzymatic reactions. 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. Using single-molecule fluorescence spectroscopy, we have probed T4 lysozyme conformational motions under the hydrolysis reaction of a polysaccharide of E. coli B cell walls by monitoring the fluorescence resonant energy transfer (FRET) between a donor-acceptor probe pair tethered to T4 lysozyme domains involving open-close hinge-bending motions. Based on the single-molecule spectroscopic results, molecular dynamics simulation, a random walk model analysis, and a novel 2D statistical correlation analysis, we have revealed a time bunching effect in protein conformational motion dynamics that is critical to enzymatic functions. Bunching effect implies that conformational motion times tend to bunch in a finite and narrow time window. We show that convoluted multiple Poisson rate processes give rise to the bunching effect in the enzymatic reaction dynamics. Evidently, the bunching effect is likely common in protein conformational dynamics involving in conformation-gated protein functions. In this perspective, we will also discuss a new approach of 2D regional correlation analysis capable of analyzing fluctuation dynamics of complex multiple correlated and anti-correlated fluctuations under a non-correlated noise background. Using this new method, we are able to map out any defined segments along the fluctuation trajectories and determine whether they are correlated, anti-correlated, or non-correlated; after which, a

The Use of Ultrashort Picosecond Laser Pulses to Generate Quantum Optical Properties of Single Molecules in Biophysics

Science.gov (United States)

Ly, Sonny

Generation of quantum optical states from ultrashort laser-molecule interactions have led to fascinating discoveries in physics and chemistry. In recent years, these interactions have been extended to probe phenomena in single molecule biophysics. Photons emitted from a single fluorescent molecule contains important properties about how the molecule behave and function in that particular environment. Analysis of the second order coherence function through fluorescence correlation spectroscopy plays a pivotal role in quantum optics. At very short nanosecond timescales, the coherence function predicts photon antibunching, a purely quantum optical phenomena which states that a single molecule can only emit one photon at a time. Photon antibunching is the only direct proof of single molecule emission. From the nanosecond to microsecond timescale, the coherence function gives information about rotational diffusion coefficients, and at longer millisecond timescales, gives information regarding the translational diffusion coefficients. In addition, energy transfer between molecules from dipole-dipole interaction results in FRET, a highly sensitive method to probe conformational dynamics at nanometer distances. Here I apply the quantum optical techniques of photon antibunching, fluorescence correlation spectroscopy and FRET to probe how lipid nanodiscs form and function at the single molecule level. Lipid nanodiscs are particles that contain two apolipoprotein (apo) A-I circumventing a lipid bilayer in a belt conformation. From a technological point of view, nanodiscs mimics a patch of cell membrane that have recently been used to reconstitute a variety of membrane proteins including cytochrome P450 and bacteriorhodopsin. They are also potential drug transport vehicles due to its small and stable 10nm diameter size. Biologically, nanodiscs resemble to high degree, high density lipoproteins (HDL) in our body and provides a model platform to study lipid-protein interactions

Making ultracold molecules in a two-color pump-dump photoassociation scheme using chirped pulses

International Nuclear Information System (INIS)

Koch, Christiane P.; Luc-Koenig, Eliane; Masnou-Seeuws, Francoise

2006-01-01

This theoretical paper investigates the formation of ground state molecules from ultracold cesium atoms in a two-color scheme. Following previous work on photoassociation with chirped picosecond pulses [Luc-Koenig et al., Phys. Rev. A, 70, 033414 (2004)], we investigate stabilization by a second (dump) pulse. By appropriately choosing the dump pulse parameters and time delay with respect to the photoassociation pulse, we show that a large number of deeply bound molecules are created in the ground triplet state. We discuss (i) broad-bandwidth dump pulses which maximize the probability to form molecules while creating a broad vibrational distribution as well as (ii) narrow-bandwidth pulses populating a single vibrational ground state level, bound by 113 cm -1 . The use of chirped pulses makes the two-color scheme robust, simple, and efficient

Electrochemically-gated single-molecule electrical devices

International Nuclear Information System (INIS)

Guo, Shaoyin; Artés, Juan Manuel; Díez-Pérez, Ismael

2013-01-01

In the last decade, single-molecule electrical contacts have emerged as a new experimental platform that allows exploring charge transport phenomena in individual molecular blocks. This novel tool has evolved into an essential element within the Molecular Electronics field to understand charge transport processes in hybrid (bio)molecule/electrode interfaces at the nanoscale, and prospect the implementation of active molecular components into functional nanoscale optoelectronic devices. Within this area, three-terminal single-molecule devices have been sought, provided that they are highly desired to achieve full functionality in logic electronic circuits. Despite the latest experimental developments offer consistent methods to bridge a molecule between two electrodes (source and drain in a transistor notation), placing a third electrode (gate) close to the single-molecule electrical contact is still technically challenging. In this vein, electrochemically-gated single-molecule devices have emerged as an experimentally affordable alternative to overcome these technical limitations. In this review, the operating principle of an electrochemically-gated single-molecule device is presented together with the latest experimental methodologies to built them and characterize their charge transport characteristics. Then, an up-to-date comprehensive overview of the most prominent examples will be given, emphasizing on the relationship between the molecular structure and the final device electrical behaviour

The Conformational Dynamics of Cas9 Governing DNA Cleavage Are Revealed by Single-Molecule FRET.

Science.gov (United States)

Yang, Mengyi; Peng, Sijia; Sun, Ruirui; Lin, Jingdi; Wang, Nan; Chen, Chunlai

2018-01-09

Off-target binding and cleavage by Cas9 pose major challenges in its application. How the conformational dynamics of Cas9 govern its nuclease activity under on- and off-target conditions remains largely unknown. Here, using intra-molecular single-molecule fluorescence resonance energy transfer measurements, we revealed that Cas9 in apo, sgRNA-bound, and dsDNA/sgRNA-bound forms spontaneously transits among three major conformational states, mainly reflecting significant conformational mobility of the catalytic HNH domain. We also uncovered surprising long-range allosteric communication between the HNH domain and the RNA/DNA heteroduplex at the PAM-distal end to ensure correct positioning of the catalytic site, which demonstrated that a unique proofreading mechanism served as the last checkpoint before DNA cleavage. Several Cas9 residues were likely to mediate the allosteric communication and proofreading step. Modulating interactions between Cas9 and heteroduplex at the PAM-distal end by introducing mutations on these sites provides an alternative route to improve and optimize the CRISPR/Cas9 toolbox. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Highly sensitive and quantitative FRET-FLIM imaging in single dendritic spines using improved non-radiative YFP.

Science.gov (United States)

Murakoshi, Hideji; Lee, Seok-Jin; Yasuda, Ryohei

2008-08-01

Two-photon fluorescence lifetime imaging microscopy (TPFLIM) enables the quantitative measurements of fluorescence resonance energy transfer (FRET) in small subcellular compartments in light scattering tissue. We evaluated and optimized the FRET pair of mEGFP (monomeric EGFP with the A206K mutation) and REACh (non-radiative YFP variants) for TPFLIM. We characterized several mutants of REACh in terms of their "darkness," and their ability to act as a FRET acceptor for mEGFP in HeLa cells and hippocampal neurons. Since the commonly used monomeric mutation A206K increases the brightness of REACh, we introduced a different monomeric mutation (F223R) which does not affect the brightness. Also, we found that the folding efficiency of original REACh, as measured by the fluorescence lifetime of a mEGFP-REACh tandem dimer, was low and variable from cell to cell. Introducing two folding mutations (F46L, Q69M) into REACh increased the folding efficiency by approximately 50%, and reduced the variability of FRET signal. Pairing mEGFP with the new REACh (super-REACh, or sREACh) improved the signal-to-noise ratio compared to the mEGFP-mRFP or mEGFP-original REACh pair by approximately 50%. Using this new pair, we demonstrated that the fraction of actin monomers in filamentous and globular forms in single dendritic spines can be quantitatively measured with high sensitivity. Thus, the mEGFP-sREACh pair is suited for quantitative FRET measurement by TPFLIM, and enables us to measure protein-protein interactions in individual dendritic spines in brain slices with high sensitivity.

Simulation of FRET dyes allows quantitative comparison against experimental data

Science.gov (United States)

Reinartz, Ines; Sinner, Claude; Nettels, Daniel; Stucki-Buchli, Brigitte; Stockmar, Florian; Panek, Pawel T.; Jacob, Christoph R.; Nienhaus, Gerd Ulrich; Schuler, Benjamin; Schug, Alexander

2018-03-01

Fully understanding biomolecular function requires detailed insight into the systems' structural dynamics. Powerful experimental techniques such as single molecule Förster Resonance Energy Transfer (FRET) provide access to such dynamic information yet have to be carefully interpreted. Molecular simulations can complement these experiments but typically face limits in accessing slow time scales and large or unstructured systems. Here, we introduce a coarse-grained simulation technique that tackles these challenges. While requiring only few parameters, we maintain full protein flexibility and include all heavy atoms of proteins, linkers, and dyes. We are able to sufficiently reduce computational demands to simulate large or heterogeneous structural dynamics and ensembles on slow time scales found in, e.g., protein folding. The simulations allow for calculating FRET efficiencies which quantitatively agree with experimentally determined values. By providing atomically resolved trajectories, this work supports the planning and microscopic interpretation of experiments. Overall, these results highlight how simulations and experiments can complement each other leading to new insights into biomolecular dynamics and function.

APPL proteins FRET at the BAR: direct observation of APPL1 and APPL2 BAR domain-mediated interactions on cell membranes using FRET microscopy.

Directory of Open Access Journals (Sweden)

Heidi J Chial

2010-08-01

Full Text Available Human APPL1 and APPL2 are homologous RAB5 effectors whose binding partners include a diverse set of transmembrane receptors, signaling proteins, and phosphoinositides. APPL proteins associate dynamically with endosomal membranes and are proposed to function in endosome-mediated signaling pathways linking the cell surface to the cell nucleus. APPL proteins contain an N-terminal Bin/Amphiphysin/Rvs (BAR domain, a central pleckstrin homology (PH domain, and a C-terminal phosphotyrosine binding (PTB domain. Previous structural and biochemical studies have shown that the APPL BAR domains mediate homotypic and heterotypic APPL-APPL interactions and that the APPL1 BAR domain forms crescent-shaped dimers. Although previous studies have shown that APPL minimal BAR domains associate with curved cell membranes, direct interaction between APPL BAR domains on cell membranes in vivo has not been reported.Herein, we used a laser-scanning confocal microscope equipped with a spectral detector to carry out fluorescence resonance energy transfer (FRET experiments with cyan fluorescent protein/yellow fluorescent protein (CFP/YFP FRET donor/acceptor pairs to examine interactions between APPL minimal BAR domains at the subcellular level. This comprehensive approach enabled us to evaluate FRET levels in a single cell using three methods: sensitized emission, standard acceptor photobleaching, and sequential acceptor photobleaching. We also analyzed emission spectra to address an outstanding controversy regarding the use of CFP donor/YFP acceptor pairs in FRET acceptor photobleaching experiments, based on reports that photobleaching of YFP converts it into a CFP-like species.All three methods consistently showed significant FRET between APPL minimal BAR domain FRET pairs, indicating that they interact directly in a homotypic (i.e., APPL1-APPL1 and APPL2-APPL2 and heterotypic (i.e., APPL1-APPL2 manner on curved cell membranes. Furthermore, the results of our experiments

Single molecule force spectroscopy: methods and applications in biology

International Nuclear Information System (INIS)

Shen Yi; Hu Jun

2012-01-01

Single molecule measurements have transformed our view of biomolecules. Owing to the ability of monitoring the activity of individual molecules, we now see them as uniquely structured, fluctuating molecules that stochastically transition between frequently many substrates, as two molecules do not follow precisely the same trajectory. Indeed, it is this discovery of critical yet short-lived substrates that were often missed in ensemble measurements that has perhaps contributed most to the better understanding of biomolecular functioning resulting from single molecule experiments. In this paper, we give a review on the three major techniques of single molecule force spectroscopy, and their applications especially in biology. The single molecular study of biotin-streptavidin interactions is introduced as a successful example. The problems and prospects of the single molecule force spectroscopy are discussed, too. (authors)

Surveillance of siRNA integrity by FRET imaging

Science.gov (United States)

Järve, Anne; Müller, Julius; Kim, Il-Han; Rohr, Karl; MacLean, Caroline; Fricker, Gert; Massing, Ulrich; Eberle, Florian; Dalpke, Alexander; Fischer, Roger; Trendelenburg, Michael F.; Helm, Mark

2007-01-01

Techniques for investigation of exogenous small interfering RNA (siRNA) after penetration of the cell are of substantial interest to the development of efficient transfection methods as well as to potential medical formulations of siRNA. A FRET-based visualization method including the commonplace dye labels fluorescein and tetramethylrhodamin (TMR) on opposing strands of siRNA was found compatible with RNA interference (RNAi). Investigation of spectral properties of three labelled siRNAs with differential FRET efficiencies in the cuvette, including pH dependence and FRET efficiency in lipophilic environments, identified the ratio of red and green fluorescence (R/G-ratio) as a sensitive parameter, which reliably identifies samples containing >90% un-degraded siRNA. Spectral imaging of siRNAs microinjected into cells showed emission spectra indistinguishable from those measured in the cuvette. These were used to establish a calibration curve for assessing the degradation state of siRNA in volume elements inside cells. An algorithm, applied to fluorescence images recorded in standard green and red fluorescence channels, produces R/G-ratio images of high spatial resolution, identifying volume elements in the cell with high populations of intact siRNA with high fidelity. To demonstrate the usefulness of this technique, the movement of intact siRNA molecules are observed after introduction into the cytosol by microinjection, standard transfection and lipofection with liposomes. PMID:17890733

Using Single Colors and Color Pairs to Communicate Basic Tastes II: Foreground-Background Color Combinations.

Science.gov (United States)

Woods, Andy T; Marmolejo-Ramos, Fernando; Velasco, Carlos; Spence, Charles

2016-01-01

People associate basic tastes (e.g., sweet, sour, bitter, and salty) with specific colors (e.g., pink or red, green or yellow, black or purple, and white or blue). In the present study, we investigated whether a color bordered by another color (either the same or different) would give rise to stronger taste associations relative to a single patch of color. We replicate previous findings, highlighting the existence of a robust crossmodal correspondence between individual colors and basic tastes. On occasion, color pairs were found to communicate taste expectations more consistently than were single color patches. Furthermore, and in contrast to a recent study in which the color pairs were shown side-by-side, participants took no longer to match the color pairs with tastes than the single colors (they had taken twice as long to respond to the color pairs in the previous study). Possible reasons for these results are discussed, and potential applications for the results, and for the testing methodology developed, are outlined.

Single molecules and nanotechnology

CERN Document Server

Vogel, Horst

2007-01-01

This book focuses on recent advances in the rapidly evolving field of single molecule research. These advances are of importance for the investigation of biopolymers and cellular biochemical reactions, and are essential to the development of quantitative biology. Written by leading experts in the field, the articles cover a broad range of topics, including: quantum photonics of organic dyes and inorganic nanoparticles their use in detecting properties of single molecules the monitoring of single molecule (enzymatic) reactions single protein (un)folding in nanometer-sized confined volumes the dynamics of molecular interactions in biological cells The book is written for advanced students and scientists who wish to survey the concepts, techniques and results of single molecule research and assess them for their own scientific activities.

Using Single Colors and Color Pairs to Communicate Basic Tastes

Directory of Open Access Journals (Sweden)

Andy T. Woods

2016-07-01

Full Text Available Recently, it has been demonstrated that people associate each of the basic tastes (e.g., sweet, sour, bitter, and salty with specific colors (e.g., red, green, black, and white. In the present study, we investigated whether pairs of colors (both associated with a particular taste or taste word would give rise to stronger associations relative to pairs of colors that were associated with different tastes. We replicate the findings of previous studies highlighting the existence of a robust crossmodal correspondence between individual colors and basic tastes. However, while there was evidence that pairs of colors could indeed communicate taste information more consistently than single colors, our participants took more than twice as long to match the color pairs with tastes than the single colors. Possible reasons for these results are discussed.

Using Single Colors and Color Pairs to Communicate Basic Tastes.

Science.gov (United States)

Woods, Andy T; Spence, Charles

2016-01-01

Recently, it has been demonstrated that people associate each of the basic tastes (e.g., sweet, sour, bitter, and salty) with specific colors (e.g., red, green, black, and white). In the present study, we investigated whether pairs of colors (both associated with a particular taste or taste word) would give rise to stronger associations relative to pairs of colors that were associated with different tastes. We replicate the findings of previous studies highlighting the existence of a robust crossmodal correspondence between individual colors and basic tastes. However, while there was evidence that pairs of colors could indeed communicate taste information more consistently than single colors, our participants took more than twice as long to match the color pairs with tastes than the single colors. Possible reasons for these results are discussed.

Using Single Colors and Color Pairs to Communicate Basic Tastes II: Foreground–Background Color Combinations

Science.gov (United States)

Marmolejo-Ramos, Fernando; Velasco, Carlos; Spence, Charles

2016-01-01

People associate basic tastes (e.g., sweet, sour, bitter, and salty) with specific colors (e.g., pink or red, green or yellow, black or purple, and white or blue). In the present study, we investigated whether a color bordered by another color (either the same or different) would give rise to stronger taste associations relative to a single patch of color. We replicate previous findings, highlighting the existence of a robust crossmodal correspondence between individual colors and basic tastes. On occasion, color pairs were found to communicate taste expectations more consistently than were single color patches. Furthermore, and in contrast to a recent study in which the color pairs were shown side-by-side, participants took no longer to match the color pairs with tastes than the single colors (they had taken twice as long to respond to the color pairs in the previous study). Possible reasons for these results are discussed, and potential applications for the results, and for the testing methodology developed, are outlined. PMID:27708752

Precision and accuracy in smFRET based structural studies—A benchmark study of the Fast-Nano-Positioning System

Science.gov (United States)

Nagy, Julia; Eilert, Tobias; Michaelis, Jens

2018-03-01

Modern hybrid structural analysis methods have opened new possibilities to analyze and resolve flexible protein complexes where conventional crystallographic methods have reached their limits. Here, the Fast-Nano-Positioning System (Fast-NPS), a Bayesian parameter estimation-based analysis method and software, is an interesting method since it allows for the localization of unknown fluorescent dye molecules attached to macromolecular complexes based on single-molecule Förster resonance energy transfer (smFRET) measurements. However, the precision, accuracy, and reliability of structural models derived from results based on such complex calculation schemes are oftentimes difficult to evaluate. Therefore, we present two proof-of-principle benchmark studies where we use smFRET data to localize supposedly unknown positions on a DNA as well as on a protein-nucleic acid complex. Since we use complexes where structural information is available, we can compare Fast-NPS localization to the existing structural data. In particular, we compare different dye models and discuss how both accuracy and precision can be optimized.

Single Molecules as Optical Probes for Structure and Dynamics

Science.gov (United States)

Orrit, Michel

Single molecules and single nanoparticles are convenient links between the nanoscale world and the laboratory. We discuss the limits for their optical detection by three different methods: fluorescence, direct absorption, and photothermal detection. We briefly review some recent illustrations of qualitatively new information gathered from single-molecule signals: intermittency of the fluorescence intensity, acoustic vibrations of nanoparticles (1-100 GHz) or of extended defects in molecular crystals (0.1-1 MHz), and dynamical heterogeneity in glass-forming molecular liquids. We conclude with an outlook of future uses of single-molecule methods in physical chemistry, soft matter, and material science.

Simple single-emitting layer hybrid white organic light emitting with high color stability

Science.gov (United States)

Nguyen, C.; Lu, Z. H.

2017-10-01

Simultaneously achieving a high efficiency and color quality at luminance levels required for solid-state lighting has been difficult for white organic light emitting diodes (OLEDs). Single-emitting layer (SEL) white OLEDs, in particular, exhibit a significant tradeoff between efficiency and color stability. Furthermore, despite the simplicity of SEL white OLEDs being its main advantage, the reported device structures are often complicated by the use of multiple blocking layers. In this paper, we report a highly simplified three-layered white OLED that achieves a low turn-on voltage of 2.7 V, an external quantum efficiency of 18.9% and power efficiency of 30 lm/W at 1000 cd/cm2. This simple white OLED also shows good color quality with a color rendering index of 75, CIE coordinates (0.42, 0.46), and little color shifting at high luminance. The device consists of a SEL sandwiched between a hole transport layer and an electron transport layer. The SEL comprises a thermally activated delayer fluorescent molecule having dual functions as a blue emitter and as a host for other lower energy emitters. The improved color stability and efficiency in such a simple device structure is explained as due to the elimination of significant energy barriers at various organic-organic interfaces in the traditional devices having multiple blocking layers.

Förster resonance energy transfer: Role of diffusion of fluorophore orientation and separation in observed shifts of FRET efficiency.

Directory of Open Access Journals (Sweden)

Bram Wallace

Full Text Available Förster resonance energy transfer (FRET is a widely used single-molecule technique for measuring nanoscale distances from changes in the non-radiative transfer of energy between donor and acceptor fluorophores. For macromolecules and complexes this observed transfer efficiency is used to infer changes in molecular conformation under differing experimental conditions. However, sometimes shifts are observed in the FRET efficiency even when there is strong experimental evidence that the molecular conformational state is unchanged. We investigate ways in which such discrepancies can arise from kinetic effects. We show that significant shifts can arise from the interplay between excitation kinetics, orientation diffusion of fluorophores, separation diffusion of fluorophores, and non-emitting quenching.

Single Molecule Electronics and Devices

Science.gov (United States)

Tsutsui, Makusu; Taniguchi, Masateru

2012-01-01

The manufacture of integrated circuits with single-molecule building blocks is a goal of molecular electronics. While research in the past has been limited to bulk experiments on self-assembled monolayers, advances in technology have now enabled us to fabricate single-molecule junctions. This has led to significant progress in understanding electron transport in molecular systems at the single-molecule level and the concomitant emergence of new device concepts. Here, we review recent developments in this field. We summarize the methods currently used to form metal-molecule-metal structures and some single-molecule techniques essential for characterizing molecular junctions such as inelastic electron tunnelling spectroscopy. We then highlight several important achievements, including demonstration of single-molecule diodes, transistors, and switches that make use of electrical, photo, and mechanical stimulation to control the electron transport. We also discuss intriguing issues to be addressed further in the future such as heat and thermoelectric transport in an individual molecule. PMID:22969345

Roughness Effects on Fretting Fatigue

Science.gov (United States)

Yue, Tongyan; Abdel Wahab, Magd

2017-05-01

Fretting is a small oscillatory relative motion between two normal loaded contact surfaces. It may cause fretting fatigue, fretting wear and/or fretting corrosion damage depending on various fretting couples and working conditions. Fretting fatigue usually occurs at partial slip condition, and results in catastrophic failure at the stress levels below the fatigue limit of the material. Many parameters may affect fretting behaviour, including the applied normal load and displacement, material properties, roughness of the contact surfaces, frequency, etc. Since fretting damage is undesirable due to contacting, the effect of rough contact surfaces on fretting damage has been studied by many researchers. Experimental method on this topic is usually focusing on rough surface effects by finishing treatment and random rough surface effects in order to increase fretting fatigue life. However, most of numerical models on roughness are based on random surface. This paper reviewed both experimental and numerical methodology on the rough surface effects on fretting fatigue.

Ordered array of CoPc-vacancies filled with single-molecule rotors

Science.gov (United States)

Xie, Zheng-Bo; Wang, Ya-Li; Tao, Min-Long; Sun, Kai; Tu, Yu-Bing; Yuan, Hong-Kuan; Wang, Jun-Zhong

2018-05-01

We report the highly ordered array of CoPc-vacancies and the single-molecule rotors inside the vacancies. When CoPc molecules are deposited on Cd(0001) at low-temperature, three types of molecular vacancies appeared randomly in the CoPc monolayer. Annealing the sample to higher temperature leads to the spontaneous phase separation and self-organized arrangement of the vacancies. Highly ordered arrays of two-molecule vacancies and single-molecule vacancies have been obtained. In particular, there is a rotating CoPc molecule inside each single-molecule vacancy, which constitutes the array of single-molecule rotors. These results provide a new routine to fabricate the nano-machines on a large scale.

Single Molecule Analysis Research Tool (SMART: an integrated approach for analyzing single molecule data.

Directory of Open Access Journals (Sweden)

Max Greenfeld

Full Text Available Single molecule studies have expanded rapidly over the past decade and have the ability to provide an unprecedented level of understanding of biological systems. A common challenge upon introduction of novel, data-rich approaches is the management, processing, and analysis of the complex data sets that are generated. We provide a standardized approach for analyzing these data in the freely available software package SMART: Single Molecule Analysis Research Tool. SMART provides a format for organizing and easily accessing single molecule data, a general hidden Markov modeling algorithm for fitting an array of possible models specified by the user, a standardized data structure and graphical user interfaces to streamline the analysis and visualization of data. This approach guides experimental design, facilitating acquisition of the maximal information from single molecule experiments. SMART also provides a standardized format to allow dissemination of single molecule data and transparency in the analysis of reported data.

Fuel-element vibration and bearing pad to pressure tube fretting

International Nuclear Information System (INIS)

Fisher, N.J.; Taylor, C.E.; Pettigrew, M.J.

1990-08-01

Fuel channel operation under boiling condition results in increased flow velocities, which may lead to unacceptable fuel-element vibration and bearing pad to pressure tube fretting. The existing endurance test database does not fully cover the range of future channel operating conditions. In particular, after refuelling, some channels for future designs may operate with two-phase flow conditions outside the range of endurance test conditions. Full-scale endurance testing at realistic steam-water conditions involves substantial energy costs. Therefore, fundamental laboratory investigations were conducted to define and endurance test matrix which adequately envelops the future range of operating conditions while minimizing both the number of tests and the energy requirement of individual tests. The main focus of the laboratory investigations was to establish the relationships between: fuel channel flow conditions and fuel-element vibration; and fuel-element vibration and bearing pad to pressure tube fretting. The vibration response of a single fuel element was measured over a wide range of operating conditions covering realistic fuel channel conditions and simulated endurance testing conditions. For higher void fractions, the vibration amplitudes measured in air/water were much higher than in steam/water, while for low void fractions, the amplitudes were similar. The measured amplitudes in steam/water varied very little over the range of temperature and pressure investigated. The effects of temperature, pressure tube oxide thickness, vibration amplitude and bearing pad manufacturer on pressure tube fretting were investigated. The fretting rate is extremely temperature dependent. For vibration amplitudes about three or four times greater than expected in-reactor conditions, peak fretting rates were observed in the 225 to 286 degrees C temperature range. Fretting rates were seven times less at the higher temperatures of 300 and 315 degrees C, and the lower temperatures

Single-molecule dynamics in nanofabricated traps

Science.gov (United States)

Cohen, Adam

2009-03-01

The Anti-Brownian Electrokinetic trap (ABEL trap) provides a means to immobilize a single fluorescent molecule in solution, without surface attachment chemistry. The ABEL trap works by tracking the Brownian motion of a single molecule, and applying feedback electric fields to induce an electrokinetic motion that approximately cancels the Brownian motion. We present a new design for the ABEL trap that allows smaller molecules to be trapped and more information to be extracted from the dynamics of a single molecule than was previously possible. In particular, we present strategies for extracting dynamically fluctuating mobilities and diffusion coefficients, as a means to probe dynamic changes in molecular charge and shape. If one trapped molecule is good, many trapped molecules are better. An array of single molecules in solution, each immobilized without surface attachment chemistry, provides an ideal test-bed for single-molecule analyses of intramolecular dynamics and intermolecular interactions. We present a technology for creating such an array, using a fused silica plate with nanofabricated dimples and a removable cover for sealing single molecules within the dimples. With this device one can watch the shape fluctuations of single molecules of DNA or study cooperative interactions in weakly associating protein complexes.

Single-photon sources based on single molecules in solids

International Nuclear Information System (INIS)

Moerner, W E

2004-01-01

Single molecules in suitable host crystals have been demonstrated to be useful single-photon emitters both at liquid-helium temperatures and at room temperature. The low-temperature source achieved controllable emission of single photons from a single terrylene molecule in p-terphenyl by an adiabatic rapid passage technique. In contrast with almost all other single-molecule systems, terrylene single molecules show extremely high photostability under continuous, high-intensity irradiation. A room-temperature source utilizing this material has been demonstrated, in which fast pumping into vibrational sidebands of the electronically excited state achieved efficient inversion of the emissive level. This source yielded a single-photon emission probability p(1) of 0.86 at a detected count rate near 300 000 photons s -1 , with very small probability of emission of more than one photon. Thus, single molecules in solids can be considered as contenders for applications of single-photon sources such as quantum key distribution

Single Molecule Nanoelectrochemistry in Electrical Junctions.

Science.gov (United States)

Nichols, Richard J; Higgins, Simon J

2016-11-15

gating. This has been referred to as to a "single molecule transistor configuration" with the gate voltage being provided by the controllable potential achieved through the electrochemical double layer. It is shown how the electrolyte medium can control such gating, with ionic liquids providing more efficient gate coupling than aqueous electrolytes. Control of the conductance of viologen molecular wires can also be achieved by encapsulating the viologen redox moiety within a molecular cage, thereby controlling its immediate environment. Molecular conductance can also be gated through multiple redox states. This has been shown for the redox moiety pyrrolo-tetrathiafulvalene, which undergoes single molecule electrochemical transistor gating through three redox states in molecular junctions. Charge transport through this junction follows a two-step hopping mechanism, demonstrating the role of the redox center in electron transfer across the molecular bridge. Recent electrolyte gating studies of rigid, conjugated redox-active metal complexes with tailored terpyridine coordinating ligands and anchors are also presented. These aforementioned studies have all been performed with gold electrode contacts. The Account concludes with recent data showing that it is now possible to study single molecule electrochemical gating with nickel electrodes. This opens up new perspectives for studying interfacial charge transfer with a wide variety of other electrode materials including semiconductor electrodes and also points toward future opportunities for coupling molecular spintronics and nanoelectrochemistry.

FRET-based biosensors for the detection and quantification of AI-2 class of quorum sensing compounds.

Science.gov (United States)

Rajamani, Sathish; Sayre, Richard

2011-01-01

Intercellular small molecular weight signaling molecules modulate a variety of biological functions in bacteria. One of the more complex behaviors mediated by intercellular signaling molecules is the suite of activities regulated by quorum sensing molecules. These molecules mediate a variety of population-dependent responses, including the expression of genes that regulate bioluminescence, type III secretion, siderophore production, colony morphology, biofilm formation, and metalloprotease production. Given their central role in regulating these responses, the detection and quantification of QS molecules has important practical implications. Until recently, the detection of QS molecules from Gram-negative bacteria has relied primarily on bacterial reporter systems. These bioassays though immensely useful are subject to interference by compounds that affect bacterial growth and metabolism. In addition, the reporter response is highly dependent on culture age and cell population density. To overcome such limitations, we developed an in vitro protein-based assay system for the rapid detection and quantification of the furanosyl borate diester (BAI-2) subclass of autoinducer-2 (AI-2) QS molecules. The biosensor is based on the interaction of BAI-2 with the Vibrio harveyi QS receptor LuxP. Conformation changes associated with BAI-2 binding to the LuxP receptor change the orientation of cyan and yellow variants of GFP (CFP and YFP) fused the N- and C-termini, respectively, of the LuxP receptor. LuxP-BAI2 binding induces changes in fluorescence resonance energy transfer (FRET) between CFP and YFP, whose magnitude of change is ligand concentration dependent. A set of ligand-insensitive LuxP-mutant FRET protein sensor was also developed for use as control biosensors. The FRET-based BAI-2 biosensor responds selectively to both synthetic and biologically derived BAI-2compounds. This report describes the use of the LuxP-FRET biosensor for the detection and quantification of

Single-Molecule Spectroscopy

Indian Academy of Sciences (India)

Home; Journals; Resonance – Journal of Science Education; Volume 20; Issue 2. Single-Molecule Spectroscopy: Every Molecule is Different! Kankan Bhattacharyya. General Article Volume 20 Issue 2 February 2015 pp 151-164. Fulltext. Click here to view fulltext PDF. Permanent link:

Preface: Special Topic on Single-Molecule Biophysics.

Science.gov (United States)

Makarov, Dmitrii E; Schuler, Benjamin

2018-03-28

Single-molecule measurements are now almost routinely used to study biological systems and processes. The scope of this special topic emphasizes the physics side of single-molecule observations, with the goal of highlighting new developments in physical techniques as well as conceptual insights that single-molecule measurements bring to biophysics. This issue also comprises recent advances in theoretical physical models of single-molecule phenomena, interpretation of single-molecule signals, and fundamental areas of statistical mechanics that are related to single-molecule observations. A particular goal is to illustrate the increasing synergy between theory, simulation, and experiment in single-molecule biophysics.

Single-molecule FRET unveils induced-fit mechanism for substrate selectivity in flap endonuclease 1

KAUST Repository

Rashid, Fahad

2017-02-23

Human flap endonuclease 1 (FEN1) and related structure-specific 5\\'nucleases precisely identify and incise aberrant DNA structures during replication, repair and recombination to avoid genomic instability. Yet, it is unclear how the 5\\'nuclease mechanisms of DNA distortion and protein ordering robustly mediate efficient and accurate substrate recognition and catalytic selectivity. Here, single-molecule sub-millisecond and millisecond analyses of FEN1 reveal a protein-DNA induced-fit mechanism that efficiently verifies substrate and suppresses off-target cleavage. FEN1 sculpts DNA with diffusion-limited kinetics to test DNA substrate. This DNA distortion mutually \\'locks\\' protein and DNA conformation and enables substrate verification with extreme precision. Strikingly, FEN1 never misses cleavage of its cognate substrate while blocking probable formation of catalytically competent interactions with noncognate substrates and fostering their pre-incision dissociation. These findings establish FEN1 has practically perfect precision and that separate control of induced-fit substrate recognition sets up the catalytic selectivity of the nuclease active site for genome stability.

Single Molecule Screening of Disease DNA Without Amplification

Energy Technology Data Exchange (ETDEWEB)

Lee, Ji-Young [Iowa State Univ., Ames, IA (United States)

2006-01-01

The potential of single molecule detection as an analysis tool in biological and medical fields is well recognized today. This fast evolving technique will provide fundamental sensitivity to pick up individual pathogen molecules, and therefore contribute to a more accurate diagnosis and a better chance for a complete cure. Many studies are being carried out to successfully apply this technique in real screening fields. In this dissertation, several attempts are shown that have been made to test and refine the application of the single molecule technique as a clinical screening method. A basic applicability was tested with a 100% target content sample, using electrophoretic mobility and multiple colors as identification tools. Both electrophoretic and spectral information of individual molecule were collected within a second, while the molecule travels along the flow in a capillary. Insertion of a transmission grating made the recording of the whole spectrum of a dye-stained molecule possible without adding complicated instrumental components. Collecting two kinds of information simultaneously and combining them allowed more thorough identification, up to 98.8% accuracy. Probing mRNA molecules with fluorescently labeled cDNA via hybridization was also carried out. The spectral differences among target, probe, and hybrid were interpreted in terms of dispersion distances after transmission grating, and used for the identification of each molecule. The probes were designed to have the least background when they are free, but have strong fluorescence after hybridization via fluorescence resonance energy transfer. The mRNA-cDNA hybrids were further imaged in whole blood, plasma, and saliva, to test how far a crude preparation can be tolerated. Imaging was possible with up to 50% of clear bio-matrix contents, suggesting a simple lysis and dilution would be sufficient for imaging for some cells. Real pathogen DNA of human papillomavirus (HPV) type-I6 in human genomic DNA

Easy-DHPSF open-source software for three-dimensional localization of single molecules with precision beyond the optical diffraction limit.

Science.gov (United States)

Lew, Matthew D; von Diezmann, Alexander R S; Moerner, W E

2013-02-25

Automated processing of double-helix (DH) microscope images of single molecules (SMs) streamlines the protocol required to obtain super-resolved three-dimensional (3D) reconstructions of ultrastructures in biological samples by single-molecule active control microscopy. Here, we present a suite of MATLAB subroutines, bundled with an easy-to-use graphical user interface (GUI), that facilitates 3D localization of single emitters (e.g. SMs, fluorescent beads, or quantum dots) with precisions of tens of nanometers in multi-frame movies acquired using a wide-field DH epifluorescence microscope. The algorithmic approach is based upon template matching for SM recognition and least-squares fitting for 3D position measurement, both of which are computationally expedient and precise. Overlapping images of SMs are ignored, and the precision of least-squares fitting is not as high as maximum likelihood-based methods. However, once calibrated, the algorithm can fit 15-30 molecules per second on a 3 GHz Intel Core 2 Duo workstation, thereby producing a 3D super-resolution reconstruction of 100,000 molecules over a 20×20×2 μm field of view (processing 128×128 pixels × 20000 frames) in 75 min.

A large collapsed-state RNA can exhibit simple exponential single-molecule dynamics.

Science.gov (United States)

Smith, Glenna J; Lee, Kang Taek; Qu, Xiaohui; Xie, Zheng; Pesic, Jelena; Sosnick, Tobin R; Pan, Tao; Scherer, Norbert F

2008-05-09

The process of large RNA folding is believed to proceed from many collapsed structures to a unique functional structure requiring precise organization of nucleotides. The diversity of possible structures and stabilities of large RNAs could result in non-exponential folding kinetics (e.g. stretched exponential) under conditions where the molecules have not achieved their native state. We describe a single-molecule fluorescence resonance energy transfer (FRET) study of the collapsed-state region of the free energy landscape of the catalytic domain of RNase P RNA from Bacillus stearothermophilus (C(thermo)). Ensemble measurements have shown that this 260 residue RNA folds cooperatively to its native state at >or=1 mM Mg(2+), but little is known about the conformational dynamics at lower ionic strength. Our measurements of equilibrium conformational fluctuations reveal simple exponential kinetics that reflect a small number of discrete states instead of the expected inhomogeneous dynamics. The distribution of discrete dwell times, collected from an "ensemble" of 300 single molecules at each of a series of Mg(2+) concentrations, fit well to a double exponential, which indicates that the RNA conformational changes can be described as a four-state system. This finding is somewhat unexpected under [Mg(2+)] conditions in which this RNA does not achieve its native state. Observation of discrete well-defined conformations in this large RNA that are stable on the seconds timescale at low [Mg(2+)] (<0.1 mM) suggests that even at low ionic strength, with a tremendous number of possible (weak) interactions, a few critical interactions may produce deep energy wells that allow for rapid averaging of motions within each well, and yield kinetics that are relatively simple.

Applicability of out-of-pile fretting wear tests to in-reactor fretting wear-induced failure time prediction

Science.gov (United States)

Kim, Kyu-Tae

2013-02-01

In order to investigate whether or not the grid-to-rod fretting wear-induced fuel failure will occur for newly developed spacer grid spring designs for the fuel lifetime, out-of-pile fretting wear tests with one or two fuel assemblies are to be performed. In this study, the out-of-pile fretting wear tests were performed in order to compare the potential for wear-induced fuel failure in two newly-developed, Korean PWR spacer grid designs. Lasting 20 days, the tests simulated maximum grid-to-rod gap conditions and the worst flow induced vibration effects that might take place over the fuel life time. The fuel rod perforation times calculated from the out-of-pile tests are greater than 1933 days for 2 μm oxidized fuel rods with a 100 μm grid-to-rod gap, whereas those estimated from in-reactor fretting wear failure database may be about in the range of between 60 and 100 days. This large discrepancy in fuel rod perforation may occur due to irradiation-induced cladding oxide microstructure changes on the one hand and a temperature gradient-induced hydrogen content profile across the cladding metal region on the other hand, which may accelerate brittleness in the grid-contacting cladding oxide and metal regions during the reactor operation. A three-phase grid-to-rod fretting wear model is proposed to simulate in-reactor fretting wear progress into the cladding, considering the microstructure changes of the cladding oxide and the hydrogen content profile across the cladding metal region combined with the temperature gradient. The out-of-pile tests cannot be directly applicable to the prediction of in-reactor fretting wear-induced cladding perforations but they can be used only for evaluating a relative wear resistance of one grid design against the other grid design.

Single-Molecule Interfacial Electron Transfer

Energy Technology Data Exchange (ETDEWEB)

Lu, H. Peter [Bowling Green State Univ., Bowling Green, OH (United States). Dept. of Chemistry and Center for Photochemical Sciences

2017-11-28

This project is focused on the use of single-molecule high spatial and temporal resolved techniques to study molecular dynamics in condensed phase and at interfaces, especially, the complex reaction dynamics associated with electron and energy transfer rate processes. The complexity and inhomogeneity of the interfacial ET dynamics often present a major challenge for a molecular level comprehension of the intrinsically complex systems, which calls for both higher spatial and temporal resolutions at ultimate single-molecule and single-particle sensitivities. Combined single-molecule spectroscopy and electrochemical atomic force microscopy approaches are unique for heterogeneous and complex interfacial electron transfer systems because the static and dynamic inhomogeneities can be identified and characterized by studying one molecule at a specific nanoscale surface site at a time. The goal of our project is to integrate and apply these spectroscopic imaging and topographic scanning techniques to measure the energy flow and electron flow between molecules and substrate surfaces as a function of surface site geometry and molecular structure. We have been primarily focusing on studying interfacial electron transfer under ambient condition and electrolyte solution involving both single crystal and colloidal TiO₂ and related substrates. The resulting molecular level understanding of the fundamental interfacial electron transfer processes will be important for developing efficient light harvesting systems and broadly applicable to problems in fundamental chemistry and physics. We have made significant advancement on deciphering the underlying mechanism of the complex and inhomogeneous interfacial electron transfer dynamics in dyesensitized TiO₂ nanoparticle systems that strongly involves with and regulated by molecule-surface interactions. We have studied interfacial electron transfer on TiO₂ nanoparticle surfaces by using ultrafast single-molecule

SISGR: Room Temperature Single-Molecule Detection and Imaging by Stimulated Emission Microscopy

Energy Technology Data Exchange (ETDEWEB)

Xie, Xiaoliang Sunney [Harvard Univ., Cambridge, MA (United States). Dept. of Chemistry and Chemical Biology

2017-03-13

specificity than absorption and fluorescence. Current sensitivity limit of SRS microscopy has not yet reached single molecule detection. We proposed to capitalize on our state-of-the-art SRS microscopy and develop near-resonance enhanced SRS for single molecule detection of carotenoids and heme proteins. The specific aims we pursued are: (1) building the next SRS generation microscope that utilizes near resonance enhancement to allow detection and imaging of single molecules with undetectable fluorescence, such as -carotene. (2) using near-resonance SRS as a contrast mechanism to study dye-sensitize semiconductor interface, elucidating the heterogeneous electron ejection kinetics with high spatial and temporal resolution. (3) studying the binding and unbinding of oxygen in single hemoglobin molecules in order to gain molecular level understanding of the long-standing issue of cooperativity. The new methods developed in the fund period of this grant have advanced the detection sensitivity in many aspects. Near-resonance SRS improved the signal by using shorter wavelengths for SRS microscopy. Frequency modulation and multi-color SRS target the reduction of background to improve the chemical specificity of SRS while maintaining the high imaging speed. Time-domain coherent Raman scattering microscopy targets to reduce the noise floor of coherent Raman microscopy. These methods have already demonstrated first-of-a-kind new applications in biology and medical research. However, we are still one order of magnitude away from single molecule limit. It is important to continue to improve the laser specification and develop new imaging methods to finally achieve label-free single molecule microscopy.

Quantifying the Assembly of Multicomponent Molecular Machines by Single-Molecule Total Internal Reflection Fluorescence Microscopy.

Science.gov (United States)

Boehm, E M; Subramanyam, S; Ghoneim, M; Washington, M Todd; Spies, M

2016-01-01

Large, dynamic macromolecular complexes play essential roles in many cellular processes. Knowing how the components of these complexes associate with one another and undergo structural rearrangements is critical to understanding how they function. Single-molecule total internal reflection fluorescence (TIRF) microscopy is a powerful approach for addressing these fundamental issues. In this article, we first discuss single-molecule TIRF microscopes and strategies to immobilize and fluorescently label macromolecules. We then review the use of single-molecule TIRF microscopy to study the formation of binary macromolecular complexes using one-color imaging and inhibitors. We conclude with a discussion of the use of TIRF microscopy to examine the formation of higher-order (i.e., ternary) complexes using multicolor setups. The focus throughout this article is on experimental design, controls, data acquisition, and data analysis. We hope that single-molecule TIRF microscopy, which has largely been the province of specialists, will soon become as common in the tool box of biophysicists and biochemists as structural approaches have become today. © 2016 Elsevier Inc. All rights reserved.

Single-molecule dataset (SMD): a generalized storage format for raw and processed single-molecule data.

Science.gov (United States)

Greenfeld, Max; van de Meent, Jan-Willem; Pavlichin, Dmitri S; Mabuchi, Hideo; Wiggins, Chris H; Gonzalez, Ruben L; Herschlag, Daniel

2015-01-16

Single-molecule techniques have emerged as incisive approaches for addressing a wide range of questions arising in contemporary biological research [Trends Biochem Sci 38:30-37, 2013; Nat Rev Genet 14:9-22, 2013; Curr Opin Struct Biol 2014, 28C:112-121; Annu Rev Biophys 43:19-39, 2014]. The analysis and interpretation of raw single-molecule data benefits greatly from the ongoing development of sophisticated statistical analysis tools that enable accurate inference at the low signal-to-noise ratios frequently associated with these measurements. While a number of groups have released analysis toolkits as open source software [J Phys Chem B 114:5386-5403, 2010; Biophys J 79:1915-1927, 2000; Biophys J 91:1941-1951, 2006; Biophys J 79:1928-1944, 2000; Biophys J 86:4015-4029, 2004; Biophys J 97:3196-3205, 2009; PLoS One 7:e30024, 2012; BMC Bioinformatics 288 11(8):S2, 2010; Biophys J 106:1327-1337, 2014; Proc Int Conf Mach Learn 28:361-369, 2013], it remains difficult to compare analysis for experiments performed in different labs due to a lack of standardization. Here we propose a standardized single-molecule dataset (SMD) file format. SMD is designed to accommodate a wide variety of computer programming languages, single-molecule techniques, and analysis strategies. To facilitate adoption of this format we have made two existing data analysis packages that are used for single-molecule analysis compatible with this format. Adoption of a common, standard data file format for sharing raw single-molecule data and analysis outcomes is a critical step for the emerging and powerful single-molecule field, which will benefit both sophisticated users and non-specialists by allowing standardized, transparent, and reproducible analysis practices.

Functional Dynamics within the Human Ribosome Regulate the Rate of Active Protein Synthesis.

Science.gov (United States)

Ferguson, Angelica; Wang, Leyi; Altman, Roger B; Terry, Daniel S; Juette, Manuel F; Burnett, Benjamin J; Alejo, Jose L; Dass, Randall A; Parks, Matthew M; Vincent, C Theresa; Blanchard, Scott C

2015-11-05

The regulation of protein synthesis contributes to gene expression in both normal physiology and disease, yet kinetic investigations of the human translation mechanism are currently lacking. Using single-molecule fluorescence imaging methods, we have quantified the nature and timing of structural processes in human ribosomes during single-turnover and processive translation reactions. These measurements reveal that functional complexes exhibit dynamic behaviors and thermodynamic stabilities distinct from those observed for bacterial systems. Structurally defined sub-states of pre- and post-translocation complexes were sensitive to specific inhibitors of the eukaryotic ribosome, demonstrating the utility of this platform to probe drug mechanism. The application of three-color single-molecule fluorescence resonance energy transfer (smFRET) methods further revealed a long-distance allosteric coupling between distal tRNA binding sites within ribosomes bearing three tRNAs, which contributed to the rate of processive translation. Copyright © 2015 Elsevier Inc. All rights reserved.

Fluorescent Biosensors Based on Single-Molecule Counting.

Science.gov (United States)

Ma, Fei; Li, Ying; Tang, Bo; Zhang, Chun-Yang

2016-09-20

Biosensors for highly sensitive, selective, and rapid quantification of specific biomolecules make great contributions to biomedical research, especially molecular diagnostics. However, conventional methods for biomolecular assays often suffer from insufficient sensitivity and poor specificity. In some case (e.g., early disease diagnostics), the concentration of target biomolecules is too low to be detected by these routine approaches, and cumbersome procedures are needed to improve the detection sensitivity. Therefore, there is an urgent need for rapid and ultrasensitive analytical tools. In this respect, single-molecule fluorescence approaches may well satisfy the requirement and hold promising potential for the development of ultrasensitive biosensors. Encouragingly, owing to the advances in single-molecule microscopy and spectroscopy over past decades, the detection of single fluorescent molecule comes true, greatly boosting the development of highly sensitive biosensors. By in vitro/in vivo labeling of target biomolecules with proper fluorescent tags, the quantification of certain biomolecule at the single-molecule level is achieved. In comparison with conventional ensemble measurements, single-molecule detection-based analytical methods possess the advantages of ultrahigh sensitivity, good selectivity, rapid analysis time, and low sample consumption. Consequently, single-molecule detection may be potentially employed as an ideal analytical approach to quantify low-abundant biomolecules with rapidity and simplicity. In this Account, we will summarize our efforts for developing a series of ultrasensitive biosensors based on single-molecule counting. Single-molecule counting is a member of single-molecule detection technologies and may be used as a very simple and ultrasensitive method to quantify target molecules by simply counting the individual fluorescent bursts. In the fluorescent sensors, the signals of target biomolecules may be translated to the

Extracting Models in Single Molecule Experiments

Science.gov (United States)

Presse, Steve

2013-03-01

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.

Electron transfer dynamics of bistable single-molecule junctions

DEFF Research Database (Denmark)

Danilov, A.V; Kubatkin, S.; Kafanov, S. G.

2006-01-01

We present transport measurements of single-molecule junctions bridged by a molecule with three benzene rings connected by two double bonds and with thiol end-groups that allow chemical binding to gold electrodes. The I-V curves show switching behavior between two distinct states. By statistical ...... analysis of the switching events, we show that a 300 meV mode mediates the transition between the two states. We propose that breaking and reformation of a S-H bond in the contact zone between molecule and electrode explains the observed bistability....

Standard guide for fretting fatigue testing

CERN Document Server

American Society for Testing and Materials. Philadelphia

2010-01-01

1.1 This guide defines terminology and covers general requirements for conducting fretting fatigue tests and reporting the results. It describes the general types of fretting fatigue tests and provides some suggestions on developing and conducting fretting fatigue test programs. 1.2 Fretting fatigue tests are designed to determine the effects of mechanical and environmental parameters on the fretting fatigue behavior of metallic materials. This guide is not intended to establish preference of one apparatus or specimen design over others, but will establish guidelines for adherence in the design, calibration, and use of fretting fatigue apparatus and recommend the means to collect, record, and reporting of the data. 1.3 The number of cycles to form a fretting fatigue crack is dependent on both the material of the fatigue specimen and fretting pad, the geometry of contact between the two, and the method by which the loading and displacement are imposed. Similar to wear behavior of materials, it is important t...

Investigation of sliding DNA clamp dynamics by single-molecule fluorescence, mass spectrometry and structure-based modeling

Science.gov (United States)

Gadkari, Varun V; Harvey, Sophie R; Raper, Austin T; Chu, Wen-Ting; Wang, Jin; Wysocki, Vicki H; Suo, Zucai

2018-01-01

Abstract Proliferating cell nuclear antigen (PCNA) is a trimeric ring-shaped clamp protein that encircles DNA and interacts with many proteins involved in DNA replication and repair. Despite extensive structural work to characterize the monomeric, dimeric, and trimeric forms of PCNA alone and in complex with interacting proteins, no structure of PCNA in a ring-open conformation has been published. Here, we use a multidisciplinary approach, including single-molecule Förster resonance energy transfer (smFRET), native ion mobility-mass spectrometry (IM-MS), and structure-based computational modeling, to explore the conformational dynamics of a model PCNA from Sulfolobus solfataricus (Sso), an archaeon. We found that Sso PCNA samples ring-open and ring-closed conformations even in the absence of its clamp loader complex, replication factor C, and transition to the ring-open conformation is modulated by the ionic strength of the solution. The IM-MS results corroborate the smFRET findings suggesting that PCNA dynamics are maintained in the gas phase and further establishing IM-MS as a reliable strategy to investigate macromolecular motions. Our molecular dynamic simulations agree with the experimental data and reveal that ring-open PCNA often adopts an out-of-plane left-hand geometry. Collectively, these results implore future studies to define the roles of PCNA dynamics in DNA loading and other PCNA-mediated interactions. PMID:29529283

Sensitivity-Enhancement of FRET Immunoassays by Multiple-Antibody Conjugation on Quantum Dots.

Science.gov (United States)

Annio, Giacomo; Jennings, Travis; Tagit, Oya; Hildebrandt, Niko

2018-05-23

Quantum dots (QDs) are not only advantageous for color-tuning, improved brightness, and high stability, but their nanoparticle surfaces also allow for the attachment of many biomolecules. Because IgG antibodies (ABs) are in the same size range of biocompatible QDs and the AB orientation after conjugation to the QD is often random, it is difficult to predict if few or many ABs per QD will lead to an efficient AB-QD conjugate. This is particularly true for homogeneous Förster resonance energy transfer (FRET) sandwich immunoassays, for which the ABs on the QD must bind a biomarker that needs to bind a second AB-FRET-conjugate. Here, we investigate the performance of Tb-to-QD FRET immunoassays against total prostate specific antigen (TPSA) by changing the number of ABs per QD while leaving all the other assay components unchanged. We first characterize the AB-QD conjugation by various spectroscopic, microscopic, and chromatographic techniques and then quantify the TPSA immunoassay performance regarding sensitivity, limit of detection, and dynamic range. Our results show that an increasing conjugation ratio leads to significantly enhanced FRET immunoassays. These findings will be highly important for developing QD-based immunoassays in which the concentrations of both ABs and QDs can significantly influence the assay performance.

Single-exposure color digital holography

Science.gov (United States)

Feng, Shaotong; Wang, Yanhui; Zhu, Zhuqing; Nie, Shouping

2010-11-01

In this paper, we report a method for color image reconstruction by recording only one single multi-wavelength hologram. In the recording process, three lasers of different wavelengths emitting in the red, green and blue regions are used for illuminating on the object and the object diffraction fields will arrive at the hologram plane simultaneously. Three reference beams with different spatial angles will interfere with the corresponding object diffraction fields on the hologram plane, respectively. Finally, a series of sub-holograms incoherently overlapped on the CCD to be recorded as a multi-wavelength hologram. Angular division multiplexing is employed to reference beams so that the spatial spectra of the multiple recordings will be separated in the Fourier plane. In the reconstruction process, the multi-wavelength hologram will be Fourier transformed into its Fourier plane, where the spatial spectra of different wavelengths are separated and can be easily extracted by employing frequency filtering. The extracted spectra are used to reconstruct the corresponding monochromatic complex amplitudes, which will be synthesized to reconstruct the color image. For singleexposure recording technique, it is convenient for applications on the real-time image processing fields. However, the quality of the reconstructed images is affected by speckle noise. How to improve the quality of the images needs for further research.

Developing DNA nanotechnology using single-molecule fluorescence.

Science.gov (United States)

Tsukanov, Roman; Tomov, Toma E; Liber, Miran; Berger, Yaron; Nir, Eyal

2014-06-17

CONSPECTUS: An important effort in the DNA nanotechnology field is focused on the rational design and manufacture of molecular structures and dynamic devices made of DNA. As is the case for other technologies that deal with manipulation of matter, rational development requires high quality and informative feedback on the building blocks and final products. For DNA nanotechnology such feedback is typically provided by gel electrophoresis, atomic force microscopy (AFM), and transmission electron microscopy (TEM). These analytical tools provide excellent structural information; however, usually they do not provide high-resolution dynamic information. For the development of DNA-made dynamic devices such as machines, motors, robots, and computers this constitutes a major problem. Bulk-fluorescence techniques are capable of providing dynamic information, but because only ensemble averaged information is obtained, the technique may not adequately describe the dynamics in the context of complex DNA devices. The single-molecule fluorescence (SMF) technique offers a unique combination of capabilities that make it an excellent tool for guiding the development of DNA-made devices. The technique has been increasingly used in DNA nanotechnology, especially for the analysis of structure, dynamics, integrity, and operation of DNA-made devices; however, its capabilities are not yet sufficiently familiar to the community. The purpose of this Account is to demonstrate how different SMF tools can be utilized for the development of DNA devices and for structural dynamic investigation of biomolecules in general and DNA molecules in particular. Single-molecule diffusion-based Förster resonance energy transfer and alternating laser excitation (sm-FRET/ALEX) and immobilization-based total internal reflection fluorescence (TIRF) techniques are briefly described and demonstrated. To illustrate the many applications of SMF to DNA nanotechnology, examples of SMF studies of DNA hairpins and

FRET microscopy autologous tumor lysate processing in mature dendritic cell vaccine therapy

Directory of Open Access Journals (Sweden)

Ridolfi Ruggero

2010-06-01

Full Text Available Abstract Background Antigen processing by dendritic cells (DC exposed to specific stimuli has been well characterized in biological studies. Nonetheless, the question of whether autologous whole tumor lysates (as used in clinical trials are similarly processed by these cells has not yet been resolved. Methods In this study, we examined the transfer of peptides from whole tumor lysates to major histocompatibility complex class II molecules (MHC II in mature dendritic cells (mDC from a patient with advanced melanoma. Tumor antigenic peptides-MHC II proximity was revealed by Förster Resonance Energy Transfer (FRET measurements, which effectively extends the application of fluorescence microscopy to the molecular level ( Results We detected significant energy transfer between donor and acceptor-labelled antibodies against HLA-DR at the membrane surface of mDC. FRET data indicated that fluorescent peptide-loaded MHC II molecules start to accumulate on mDC membranes at 16 hr from the maturation stimulus, steeply increasing at 22 hr with sustained higher FRET detected up to 46 hr. Conclusions The results obtained imply that the patient mDC correctly processed the tumor specific antigens and their display on the mDC surface may be effective for several days. These observations support the rationale for immunogenic efficacy of autologous tumor lysates.

Understanding and modeling Förster-type resonance energy transfer (FRET) introduction to FRET

CERN Document Server

Govorov, Alexander; Demir, Hilmi Volkan

2016-01-01

This Brief presents a historical overview of the Förster-type nonradiative energy transfer and a compilation of important progress in FRET research, starting from Förster until today, along with a summary of the current state-of-the-art. Here the objective is to provide the reader with a complete account of important milestones in FRET studies and FRET applications as well as a picture of the current status.

Nano-manipulation of single DNA molecules

International Nuclear Information System (INIS)

Hu Jun; Shanghai Jiaotong Univ., Shanghai; Lv Junhong; Wang Guohua; Wang Ying; Li Minqian; Zhang Yi; Li Bin; Li Haikuo; An Hongjie

2004-01-01

Nano-manipulation of single atoms and molecules is a critical technique in nanoscience and nanotechnology. This review paper will focus on the recent development of the manipulation of single DNA molecules based on atomic force microscopy (AFM). Precise manipulation has been realized including varied manipulating modes such as 'cutting', 'pushing', 'folding', 'kneading', 'picking up', 'dipping', etc. The cutting accuracy is dominated by the size of the AFM tip, which is usually 10 nm or less. Single DNA fragments can be cut and picked up and then amplified by single molecule PCR. Thus positioning isolation and sequencing can be performed. (authors)

Effect of mixed alloy combinations on fretting corrosion performance of spinal screw and rod implants.

Science.gov (United States)

Mali, Sachin A; Singh, Vaneet; Gilbert, Jeremy L

2017-07-01

Spinal implants are made from a variety of materials to meet the unique mechanical demands of each application. However, the medical device community has raised concern about mixing dissimilar metals in an implant because of fear of inducing corrosion. There is a lack of systematic studies on the effects of mixing metals on performance of spinal implants, especially in fretting corrosion conditions. Hence, the goal was to determine whether mixing stainless steel (SS316L), titanium alloy (Ti6Al4V) and cobalt chromium (CoCrMo) alloy components in a spinal implant leads to any increased risk of corrosion degradation. Spinal constructs consisting of single assembly screw-connector-rod components were tested using a novel short-term cyclic fretting corrosion test method. A total of 17 alloy component combinations (comprised of SS316L, Ti6Al4V-anodized and CoCrMo alloy for rod, screws and connectors) were tested under three anatomic orientations. Spinal constructs having all SS316L were most susceptible to fretting-initiated crevice corrosion attack and showed higher average fretting currents (∼25 - 30 µA), whereas constructs containing all Ti6Al4V components were less susceptible to fretting corrosion with average fretting currents in the range of 1 - 6 µA. Mixed groups showed evidence of fretting corrosion but they were not as severe as all SS316L group. SEM results showed evidence of severe corrosion attack in constructs having SS316L components. There also did not appear to be any galvanic effects of combining alloys together. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1169-1177, 2017. © 2016 Wiley Periodicals, Inc.

Entangled photons from single atoms and molecules

Science.gov (United States)

Nordén, Bengt

2018-05-01

The first two-photon entanglement experiment performed 50 years ago by Kocher and Commins (KC) provided isolated pairs of entangled photons from an atomic three-state fluorescence cascade. In view of questioning of Bell's theorem, data from these experiments are re-analyzed and shown sufficiently precise to confirm quantum mechanical and dismiss semi-classical theory without need for Bell's inequalities. Polarization photon correlation anisotropy (A) is useful: A is near unity as predicted quantum mechanically and well above the semi-classic range, 0 ⩽ A ⩽ 1 / 2 . Although yet to be found, one may envisage a three-state molecule emitting entangled photon pairs, in analogy with the KC atomic system. Antibunching in fluorescence from single molecules in matrix and entangled photons from quantum dots promise it be possible. Molecules can have advantages to parametric down-conversion as the latter photon distribution is Poissonian and unsuitable for producing isolated pairs of entangled photons. Analytical molecular applications of entangled light are also envisaged.

Electrochemistry and bioelectrochemistry towards the single-molecule level: Theoretical notions and systems

International Nuclear Information System (INIS)

Zhang Jingdong; Chi Qijin; Albrecht, Tim; Kuznetsov, Alexander M.; Grubb, Mikala; Hansen, Allan G.; Wackerbarth, Hainer; Welinder, Anne C.; Ulstrup, Jens

2005-01-01

Surface structures controlled at the nanometer and single-molecule levels, with functions crucially determined by interfacial electron transfer (ET) are broadly reported in recent years, with different kinds of electrochemically controlled nanoscale/single molecule systems. One is the broad class of metallic and semiconductor-based nanoparticles, nano-arrays, nanotubes, and nanopits. Others are based on self-assembled molecular monolayers. The latter extend to bioelectrochemical systems with redox metalloproteins and DNA-based molecules as targets. We overview here some recent achievements in areas of interfacial electrochemical ET systems, mapped to the nanoscale and single-molecule levels. Focus is on both experimental and theoretical studies in our group. Systems addressed are organized monolayers of redox active transition metal complexes, and metalloproteins and metalloenzymes on single-crystal Au(1 1 1)-electrode surfaces. These systems have been investigated by voltammetry, spectroscopy, microcantilever technology, and scanning probe microscopy. A class of Os-complexes has shown suitable as targets for electrochemical in situ scanning tunnelling microscopy (STM), with close to single-molecule scanning tunnelling spectroscopic (STS) features. Mapping of redox metalloproteins from the three major classes, i.e. blue copper proteins, heme proteins, and iron-sulfur proteins, at the monolayer and single-molecule levels have also been achieved. In situ STM and spectroscopy of redox molecules and biomolecules have been supported by new theoretical frames, which extend established theory of interfacial electrochemical ET. The electrochemical nanoscale and single-molecule systems discussed are compared with other recent nanoscale and single-molecule systems with conspicuous device-like properties, particularly unimolecular rectifiers and single-molecule transistors. Both of these show analogies to electrochemical in situ STM features of redox molecules and

Electric-Field Control of Interfering Transport Pathways in a Single-Molecule Anthraquinone Transistor

NARCIS (Netherlands)

Koole, Max; Thijssen, Jos M.; Valkenier, Hennie; Hummelen, Jan C.; van der Zant, Herre S. J.

It is understood that molecular conjugation plays an important role in charge transport through single-molecule junctions. Here, we investigate electron transport through an anthraquinone based single-molecule three-terminal device. With the use of an electric-field induced by a gate electrode, the

One-step synthesis of DNA functionalized cadmium-free quantum dots and its application in FRET-based protein sensing

Energy Technology Data Exchange (ETDEWEB)

Zhang, Cuiling, E-mail: clzhang@chem.ecnu.edu.cn [Department of Chemistry, School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200241 (China); Ding, Caiping [Department of Chemistry, School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200241 (China); Zhou, Guohua [School of Chemistry and Chemical Engineering, Lingnan Normal University, Zhanjiang, 524048 (China); Xue, Qin [Department of Chemistry, School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200241 (China); Xian, Yuezhong, E-mail: yzxian@chem.ecnu.edu.cn [Department of Chemistry, School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200241 (China)

2017-03-08

DNA functionalized quantum dots (QDs) are promising nanoprobes for the fluorescence resonance energy transfer (FRET)-based biosensing. Herein, cadmium-free DNA functionalized Mn-doped ZnS (DNA-ZnS:Mn{sup 2+}) QDs were successfully synthesized by one-step route. As-synthesized QDs show excellent photo-stability with the help of PAA and DNA. Then, we constructed a novel FRET model based on the QDs and WS{sub 2} nanosheets as the energy donor-acceptor pairs, which was successfully applied for the protein detection through the terminal protection of small molecule-linked DNA assay. This work not only explores the potential bioapplication of the DNA-ZnS:Mn{sup 2+} QDs, but also provides a platform for the investigation of small molecule-protein interaction. - Highlights: • The stable and cadmium-free DNA functionalized ZnS:Mn{sup 2+} QDs were successfully synthesized through a facile one-step route. • We constructed a novel FRET system based on one-step synthesized DNA-ZnS:Mn{sup 2+} QDs (donor) and WS{sub 2} nanosheets (acceptor). • The FRET-based strategy was applied for the detection of streptavidin and folate receptor by combining TPSMLD and Exo III.

Single Molecule Spectroscopy of Electron Transfer

International Nuclear Information System (INIS)

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

2009-01-01

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.

Fast recognition of single molecules based on single-event photon statistics

International Nuclear Information System (INIS)

Dong Shuangli; Huang Tao; Liu Yuan; Wang Jun; Zhang Guofeng; Xiao Liantuan; Jia Suotang

2007-01-01

Mandel's Q parameter, which is determined from single-event photon statistics, provides an alternative way to recognize single molecules with fluorescence detection, other than the second-order correlation function. It is shown that the Q parameter of an assumed ideal double-molecule fluorescence with the same average photon number as that of the sample fluorescence can act as the criterion for single-molecule recognition. The influence of signal-to-background ratio and the error estimates for photon statistics are also presented. We have applied this method to ascertain single Cy5 dye molecules within hundreds of milliseconds

A Brief Introduction to Single-Molecule Fluorescence Methods.

Science.gov (United States)

van den Wildenberg, Siet M J L; Prevo, Bram; Peterman, Erwin J G

2018-01-01

One of the more popular single-molecule approaches in biological science is single-molecule fluorescence microscopy, which will be the subject of the following section of this volume. Fluorescence methods provide the sensitivity required to study biology on the single-molecule level, but they also allow access to useful measurable parameters on time and length scales relevant for the biomolecular world. Before several detailed experimental approaches will be addressed, we will first give a general overview of single-molecule fluorescence microscopy. We start with discussing the phenomenon of fluorescence in general and the history of single-molecule fluorescence microscopy. Next, we will review fluorescent probes in more detail and the equipment required to visualize them on the single-molecule level. We will end with a description of parameters measurable with such approaches, ranging from protein counting and tracking, single-molecule localization super-resolution microscopy, to distance measurements with Förster Resonance Energy Transfer and orientation measurements with fluorescence polarization.

Single-Molecule Analysis of Pre-mRNA Splicing with Colocalization Single-Molecule Spectroscopy (CoSMoS).

Science.gov (United States)

Braun, Joerg E; Serebrov, Victor

2017-01-01

Recent development of single-molecule techniques to study pre-mRNA splicing has provided insights into the dynamic nature of the spliceosome. Colocalization single-molecule spectroscopy (CoSMoS) allows following spliceosome assembly in real time at single-molecule resolution in the full complexity of cellular extracts. A detailed protocol of CoSMoS has been published previously (Anderson and Hoskins, Methods Mol Biol 1126:217-241, 2014). Here, we provide an update on the technical advances since the first CoSMoS studies including slide surface treatment, data processing, and representation. We describe various labeling strategies to generate RNA reporters with multiple dyes (or other moieties) at specific locations.

Real-time three-dimensional surface measurement by color encoded light projection

International Nuclear Information System (INIS)

Chen, S. Y.; Li, Y. F.; Guan, Q.; Xiao, G.

2006-01-01

Existing noncontact methods for surface measurement suffer from the disadvantages of poor reliability, low scanning speed, or high cost. The authors present a method for real-time three-dimensional data acquisition by a color-coded vision sensor composed of common components. The authors use a digital projector controlled by computer to generate desired color light patterns. The unique indexing of the light codes is a key problem and is solved in this study so that surface perception can be performed with only local pattern analysis of the neighbor color codes in a single image. Experimental examples and performance analysis are provided

Partially reduced graphene oxide based FRET on fiber-optic interferometer for biochemical detection.

Science.gov (United States)

Yao, B C; Wu, Y; Yu, C B; He, J R; Rao, Y J; Gong, Y; Fu, F; Chen, Y F; Li, Y R

2016-03-24

Fluorescent resonance energy transfer (FRET) with naturally exceptional selectivity is a powerful technique and widely used in chemical and biomedical analysis. However, it is still challenging for conventional FRET to perform as a high sensitivity compact sensor. Here we propose a novel 'FRET on Fiber' concept, in which a partially reduced graphene oxide (prGO) film is deposited on a fiber-optic modal interferometer, acting as both the fluorescent quencher for the FRET and the sensitive cladding for optical phase measurement due to refractive index changes in biochemical detection. The target analytes induced fluorescence recovery with good selectivity and optical phase shift with high sensitivity are measured simultaneously. The functionalized prGO film coated on the fiber-optic interferometer shows high sensitivities for the detections of metal ion, dopamine and single-stranded DNA (ssDNA), with detection limits of 1.2 nM, 1.3 μM and 1 pM, respectively. Such a prGO based 'FRET on fiber' configuration, bridging the FRET and the fiber-optic sensing technology, may serve as a platform for the realization of series of integrated 'FRET on Fiber' sensors for on-line environmental, chemical, and biomedical detection, with excellent compactness, high sensitivity, good selectivity and fast response.

Loading dynamics of a sliding DNA clamp.

KAUST Repository

Cho, Won-Ki

2014-05-22

Sliding DNA clamps are loaded at a ss/dsDNA junction by a clamp loader that depends on ATP binding for clamp opening. Sequential ATP hydrolysis results in closure of the clamp so that it completely encircles and diffuses on dsDNA. We followed events during loading of an E. coli β clamp in real time by using single-molecule FRET (smFRET). Three successive FRET states were retained for 0.3 s, 0.7 s, and 9 min: Hydrolysis of the first ATP molecule by the γ clamp loader resulted in closure of the clamp in 0.3 s, and after 0.7 s in the closed conformation, the clamp was released to diffuse on the dsDNA for at least 9 min. An additional single-molecule polarization study revealed that the interfacial domain of the clamp rotated in plane by approximately 8° during clamp closure. The single-molecule polarization and FRET studies thus revealed the real-time dynamics of the ATP-hydrolysis-dependent 3D conformational change of the β clamp during loading at a ss/dsDNA junction.

DNA origami-based shape IDs for single-molecule nanomechanical genotyping

Science.gov (United States)

Zhang, Honglu; Chao, Jie; Pan, Dun; Liu, Huajie; Qiang, Yu; Liu, Ke; Cui, Chengjun; Chen, Jianhua; Huang, Qing; Hu, Jun; Wang, Lianhui; Huang, Wei; Shi, Yongyong; Fan, Chunhai

2017-04-01

Variations on DNA sequences profoundly affect how we develop diseases and respond to pathogens and drugs. Atomic force microscopy (AFM) provides a nanomechanical imaging approach for genetic analysis with nanometre resolution. However, unlike fluorescence imaging that has wavelength-specific fluorophores, the lack of shape-specific labels largely hampers widespread applications of AFM imaging. Here we report the development of a set of differentially shaped, highly hybridizable self-assembled DNA origami nanostructures serving as shape IDs for magnified nanomechanical imaging of single-nucleotide polymorphisms. Using these origami shape IDs, we directly genotype single molecules of human genomic DNA with an ultrahigh resolution of ~10 nm and the multiplexing ability. Further, we determine three types of disease-associated, long-range haplotypes in samples from the Han Chinese population. Single-molecule analysis allows robust haplotyping even for samples with low labelling efficiency. We expect this generic shape ID-based nanomechanical approach to hold great potential in genetic analysis at the single-molecule level.

Nanogap Electrodes towards Solid State Single-Molecule Transistors.

Science.gov (United States)

Cui, Ajuan; Dong, Huanli; Hu, Wenping

2015-12-01

With the establishment of complementary metal-oxide-semiconductor (CMOS)-based integrated circuit technology, it has become more difficult to follow Moore's law to further downscale the size of electronic components. Devices based on various nanostructures were constructed to continue the trend in the minimization of electronics, and molecular devices are among the most promising candidates. Compared with other candidates, molecular devices show unique superiorities, and intensive studies on molecular devices have been carried out both experimentally and theoretically at the present time. Compared to two-terminal molecular devices, three-terminal devices, namely single-molecule transistors, show unique advantages both in fundamental research and application and are considered to be an essential part of integrated circuits based on molecular devices. However, it is very difficult to construct them using the traditional microfabrication techniques directly, thus new fabrication strategies are developed. This review aims to provide an exclusive way of manufacturing solid state gated nanogap electrodes, the foundation of constructing transistors of single or a few molecules. Such single-molecule transistors have the potential to be used to build integrated circuits. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A brief introduction to single-molecule fluorescence methods

NARCIS (Netherlands)

Wildenberg, S.M.J.L.; Prevo, B.; Peterman, E.J.G.; Peterman, EJG; Wuite, GJL

2011-01-01

One of the more popular single-molecule approaches in biological science is single-molecule fluorescence microscopy, which is the subject of the following section of this volume. Fluorescence methods provide the sensitivity required to study biology on the single-molecule level, but they also allow

A brief introduction to single-molecule fluorescence methods

NARCIS (Netherlands)

van den Wildenberg, Siet M.J.L.; Prevo, Bram; Peterman, Erwin J.G.

2018-01-01

One of the more popular single-molecule approaches in biological science is single-molecule fluorescence microscopy, which will be the subject of the following section of this volume. Fluorescence methods provide the sensitivity required to study biology on the single-molecule level, but they also

Characteristic of fretting damage in metal material

Energy Technology Data Exchange (ETDEWEB)

Li, D.; Zhi, F.

1988-10-01

The fretting fatigue experiment of LC4 high strength aluminum alloy is described. An SEM examination of the fractology and morphology of fretting damage is carried out as well as an EDAX analysis of the chemical composition of fretting particles. The results show that many loose oxide particles were produced and accumulated in the fretting damage region. 10 references.

Direct single-molecule dynamic detection of chemical reactions.

Science.gov (United States)

Guan, Jianxin; Jia, Chuancheng; Li, Yanwei; Liu, Zitong; Wang, Jinying; Yang, Zhongyue; Gu, Chunhui; Su, Dingkai; Houk, Kendall N; Zhang, Deqing; Guo, Xuefeng

2018-02-01

Single-molecule detection can reveal time trajectories and reaction pathways of individual intermediates/transition states in chemical reactions and biological processes, which is of fundamental importance to elucidate their intrinsic mechanisms. We present a reliable, label-free single-molecule approach that allows us to directly explore the dynamic process of basic chemical reactions at the single-event level by using stable graphene-molecule single-molecule junctions. These junctions are constructed by covalently connecting a single molecule with a 9-fluorenone center to nanogapped graphene electrodes. For the first time, real-time single-molecule electrical measurements unambiguously show reproducible large-amplitude two-level fluctuations that are highly dependent on solvent environments in a nucleophilic addition reaction of hydroxylamine to a carbonyl group. Both theoretical simulations and ensemble experiments prove that this observation originates from the reversible transition between the reactant and a new intermediate state within a time scale of a few microseconds. These investigations open up a new route that is able to be immediately applied to probe fast single-molecule physics or biophysics with high time resolution, making an important contribution to broad fields beyond reaction chemistry.

The Control of Single-color and Multiple-color Visual Search by Attentional Templates in Working Memory and in Long-term Memory.

Science.gov (United States)

Grubert, Anna; Carlisle, Nancy B; Eimer, Martin

2016-12-01

The question whether target selection in visual search can be effectively controlled by simultaneous attentional templates for multiple features is still under dispute. We investigated whether multiple-color attentional guidance is possible when target colors remain constant and can thus be represented in long-term memory but not when they change frequently and have to be held in working memory. Participants searched for one, two, or three possible target colors that were specified by cue displays at the start of each trial. In constant-color blocks, the same colors remained task-relevant throughout. In variable-color blocks, target colors changed between trials. The contralateral delay activity (CDA) to cue displays increased in amplitude as a function of color memory load in variable-color blocks, which indicates that cued target colors were held in working memory. In constant-color blocks, the CDA was much smaller, suggesting that color representations were primarily stored in long-term memory. N2pc components to targets were measured as a marker of attentional target selection. Target N2pcs were attenuated and delayed during multiple-color search, demonstrating less efficient attentional deployment to color-defined target objects relative to single-color search. Importantly, these costs were the same in constant-color and variable-color blocks. These results demonstrate that attentional guidance by multiple-feature as compared with single-feature templates is less efficient both when target features remain constant and can be represented in long-term memory and when they change across trials and therefore have to be maintained in working memory.

Challenges for single molecule electronic devices with nanographene and organic molecules. Do single molecules offer potential as elements of electronic devices in the next generation?

Science.gov (United States)

Enoki, Toshiaki; Kiguchi, Manabu

2018-03-01

Interest in utilizing organic molecules to fabricate electronic materials has existed ever since organic (molecular) semiconductors were first discovered in the 1950s. Since then, scientists have devoted serious effort to the creation of various molecule-based electronic systems, such as molecular metals and molecular superconductors. Single-molecule electronics and the associated basic science have emerged over the past two decades and provided hope for the development of highly integrated molecule-based electronic devices in the future (after the Si-based technology era has ended). Here, nanographenes (nano-sized graphene) with atomically precise structures are among the most promising molecules that can be utilized for electronic/spintronic devices. To manipulate single small molecules for an electronic device, a single molecular junction has been developed. It is a powerful tool that allows even small molecules to be utilized. External electric, magnetic, chemical, and mechanical perturbations can change the physical and chemical properties of molecules in a way that is different from bulk materials. Therefore, the various functionalities of molecules, along with changes induced by external perturbations, allows us to create electronic devices that we cannot create using current top-down Si-based technology. Future challenges that involve the incorporation of condensed matter physics, quantum chemistry calculations, organic synthetic chemistry, and electronic device engineering are expected to open a new era in single-molecule device electronic technology.

Single-Molecule Electronics: Chemical and Analytical Perspectives.

Science.gov (United States)

Nichols, Richard J; Higgins, Simon J

2015-01-01

It is now possible to measure the electrical properties of single molecules using a variety of techniques including scanning probe microcopies and mechanically controlled break junctions. Such measurements can be made across a wide range of environments including ambient conditions, organic liquids, ionic liquids, aqueous solutions, electrolytes, and ultra high vacuum. This has given new insights into charge transport across molecule electrical junctions, and these experimental methods have been complemented with increasingly sophisticated theory. This article reviews progress in single-molecule electronics from a chemical perspective and discusses topics such as the molecule-surface coupling in electrical junctions, chemical control, and supramolecular interactions in junctions and gating charge transport. The article concludes with an outlook regarding chemical analysis based on single-molecule conductance.

Likelihood functions for the analysis of single-molecule binned photon sequences

Energy Technology Data Exchange (ETDEWEB)

Gopich, Irina V., E-mail: irinag@niddk.nih.gov [Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892 (United States)

2012-03-02

Graphical abstract: Folding of a protein with attached fluorescent dyes, the underlying conformational trajectory of interest, and the observed binned photon trajectory. Highlights: Black-Right-Pointing-Pointer A sequence of photon counts can be analyzed using a likelihood function. Black-Right-Pointing-Pointer The exact likelihood function for a two-state kinetic model is provided. Black-Right-Pointing-Pointer Several approximations are considered for an arbitrary kinetic model. Black-Right-Pointing-Pointer Improved likelihood functions are obtained to treat sequences of FRET efficiencies. - Abstract: We consider the analysis of a class of experiments in which the number of photons in consecutive time intervals is recorded. Sequence of photon counts or, alternatively, of FRET efficiencies can be studied using likelihood-based methods. For a kinetic model of the conformational dynamics and state-dependent Poisson photon statistics, the formalism to calculate the exact likelihood that this model describes such sequences of photons or FRET efficiencies is developed. Explicit analytic expressions for the likelihood function for a two-state kinetic model are provided. The important special case when conformational dynamics are so slow that at most a single transition occurs in a time bin is considered. By making a series of approximations, we eventually recover the likelihood function used in hidden Markov models. In this way, not only is insight gained into the range of validity of this procedure, but also an improved likelihood function can be obtained.

Single Molecule Biophysics Experiments and Theory

CERN Document Server

Komatsuzaki, Tamiki; Takahashi, Satoshi; Yang, Haw; Silbey, Robert J; Rice, Stuart A; Dinner, Aaron R

2011-01-01

Discover the experimental and theoretical developments in optical single-molecule spectroscopy that are changing the ways we think about molecules and atoms The Advances in Chemical Physics series provides the chemical physics field with a forum for critical, authoritative evaluations of advances in every area of the discipline. This latest volume explores the advent of optical single-molecule spectroscopy, and how atomic force microscopy has empowered novel experiments on individual biomolecules, opening up new frontiers in molecular and cell biology and leading to new theoretical approaches

Single molecule detection, thermal fluctuation and life

Science.gov (United States)

YANAGIDA, Toshio; ISHII, Yoshiharu

2017-01-01

Single molecule detection has contributed to our understanding of the unique mechanisms of life. Unlike artificial man-made machines, biological molecular machines integrate thermal noises rather than avoid them. For example, single molecule detection has demonstrated that myosin motors undergo biased Brownian motion for stepwise movement and that single protein molecules spontaneously change their conformation, for switching to interactions with other proteins, in response to thermal fluctuation. Thus, molecular machines have flexibility and efficiency not seen in artificial machines. PMID:28190869

On theory of single-molecule transistor

International Nuclear Information System (INIS)

Tran Tien Phuc

2009-01-01

The results of the study on single-molecule transistor are mainly investigated in this paper. The structure of constructed single-molecule transistor is similar to a conventional MOSFET. The conductive channel of the transistors is a single-molecule of halogenated benzene derivatives. The chemical simulation software CAChe was used to design and implement for the essential parameter of the molecules utilized as the conductive channel. The GUI of Matlab has been built to design its graphical interface, calculate and plot the output I-V characteristic curves for the transistor. The influence of temperature, length and width of the conductive channel, and gate voltage is considered. As a result, the simulated curves are similar to the traditional MOSFET's. The operating temperature range of the transistors is wider compared with silicon semiconductors. The supply voltage for transistors is only about 1 V. The size of transistors in this research is several nanometers.

Molecular electronics: the single molecule switch and transistor

NARCIS (Netherlands)

Sotthewes, Kai; Geskin, Victor; Heimbuch, Rene; Kumar, Avijit; Zandvliet, Henricus J.W.

2014-01-01

In order to design and realize single-molecule devices it is essential to have a good understanding of the properties of an individual molecule. For electronic applications, the most important property of a molecule is its conductance. Here we show how a single octanethiol molecule can be connected

Single molecule approaches for quantifying transcription and degradation rates in intact mammalian tissues.

Science.gov (United States)

Bahar Halpern, Keren; Itzkovitz, Shalev

2016-04-01

A key challenge in mammalian biology is to understand how rates of transcription and mRNA degradation jointly shape cellular gene expression. Powerful techniques have been developed for measuring these rates either genome-wide or at the single-molecule level, however these techniques are not applicable to assessment of cells within their native tissue microenvironment. Here we describe a technique based on single molecule Fluorescence in-situ Hybridization (smFISH) to measure transcription and degradation rates in intact mammalian tissues. The technique is based on dual-color libraries targeting the introns and exons of the genes of interest, enabling visualization and quantification of both nascent and mature mRNA. We present a software, TransQuant, that facilitates quantifying these rates from smFISH images. Our approach enables assessment of both transcription and degradation rates of any gene of interest while controlling for the inherent heterogeneity of intact tissues. Copyright © 2015 Elsevier Inc. All rights reserved.

Ultrasensitive FRET-based DNA sensor using PNA/DNA hybridization.

Science.gov (United States)

Yang, Lan-Hee; Ahn, Dong June; Koo, Eunhae

2016-12-01

In the diagnosis of genetic diseases, rapid and highly sensitive DNA detection is crucial. Therefore, many strategies for detecting target DNA have been developed, including electrical, optical, and mechanical methods. Herein, a highly sensitive FRET based sensor was developed by using PNA (Peptide Nucleic Acid) probe and QD, in which red color QDs are hybridized with capture probes, reporter probes and target DNAs by EDC-NHS coupling. The hybridized probe with target DNA gives off fluorescent signal due to the energy transfer from QD to Cy5 dye in the reporter probe. Compared to the conventional DNA sensor using DNA probes, the DNA sensor using PNA probes shows higher FRET factor and efficiency due to the higher reactivity between PNA and target DNA. In addition, to elicit the effect of the distance between the donor and the acceptor, we have investigated two types of the reporter probes having Cy5 dyes attached at the different positions of the reporter probes. Results show that the shorter the distance between QDs and Cy5s, the stronger the signal intensity. Furthermore, based on the fluorescence microscopy images using microcapillary chips, the FRET signal is enhanced to be up to 276% times stronger than the signal obtained using the cuvette by the fluorescence spectrometer. These results suggest that the PNA probe system conjugated with QDs can be used as ultrasensitive DNA nanosensors. Copyright © 2016. Published by Elsevier B.V.

Site-Selection in Single-Molecule Junction for Highly Reproducible Molecular Electronics.

Science.gov (United States)

Kaneko, Satoshi; Murai, Daigo; Marqués-González, Santiago; Nakamura, Hisao; Komoto, Yuki; Fujii, Shintaro; Nishino, Tomoaki; Ikeda, Katsuyoshi; Tsukagoshi, Kazuhito; Kiguchi, Manabu

2016-02-03

Adsorption sites of molecules critically determine the electric/photonic properties and the stability of heterogeneous molecule-metal interfaces. Then, selectivity of adsorption site is essential for development of the fields including organic electronics, catalysis, and biology. However, due to current technical limitations, site-selectivity, i.e., precise determination of the molecular adsorption site, remains a major challenge because of difficulty in precise selection of meaningful one among the sites. We have succeeded the single site-selection at a single-molecule junction by performing newly developed hybrid technique: simultaneous characterization of surface enhanced Raman scattering (SERS) and current-voltage (I-V) measurements. The I-V response of 1,4-benzenedithiol junctions reveals the existence of three metastable states arising from different adsorption sites. Notably, correlated SERS measurements show selectivity toward one of the adsorption sites: "bridge sites". This site-selectivity represents an essential step toward the reliable integration of individual molecules on metallic surfaces. Furthermore, the hybrid spectro-electric technique reveals the dependence of the SERS intensity on the strength of the molecule-metal interaction, showing the interdependence between the optical and electronic properties in single-molecule junctions.

Electric-Field Control of Interfering Transport Pathways in a Single-Molecule Anthraquinone Transistor

Science.gov (United States)

Koole, Max; Thijssen, Jos M.; Valkenier, Hennie; Hummelen, Jan C.; Zant, Herre S. J. van der

2015-08-01

It is understood that molecular conjugation plays an important role in charge transport through single-molecule junctions. Here, we investigate electron transport through an anthraquinone based single-molecule three-terminal device. With the use of an electric-field induced by a gate electrode, the molecule is reduced resulting into a ten-fold increase in the off-resonant differential conductance. Theoretical calculations link the change in differential conductance to a reduction-induced change in conjugation, thereby lifting destructive interference of transport pathways.

Single-Molecule Plasmon Sensing: Current Status and Future Prospects.

Science.gov (United States)

Taylor, Adam B; Zijlstra, Peter

2017-08-25

Single-molecule detection has long relied on fluorescent labeling with high quantum-yield fluorophores. Plasmon-enhanced detection circumvents the need for labeling by allowing direct optical detection of weakly emitting and completely nonfluorescent species. This review focuses on recent advances in single molecule detection using plasmonic metal nanostructures as a sensing platform, particularly using a single particle-single molecule approach. In the past decade two mechanisms for plasmon-enhanced single-molecule detection have been demonstrated: (1) by plasmonically enhancing the emission of weakly fluorescent biomolecules, or (2) by monitoring shifts of the plasmon resonance induced by single-molecule interactions. We begin with a motivation regarding the importance of single molecule detection, and advantages plasmonic detection offers. We describe both detection mechanisms and discuss challenges and potential solutions. We finalize by highlighting the exciting possibilities in analytical chemistry and medical diagnostics.

Observing single molecule chemical reactions on metal nanoparticles.

Energy Technology Data Exchange (ETDEWEB)

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

2001-01-01

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.

Memory effects in single-molecule spectroscopy

International Nuclear Information System (INIS)

Schmitt, Daniel T.; Schulz, Michael; Reineker, Peter

2007-01-01

From the time series of LH2 optical single-molecule fluorescence excitation spectra of Rhodospirillum molischianum the memory function of the Mori-Zwanzig equation for the optical intensity is derived numerically. We show that the time dependence of the excited states is determined by at least three different non-Markovian stochastic processes with decay constants for the Mori-Zwanzig kernel on the order of 1-5min -1 . We suggest that this decay stems from the conformational motion of the protein scaffold of LH2

Stuy on Fatigue Life of Aluminum Alloy Considering Fretting

Science.gov (United States)

Yang, Maosheng; Zhao, Hongqiang; Wang, Yunxiang; Chen, Xiaofei; Fan, Jiali

2018-01-01

To study the influence of fretting on Aluminum Alloy, a global finite element model considering fretting was performed using the commercial code ABAQUS. With which a new model for predicting fretting fatigue life has been presented based on friction work. The rationality and effectiveness of the model were validated according to the contrast of experiment life and predicting life. At last influence factor on fretting fatigue life of aerial aluminum alloy was investigated with the model. The results revealed that fretting fatigue life decreased monotonously with the increasing of normal load and then became constant at higher pressures. At low normal load, fretting fatigue life was found to increase with increase in the pad radius. At high normal load, however, the fretting fatigue life remained almost unchanged with changes in the fretting pad radius. The bulk stress amplitude had the dominant effect on fretting fatigue life. The fretting fatigue life diminished as the bulk stress amplitude increased.

Single-Molecule Transport at a Rectifying GaAs Contact.

Science.gov (United States)

Vezzoli, Andrea; Brooke, Richard J; Ferri, Nicolò; Higgins, Simon J; Schwarzacher, Walther; Nichols, Richard J

2017-02-08

In most single- or few-molecule devices, the contact electrodes are simple ohmic resistors. Here we describe a new type of single-molecule device in which metal and semiconductor contact electrodes impart a function, namely, current rectification, which is then modified by a molecule bridging the gap. We study junctions with the structure Au STM tip/X/n-GaAs substrate, where "X" is either a simple alkanedithiol or a conjugated unit bearing thiol/methylthiol contacts, and we detect current jumps corresponding to the attachment and detachment of single molecules. From the magnitudes of the current jumps we can deduce values for the conductance decay constant with molecule length that agree well with values determined from Au/molecule/Au junctions. The ability to impart functionality to a single-molecule device through the properties of the contacts as well as through the properties of the molecule represents a significant extension of the single-molecule electronics "tool-box".

Two-color studies of autoionizing states of small molecules

International Nuclear Information System (INIS)

Pratt, S.T.; Dehmer, P.M.; Dehmer, J.L.; Tomkins, F.S.; O'Halloran, M.A.

1989-01-01

Two-color, resonantly enhanced multiphoton ionization is proving to be a valuable technique for the study of autoionizing states of small molecules. In this talk, results obtained by combining REMPI, photoelectron spectroscopy, and mass spectrometry will be discussed and will be illustrated by examples from our recent studies of rotational and vibrational autoionization in molecular hydrogen and rotational autoionization in nitric oxide. 2 refs., 1 fig

Multiplexed interfacial transduction of nucleic acid hybridization using a single color of immobilized quantum dot donor and two acceptors in fluorescence resonance energy transfer.

Science.gov (United States)

Algar, W Russ; Krull, Ulrich J

2010-01-01

A multiplexed solid-phase assay for the detection of nucleic acid hybridization was developed on the basis of a single color of immobilized CdSe/ZnS quantum dot (QD) as a donor in fluorescence resonance energy transfer (FRET). This work demonstrated that two channels of detection did not necessitate two different QD donors. Two probe oligonucleotides were coimmobilized on optical fibers modified with QDs, and a sandwich assay was used to associate the acceptor dyes with interfacial hybridization events without target labeling. FRET-sensitized acceptor emission provided an analytical signal that was concentration dependent down to 10 nM. Changes in the ratio of coimmobilized probe oligonucleotides were found to yield linear changes in the relative amounts of acceptor emission. These changes were compared to previous studies that used mixed films of two QD donors for two detection channels. The analysis indicated that probe dilution effects were primarily driven by changes in acceptor number density and that QD dilution effects or changes in mean donor-acceptor distance were secondary. Hybridization kinetics were found to be consistent between different ratios of coimmobilized probes, suggesting that hybridization in this type of system occurred via the accepted model for solid-phase hybridization, where adsorption and then diffusion at the solid interface drove hybridization.

Investigating single molecule adhesion by atomic force spectroscopy.

Science.gov (United States)

Stetter, Frank W S; Kienle, Sandra; Krysiak, Stefanie; Hugel, Thorsten

2015-02-27

Atomic force spectroscopy is an ideal tool to study molecules at surfaces and interfaces. An experimental protocol to couple a large variety of single molecules covalently onto an AFM tip is presented. At the same time the AFM tip is passivated to prevent unspecific interactions between the tip and the substrate, which is a prerequisite to study single molecules attached to the AFM tip. Analyses to determine the adhesion force, the adhesion length, and the free energy of these molecules on solid surfaces and bio-interfaces are shortly presented and external references for further reading are provided. Example molecules are the poly(amino acid) polytyrosine, the graft polymer PI-g-PS and the phospholipid POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine). These molecules are desorbed from different surfaces like CH3-SAMs, hydrogen terminated diamond and supported lipid bilayers under various solvent conditions. Finally, the advantages of force spectroscopic single molecule experiments are discussed including means to decide if truly a single molecule has been studied in the experiment.

Long-Lived Feshbach Molecules in a Three-Dimensional Optical Lattice

International Nuclear Information System (INIS)

Thalhammer, G.; Winkler, K.; Lang, F.; Schmid, S.; Denschlag, J. Hecker; Grimm, R.

2006-01-01

We have created and trapped a pure sample of 87 Rb 2 Feshbach molecules in a three-dimensional optical lattice. Compared to previous experiments without a lattice, we find dramatic improvements such as long lifetimes of up to 700 ms and a near unit efficiency for converting tightly confined atom pairs into molecules. The lattice shields the trapped molecules from collisions and, thus, overcomes the problem of inelastic decay by vibrational quenching. Furthermore, we have developed an advanced purification scheme that removes residual atoms, resulting in a lattice in which individual sites are either empty or filled with a single molecule in the vibrational ground state of the lattice

Electrolytic coloration and spectral properties of hydroxyl-doped potassium chloride single crystals

International Nuclear Information System (INIS)

Gu Hongen; Wu Yanru

2011-01-01

Hydroxyl-doped potassium chloride single crystals are colored electrolytically at various temperatures and voltages using a pointed cathode and a flat anode. Characteristic OH - spectral band is observed in the absorption spectrum of uncolored single crystal. Characteristic O - , OH - , U, V 2 , V 3 , O 2- -V a + , F, R 2 and M spectral bands are observed simultaneously in absorption spectra of colored single crystals. Current-time curve for electrolytic coloration of hydroxyl-doped potassium chloride single crystal and its relationship with electrolytic coloration process are given. Production and conversion of color centers are explained. - Highlights: → Expanded the traditional electrolysis method. → Hydroxyl-doped potassium chloride crystals were colored electrolytically for the first time. → Useful V, F and F-aggregate color centers were produced in colored crystals. → V color centers were produced directly and F and F-aggregate color centers indirectly.

Computer systems for annotation of single molecule fragments

Science.gov (United States)

Schwartz, David Charles; Severin, Jessica

2016-07-19

There are provided computer systems for visualizing and annotating single molecule images. Annotation systems in accordance with this disclosure allow a user to mark and annotate single molecules of interest and their restriction enzyme cut sites thereby determining the restriction fragments of single nucleic acid molecules. The markings and annotations may be automatically generated by the system in certain embodiments and they may be overlaid translucently onto the single molecule images. An image caching system may be implemented in the computer annotation systems to reduce image processing time. The annotation systems include one or more connectors connecting to one or more databases capable of storing single molecule data as well as other biomedical data. Such diverse array of data can be retrieved and used to validate the markings and annotations. The annotation systems may be implemented and deployed over a computer network. They may be ergonomically optimized to facilitate user interactions.

Massively Parallel Single-Molecule Manipulation Using Centrifugal Force

Science.gov (United States)

Wong, Wesley; Halvorsen, Ken

2011-03-01

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. 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.

Single molecule transcription profiling with AFM

International Nuclear Information System (INIS)

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

2007-01-01

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

Optical probing of single fluorescent molecules and proteins

NARCIS (Netherlands)

Garcia Parajo, M.F.; Veerman, J.A.; Bouwhuis, R.; Bouwhuis, Rudo; van Hulst, N.F.; Vallée, R.A.L.

2001-01-01

Single-molecule detection and analysis of organic fluorescent molecules and proteins are presented, with emphasis o­n the underlying principles methodology and the application of single-molecule analysis at room temperature. This Minireview is mainly focused o­n the application of confocal and

Theoretical Investigations Regarding Single Molecules

DEFF Research Database (Denmark)

Pedersen, Kim Georg Lind

Neoclassical Valence Bond Theory, Quantum Transport, Quantum Interference, Kondo Effect, and Electron Pumping. Trap a single organic molecule between two electrodes and apply a bias voltage across this "molecular junction". When electrons pass through the molecule, the different electron paths can...... interfere destructively or constructively. Destructive interference effects in electron transport could potentially improve thermo-electrics, organic logic circuits and energy harvesting. We have investigated destructive interference in off-resonant transport through organic molecules, and have found a set...

Single Molecule Nano-Metronome

OpenAIRE

Buranachai, Chittanon; McKinney, Sean A.; Ha, Taekjip

2006-01-01

We constructed a DNA-based nano-mechanical device called the nano-metronome. Our device is made by introducing complementary single stranded overhangs at the two arms of the DNA four-way junction. The ticking rates of this stochastic metronome depend on ion concentrations and can be changed by a set of DNA-based switches to deactivate/reactivate the sticky end. Since the device displays clearly distinguishable responses even with a single basepair difference, it may lead to a single molecule ...

Investigation on Single-Molecule Junctions Based on Current–Voltage Characteristics

Directory of Open Access Journals (Sweden)

Yuji Isshiki

2018-02-01

Full Text Available The relationship between the current through an electronic device and the voltage across its terminals is a current–voltage characteristic (I–V that determine basic device performance. Currently, I–V measurement on a single-molecule scale can be performed using break junction technique, where a single molecule junction can be prepared by trapping a single molecule into a nanogap between metal electrodes. The single-molecule I–Vs provide not only the device performance, but also reflect information on energy dispersion of the electronic state and the electron-molecular vibration coupling in the junction. This mini review focuses on recent representative studies on I–Vs of the single molecule junctions that cover investigation on the single-molecule diode property, the molecular vibration, and the electronic structure as a form of transmission probability, and electronic density of states, including the spin state of the single-molecule junctions. In addition, thermoelectronic measurements based on I–Vs and identification of the charged carriers (i.e., electrons or holes are presented. The analysis in the single-molecule I–Vs provides fundamental and essential information for a better understanding of the single-molecule science, and puts the single molecule junction to more practical use in molecular devices.

Novel approaches for single molecule activation and detection

CERN Document Server

Benfenati, Fabio; Torre, Vincent

2014-01-01

How can we obtain tools able to process and exchange information at the molecular scale In order to do this, it is necessary to activate and detect single molecules under controlled conditions. This book focuses on the generation of biologically-inspired molecular devices. These devices are based on the developments of new photonic tools able to activate and stimulate single molecule machines. Additionally, new light sensitive molecules can be selectively activated by photonic tools. These technological innovations will provide a way to control activation of single light-sensitive molecules, a

CMEIAS color segmentation: an improved computing technology to process color images for quantitative microbial ecology studies at single-cell resolution.

Science.gov (United States)

Gross, Colin A; Reddy, Chandan K; Dazzo, Frank B

2010-02-01

Quantitative microscopy and digital image analysis are underutilized in microbial ecology largely because of the laborious task to segment foreground object pixels from background, especially in complex color micrographs of environmental samples. In this paper, we describe an improved computing technology developed to alleviate this limitation. The system's uniqueness is its ability to edit digital images accurately when presented with the difficult yet commonplace challenge of removing background pixels whose three-dimensional color space overlaps the range that defines foreground objects. Image segmentation is accomplished by utilizing algorithms that address color and spatial relationships of user-selected foreground object pixels. Performance of the color segmentation algorithm evaluated on 26 complex micrographs at single pixel resolution had an overall pixel classification accuracy of 99+%. Several applications illustrate how this improved computing technology can successfully resolve numerous challenges of complex color segmentation in order to produce images from which quantitative information can be accurately extracted, thereby gain new perspectives on the in situ ecology of microorganisms. Examples include improvements in the quantitative analysis of (1) microbial abundance and phylotype diversity of single cells classified by their discriminating color within heterogeneous communities, (2) cell viability, (3) spatial relationships and intensity of bacterial gene expression involved in cellular communication between individual cells within rhizoplane biofilms, and (4) biofilm ecophysiology based on ribotype-differentiated radioactive substrate utilization. The stand-alone executable file plus user manual and tutorial images for this color segmentation computing application are freely available at http://cme.msu.edu/cmeias/ . This improved computing technology opens new opportunities of imaging applications where discriminating colors really matter most

Electrolytic coloration and spectral properties of hydroxyl-doped potassium bromide single crystals

International Nuclear Information System (INIS)

Qi, Lan; Song, Cuiying; Gu, Hongen

2013-01-01

Hydroxyl-doped potassium bromide single crystals are colored electrolytically at various temperatures and voltages by using a pointed cathode and a flat anode. The characteristic OH − spectral band is observed in absorption spectrum of uncolored single crystal. The characteristic O − , OH − , U, V 2 , O 2− −V a + , M L1 , F and M spectral bands are observed simultaneously in absorption spectra of colored single crystals. Current–time curve for electrolytic coloration of hydroxyl-doped potassium bromide single crystal and its relationship with electrolytic coloration processes are given. Production and conversion of color centers are explained. - Highlights: ► We expanded the traditional electrolysis method. ► Hydroxyl-doped potassium bromide crystals were colored electrolytically for the first time. ► Useful V, F and F-aggregate color centers were produced in colored crystals. ► V color centers were produced directly and F as well as F-aggregate color centers indirectly.

A method to quantify FRET stoichiometry with phasor plot analysis and acceptor lifetime ingrowth.

Science.gov (United States)

Chen, WeiYue; Avezov, Edward; Schlachter, Simon C; Gielen, Fabrice; Laine, Romain F; Harding, Heather P; Hollfelder, Florian; Ron, David; Kaminski, Clemens F

2015-03-10

FRET is widely used for the study of protein-protein interactions in biological samples. However, it is difficult to quantify both the FRET efficiency (E) and the affinity (Kd) of the molecular interaction from intermolecular FRET signals in samples of unknown stoichiometry. Here, we present a method for the simultaneous quantification of the complete set of interaction parameters, including fractions of bound donors and acceptors, local protein concentrations, and dissociation constants, in each image pixel. The method makes use of fluorescence lifetime information from both donor and acceptor molecules and takes advantage of the linear properties of the phasor plot approach. We demonstrate the capability of our method in vitro in a microfluidic device and also in cells, via the determination of the binding affinity between tagged versions of glutathione and glutathione S-transferase, and via the determination of competitor concentration. The potential of the method is explored with simulations. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Single-base resolution and long-coverage sequencing based on single-molecule nanomanipulation

International Nuclear Information System (INIS)

An Hongjie; Huang Jiehuan; Lue Ming; Li Xueling; Lue Junhong; Li Haikuo; Zhang Yi; Li Minqian; Hu Jun

2007-01-01

We show new approaches towards a novel single-molecule sequencing strategy which consists of high-resolution positioning isolation of overlapping DNA fragments with atomic force microscopy (AFM), subsequent single-molecule PCR amplification and conventional Sanger sequencing. In this study, a DNA labelling technique was used to guarantee the accuracy in positioning the target DNA. Single-molecule multiplex PCR was carried out to test the contamination. The results showed that the two overlapping DNA fragments isolated by AFM could be successfully sequenced with high quality and perfect contiguity, indicating that single-base resolution and long-coverage sequencing have been achieved simultaneously

In vivo dynamics of enterovirus protease revealed by fluorescence resonance emission transfer (FRET) based on a novel FRET pair

International Nuclear Information System (INIS)

Hsu, Y.-Y.; Liu, Y.-N.; Wang Wenyen; Kao, Fu-Jen; Kung, S.-H.

2007-01-01

An in vivo protease assay suitable for analysis by fluorescence resonance energy transfer (FRET) was developed on the basis of a novel FRET pair. The specifically designed fusion substrate consists of green fluorescent protein 2 (GFP 2 )-peptide-red fluorescent protein 2 (DsRed2), with a cleavage motif for the enterovirus 2A protease (2A pro ) embedded within the peptide region. FRET can be readily visualized in real-time from cells expressing the fusion substrate until a proteolytic cleavage by 2A pro from the input virus. The level of FRET decay is a function of the amount and infection duration of the inoculated virus as measured by a fluorometer assay. The FRET biosensor also responded well to other related enteroviruses but not to a phylogenetically distant virus. Western blot analysis confirmed the physical cleavage of the fusion substrate upon the infections. The study provides proof of principle for applying the FRET technology to diagnostics, screening procedures, and cell biological research

Ratiometric FRET-based detection of DNA and micro-RNA in solution

International Nuclear Information System (INIS)

Matveeva, Evgenia G.; Gryczynski, Zygmunt; Stewart, Donald R.; Gryczynski, Ignacy

2009-01-01

A ratiometric method for detecting DNA oligomers in bulk solution based on Foerster resonance energy transfer (FRET) is described. The two fluorescence signals (green and red), originating from Cy3 (donor, green) and Cy5 (acceptor, red) labels, are simultaneously detected from the pre-hybridized Cy3oligomerY:Cy5oligomerX system. The ratio of red to green intensities is sensitive to the presence of the single-stranded complimentary oligomer, which replaces single-stranded Cy3oligomerY in the donor:acceptor complex and perturbs the FRET. The detection scheme is generally applicable to the detection of DNA and RNA, and particularly micro-RNA. The proposed method is applicable to various double-stranded various lengths targets (manipulation of the sample preparation conditions, such as temperature, incubation time, denaturizing agent, may be needed).

Lattice diffusion of a single molecule in solution

Science.gov (United States)

Ruggeri, Francesca; Krishnan, Madhavi

2017-12-01

The ability to trap a single molecule in an electrostatic potential well in solution has opened up new possibilities for the use of molecular electrical charge to study macromolecular conformation and dynamics at the level of the single entity. Here we study the diffusion of a single macromolecule in a two-dimensional lattice of electrostatic traps in solution. We report the ability to measure both the size and effective electrical charge of a macromolecule by observing single-molecule transport trajectories, typically a few seconds in length, using fluorescence microscopy. While, as shown previously, the time spent by the molecule in a trap is a strong function of its effective charge, we demonstrate here that the average travel time between traps in the landscape yields its hydrodynamic radius. Tailoring the pitch of the lattice thus yields two different experimentally measurable time scales that together uniquely determine both the size and charge of the molecule. Since no information is required on the location of the molecule between consecutive departure and arrival events at lattice sites, the technique is ideally suited to measurements on weakly emitting entities such as single molecules.

Putting prions into focus: application of single molecule detection to the diagnosis of prion diseases.

Science.gov (United States)

Giese, A; Bieschke, J; Eigen, M; Kretzschmar, H A

2000-01-01

Prion diseases are characterized by the cerebral deposition of an aggregated pathological isoform of the prion protein (PrP(Sc)) which constitutes the principal component of the transmissible agent termed prion. In order to develop a highly sensitive method for the detection of PrP(Sc) aggregates in biological samples such as cerebrospinal fluid (CSF), we used a method based on Fluorescence Correlation Spectroscopy (FCS), a technique which allows detection of single fluorescently labeled molecules in solution. Within the FCS setup, fluorescent photons emitted by molecules passing an open volume element defined by the beam of an excitation laser focussed into a diffraction-limited spot are imaged confocally onto a single photon counting detector. Aggregates of PrP(Sc) could be labeled by co-aggregation of probe molecules such as monomeric recombinant PrP or PrP-specific antibodies tagged with a fluorescent dye. In addition to slow diffusion, labeled aggregates are characterized by high fluorescence intensity, which allows detection and quantification by analysis of fluorescence intensity distribution. To improve detection of rare target particles, the accessible volume element was increased by scanning for intensely fluorescent targets (SIFT). To further improve sensitivity and specificity, two different probes were used simultaneously in a two-color setup. In a diagnostic model system of CSF spiked with purified prion rods, dual-color SIFT was more sensitive than Western blot analysis. In addition, a PrP(Sc)-specific signal was also detected in a number of CSF samples derived from CJD patients but not in controls.

Extending Single-Molecule Microscopy Using Optical Fourier Processing

Science.gov (United States)

2015-01-01

This article surveys the recent application of optical Fourier processing to the long-established but still expanding field of single-molecule imaging and microscopy. A variety of single-molecule studies can benefit from the additional image information that can be obtained by modulating the Fourier, or pupil, plane of a widefield microscope. After briefly reviewing several current applications, we present a comprehensive and computationally efficient theoretical model for simulating single-molecule fluorescence as it propagates through an imaging system. Furthermore, we describe how phase/amplitude-modulating optics inserted in the imaging pathway may be modeled, especially at the Fourier plane. Finally, we discuss selected recent applications of Fourier processing methods to measure the orientation, depth, and rotational mobility of single fluorescent molecules. PMID:24745862

Single Molecule Nano-Metronome

Science.gov (United States)

Buranachai, Chittanon; McKinney, Sean A.; Ha, Taekjip

2008-01-01

We constructed a DNA-based nano-mechanical device called the nano-metronome. Our device is made by introducing complementary single stranded overhangs at the two arms of the DNA four-way junction. The ticking rates of this stochastic metronome depend on ion concentrations and can be changed by a set of DNA-based switches to deactivate/reactivate the sticky end. Since the device displays clearly distinguishable responses even with a single basepair difference, it may lead to a single molecule sensor of minute sequence differences of a target DNA. PMID:16522050

Controlling single-molecule junction conductance by molecular interactions

Science.gov (United States)

Kitaguchi, Y.; Habuka, S.; Okuyama, H.; Hatta, S.; Aruga, T.; Frederiksen, T.; Paulsson, M.; Ueba, H.

2015-01-01

For the rational design of single-molecular electronic devices, it is essential to understand environmental effects on the electronic properties of a working molecule. Here we investigate the impact of molecular interactions on the single-molecule conductance by accurately positioning individual molecules on the electrode. To achieve reproducible and precise conductivity measurements, we utilize relatively weak π-bonding between a phenoxy molecule and a STM-tip to form and cleave one contact to the molecule. The anchoring to the other electrode is kept stable using a chalcogen atom with strong bonding to a Cu(110) substrate. These non-destructive measurements permit us to investigate the variation in single-molecule conductance under different but controlled environmental conditions. Combined with density functional theory calculations, we clarify the role of the electrostatic field in the environmental effect that influences the molecular level alignment. PMID:26135251

Single Fluorescent Molecules as Nano-Illuminators for Biological Structure and Function

Science.gov (United States)

Moerner, W. E.

2011-03-01

Since the first optical detection and spectroscopy of a single molecule in a solid (Phys. Rev. Lett. {62}, 2535 (1989)), much has been learned about the ability of single molecules to probe local nanoenvironments and individual behavior in biological and nonbiological materials in the absence of ensemble averaging that can obscure heterogeneity. Because each single fluorophore acts a light source roughly 1 nm in size, microscopic imaging of individual fluorophores leads naturally to superlocalization, or determination of the position of the molecule with precision beyond the optical diffraction limit, simply by digitization of the point-spread function from the single emitter. For example, the shape of single filaments in a living cell can be extracted simply by allowing a single molecule to move through the filament (PNAS {103}, 10929 (2006)). The addition of photoinduced control of single-molecule emission allows imaging beyond the diffraction limit (super-resolution) and a new array of acronyms (PALM, STORM, F-PALM etc.) and advances have appeared. We have used the native blinking and switching of a common yellow-emitting variant of green fluorescent protein (EYFP) reported more than a decade ago (Nature {388}, 355 (1997)) to achieve sub-40 nm super-resolution imaging of several protein structures in the bacterium Caulobacter crescentus: the quasi-helix of the actin-like protein MreB (Nat. Meth. {5}, 947 (2008)), the cellular distribution of the DNA binding protein HU (submitted), and the recently discovered division spindle composed of ParA filaments (Nat. Cell Biol. {12}, 791 (2010)). Even with these advances, better emitters would provide more photons and improved resolution, and a new photoactivatable small-molecule emitter has recently been synthesized and targeted to specific structures in living cells to provide super-resolution images (JACS {132}, 15099 (2010)). Finally, a new optical method for extracting three-dimensional position information based on

Prediction of fretting fatigue behavior under elastic-plastic conditions

International Nuclear Information System (INIS)

Shin, Ki Su

2009-01-01

Fretting fatigue generally leads to the degradation of the fatigue strength of a material due to cyclic micro-slip between two contacting materials. Fretting fatigue is regarded as an important issue in designing aerospace structures. While many studies have evaluated fretting fatigue behavior under elastic deformation conditions, few have focused on fretting fatigue behavior under elastic-plastic deformation conditions, especially the crack orientation and fatigue life prediction for Ti-6Al-4V. The primary goal of this study was to characterize the fretting fatigue crack initiation behavior in the presence of plasticity. Experimental tests were performed using pad configurations involving elastic-plastic deformations. To calculate stress distributions under elastic-plastic fretting fatigue conditions, FEA was also performed. Several parametric approaches were used to predict fretting fatigue life along with stress distribution resulting from FEA. However, those parameters using surface stresses were unable to establish an equivalence between elastic fretting fatigue data and elastic-plastic fretting fatigue data. Based on this observation, the critical distance methods, which are commonly used in notch analysis, were applied to the fretting fatigue problem. In conclusion, the effective strain range method when used in conjunction with the SMSSR parameter showed a good correlation of data points between the pad configurations involving elastic and elastic plastic deformations

Single-molecule conductance with nitrile and amino contacts with Ag or Cu electrodes

International Nuclear Information System (INIS)

Li, Dong-Fang; Mao, Jin-Chuan; Chen, De-Li; Chen, Fang; Ze-Wen, Hong; Zhou, Xiao-Yi; Wang, Ya-Hao; Zhou, Xiao-Shun; Niu, Zhen-Jiang; Maisonhaute, Emmanuel

2015-01-01

The single-molecule conductance of 1,4-dicyanobenzene (DCB), 1,4-benzenediamine (BDA) and 4,4'-biphenyldicarbonitrile (BPDC) with Ag and/or Cu electrodes is measured by electrochemical jump-to-contact STM-break junction. All single-molecule junctions present three sets of conductance values revealing different contact geometries. We observe that the single-molecule conductance of Ag-BDA-Ag junction is larger that of Ag-DCB-Ag junction, and DCB with Ag contacts are more conductive than that with Cu ones. This is related to a different electronic coupling between the molecules and the electrodes. Tunneling decay constants of 1.70 and 1.68 per phenyl group were found for Ag and Cu electrodes, respectively. The present study therefore shows that nitrile and amino groups can also be used as effective anchors for other metals than gold

DNA-psoralen interaction: a single molecule experiment.

Science.gov (United States)

Rocha, M S; Viana, N B; Mesquita, O N

2004-11-15

By attaching one end of a single lambda-DNA molecule to a microscope coverslip and the other end to a polystyrene microsphere trapped by an optical tweezers, we can study the entropic elasticity of the lambda-DNA by measuring force versus extension as we stretch the molecule. This powerful method permits single molecule studies. We are particularly interested in the effects of the photosensitive drug psoralen on the elasticity of the DNA molecule. We have illuminated the sample with different light sources, studying how the different wavelengths affect the psoralen-DNA linkage. To do this, we measure the persistence length of individual DNA-psoralen complexes.

Perception of color emotions for single colors in red-green defective observers.

Science.gov (United States)

Sato, Keiko; Inoue, Takaaki

2016-01-01

It is estimated that inherited red-green color deficiency, which involves both the protan and deutan deficiency types, is common in men. For red-green defective observers, some reddish colors appear desaturated and brownish, unlike those seen by normal observers. Despite its prevalence, few studies have investigated the effects that red-green color deficiency has on the psychological properties of colors (color emotions). The current study investigated the influence of red-green color deficiency on the following six color emotions: cleanliness, freshness, hardness, preference, warmth, and weight. Specifically, this study aimed to: (1) reveal differences between normal and red-green defective observers in rating patterns of six color emotions; (2) examine differences in color emotions related to the three cardinal channels in human color vision; and (3) explore relationships between color emotions and color naming behavior. Thirteen men and 10 women with normal vision and 13 men who were red-green defective performed both a color naming task and an emotion rating task with 32 colors from the Berkeley Color Project (BCP). Results revealed noticeable differences in the cleanliness and hardness ratings between the normal vision observers, particularly in women, and red-green defective observers, which appeared mainly for colors in the orange to cyan range, and in the preference and warmth ratings for colors with cyan and purple hues. Similarly, naming errors also mainly occurred in the cyan colors. A regression analysis that included the three cone-contrasts (i.e., red-green, blue-yellow, and luminance) as predictors significantly accounted for variability in color emotion ratings for the red-green defective observers as much as the normal individuals. Expressly, for warmth ratings, the weight of the red-green opponent channel was significantly lower in color defective observers than in normal participants. In addition, the analyses for individual warmth ratings in

Perception of color emotions for single colors in red-green defective observers

Directory of Open Access Journals (Sweden)

Keiko Sato

2016-12-01

Full Text Available It is estimated that inherited red-green color deficiency, which involves both the protan and deutan deficiency types, is common in men. For red-green defective observers, some reddish colors appear desaturated and brownish, unlike those seen by normal observers. Despite its prevalence, few studies have investigated the effects that red-green color deficiency has on the psychological properties of colors (color emotions. The current study investigated the influence of red-green color deficiency on the following six color emotions: cleanliness, freshness, hardness, preference, warmth, and weight. Specifically, this study aimed to: (1 reveal differences between normal and red-green defective observers in rating patterns of six color emotions; (2 examine differences in color emotions related to the three cardinal channels in human color vision; and (3 explore relationships between color emotions and color naming behavior. Thirteen men and 10 women with normal vision and 13 men who were red-green defective performed both a color naming task and an emotion rating task with 32 colors from the Berkeley Color Project (BCP. Results revealed noticeable differences in the cleanliness and hardness ratings between the normal vision observers, particularly in women, and red-green defective observers, which appeared mainly for colors in the orange to cyan range, and in the preference and warmth ratings for colors with cyan and purple hues. Similarly, naming errors also mainly occurred in the cyan colors. A regression analysis that included the three cone-contrasts (i.e., red-green, blue-yellow, and luminance as predictors significantly accounted for variability in color emotion ratings for the red-green defective observers as much as the normal individuals. Expressly, for warmth ratings, the weight of the red-green opponent channel was significantly lower in color defective observers than in normal participants. In addition, the analyses for individual warmth

Handbook of Single-Molecule Biophysics

CERN Document Server

Hinterdorfer, Peter

2009-01-01

The last decade has seen the development of a number of novel biophysical methods that allow the manipulation and study of individual biomolecules. The ability to monitor biological processes at this fundamental level of sensitivity has given rise to an improved understanding of the underlying molecular mechanisms. Through the removal of ensemble averaging, distributions and fluctuations of molecular properties can be characterized, transient intermediates identified, and catalytic mechanisms elucidated. By applying forces on biomolecules while monitoring their activity, important information can be obtained on how proteins couple function to structure. The Handbook of Single-Molecule Biophysics provides an introduction to these techniques and presents an extensive discussion of the new biological insights obtained from them. Coverage includes: Experimental techniques to monitor and manipulate individual biomolecules The use of single-molecule techniques in super-resolution and functional imaging Single-molec...

Zero-mode waveguide nanophotonic structures for single molecule characterization

Science.gov (United States)

Crouch, Garrison M.; Han, Donghoon; Bohn, Paul W.

2018-05-01

Single-molecule characterization has become a crucial research tool in the chemical and life sciences, but limitations, such as limited concentration range, inability to control molecular distributions in space, and intrinsic phenomena, such as photobleaching, present significant challenges. Recent developments in non-classical optics and nanophotonics offer promising routes to mitigating these restrictions, such that even low affinity (K D ~ mM) biomolecular interactions can be studied. Here we introduce and review specific nanophotonic devices used to support single molecule studies. Optical nanostructures, such as zero-mode waveguides (ZMWs), are usually fabricated in thin gold or aluminum films and serve to confine the observation volume of optical microspectroscopy to attoliter to zeptoliter volumes. These simple nanostructures allow individual molecules to be isolated for optical and electrochemical analysis, even when the molecules of interest are present at high concentration (µM–mM) in bulk solution. Arrays of ZMWs may be combined with optical probes such as single molecule fluorescence, single molecule fluorescence resonance energy transfer, and fluorescence correlation spectroscopy for distributed analysis of large numbers of single-molecule reactions or binding events in parallel. Furthermore, ZMWs may be used as multifunctional devices, for example by combining optical and electrochemical functions in a single discrete architecture to achieve electrochemical ZMWs. In this review, we will describe the optical properties, fabrication, and applications of ZMWs for single-molecule studies, as well as the integration of ZMWs into systems for chemical and biochemical analysis.

A Multi-Addressable Dyad with Switchable CMY Colors for Full-Color Rewritable Papers.

Science.gov (United States)

Qin, Tianyou; Han, Jiaqi; Geng, Yue; Ju, Le; Sheng, Lan; Zhang, Sean Xiao-An

2018-06-23

Reversible multicolor displays on solid media using single molecule pigments have been a long-awaited goal. Herein, a new and simple molecular dyad, which can undergo switchable CMY color changes both in solution and solid substrate upon exposure to light, water/acid, and nucleophiles, is designed and synthesized. The stimuli used in this work can be applied independent of each other, which is beneficial for color changes without mutual interference. As a comparison, the mixtures of the two molecular switching motifs forming the basis of the dyad were also studied. The dyad greatly outperforms the corresponding mixed system with respect to reversible color-switching on the paper substrate. Its potential for full-color rewritable paper with excellent reversibility has been demonstrated. Legible multicolor prints, that is, high color contrast and resolution, good dispersion, excellent reversibility, were achieved using common water-jet and light-based printers. This work provides a very promising approach for further development of full-color switchable molecules, materials and displays. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

An all-electric single-molecule motor.

Science.gov (United States)

Seldenthuis, Johannes S; Prins, Ferry; Thijssen, Joseph M; van der Zant, Herre S J

2010-11-23

Many types of molecular motors have been proposed and synthesized in recent years, displaying different kinds of motion, and fueled by different driving forces such as light, heat, or chemical reactions. We propose a new type of molecular motor based on electric field actuation and electric current detection of the rotational motion of a molecular dipole embedded in a three-terminal single-molecule device. The key aspect of this all-electronic design is the conjugated backbone of the molecule, which simultaneously provides the potential landscape of the rotor orientation and a real-time measure of that orientation through the modulation of the conductivity. Using quantum chemistry calculations, we show that this approach provides full control over the speed and continuity of motion, thereby combining electrical and mechanical control at the molecular level over a wide range of temperatures. Moreover, chemistry can be used to change all key parameters of the device, enabling a variety of new experiments on molecular motors.

Spectrally Resolved and Functional Super-resolution Microscopy via Ultrahigh-Throughput Single-Molecule Spectroscopy.

Science.gov (United States)

Yan, Rui; Moon, Seonah; Kenny, Samuel J; Xu, Ke

2018-03-20

As an elegant integration of the spatial and temporal dimensions of single-molecule fluorescence, single-molecule localization microscopy (SMLM) overcomes the diffraction-limited resolution barrier of optical microscopy by localizing single molecules that stochastically switch between fluorescent and dark states over time. While this type of super-resolution microscopy (SRM) technique readily achieves remarkable spatial resolutions of ∼10 nm, it typically provides no spectral information. Meanwhile, current scanning-based single-location approaches for mapping the positions and spectra of single molecules are limited by low throughput and are difficult to apply to densely labeled (bio)samples. In this Account, we summarize the rationale, design, and results of our recent efforts toward the integration of the spectral dimension of single-molecule fluorescence with SMLM to achieve spectrally resolved SMLM (SR-SMLM) and functional SRM ( f-SRM). By developing a wide-field scheme for spectral measurement and implementing single-molecule fluorescence on-off switching typical of SMLM, we first showed that in densely labeled (bio)samples it is possible to record the fluorescence spectra and positions of millions of single molecules synchronously within minutes, giving rise to ultrahigh-throughput single-molecule spectroscopy and SR-SMLM. This allowed us to first show statistically that for many dyes, single molecules of the same species exhibit near identical emission in fixed cells. This narrow distribution of emission wavelengths, which contrasts markedly with previous results at solid surfaces, allowed us to unambiguously identify single molecules of spectrally similar dyes. Crosstalk-free, multiplexed SRM was thus achieved for four dyes that were merely 10 nm apart in emission spectrum, with the three-dimensional SRM images of all four dyes being automatically aligned within one image channel. The ability to incorporate single-molecule fluorescence measurement with

Monitoring single membrane protein dynamics in a liposome manipulated in solution by the ABELtrap

Science.gov (United States)

Rendler, T.; Renz, M.; Hammann, E.; Ernst, S.; Zarrabi, N.; Börsch, M.

2011-02-01

FoF1-ATP synthase is the essential membrane enzyme maintaining the cellular level of adenosine triphosphate (ATP) and comprises two rotary motors. We measure subunit rotation in FoF1-ATP synthase by intramolecular Foerster resonance energy transfer (FRET) between two fluorophores at the rotor and at the stator of the enzyme. Confocal FRET measurements of freely diffusing single enzymes in lipid vesicles are limited to hundreds of milliseconds by the transit times through the laser focus. We evaluate two different methods to trap the enzyme inside the confocal volume in order to extend the observation times. Monte Carlo simulations show that optical tweezers with low laser power are not suitable for lipid vesicles with a diameter of 130 nm. A. E. Cohen (Harvard) and W. E. Moerner (Stanford) have recently developed an Anti-Brownian electrokinetic trap (ABELtrap) which is capable to apparently immobilize single molecules, proteins, viruses or vesicles in solution. Trapping of fluorescent particles is achieved by applying a real time, position-dependent feedback to four electrodes in a microfluidic device. The standard deviation from a given target position in the ABELtrap is smaller than 200 nm. We develop a combination of the ABELtrap with confocal FRET measurements to monitor single membrane enzyme dynamics by FRET for more than 10 seconds in solution.

Thousand-fold enhancement of single-molecule fluorescence near a single gold nanorod

NARCIS (Netherlands)

Yuan, H.; Khatua, S.; Zijlstra, P.; Yorulmaz, M.; Orrit, M.

2013-01-01

Single molecules: Large enhancements of single-molecule fluorescence up to 1100 times by using synthesized gold nanorods are reported (see picture). This high enhancement is achieved by selecting a dye with its adsorption and emission close to the surface plasmon resonance of the gold nanorods

Time-resolved homo-FRET studies of biotin-streptavidin complexes.

Science.gov (United States)

Andreoni, Alessandra; Nardo, Luca; Rigler, Rudolf

2016-09-01

Förster resonance energy transfer is a mechanism of fluorescence quenching that is notably useful for characterizing properties of biomolecules and/or their interactions. Here we study water-solutions of Biotin-Streptavidin complexes, in which Biotin is labeled with a rigidly-bound fluorophore that can interact by Förster resonance energy transfer with the fluorophores labeling the other, up to three, Biotins of the same complex. The fluorophore, Atto550, is a Rhodamine analogue. We detect the time-resolved fluorescence decay of the fluorophores with an apparatus endowed with single-photon sensitivity and temporal resolution of ~30ps. The decay profiles we observe for samples containing constant Biotin-Atto550 conjugates and varying Streptavidin concentrations are multi-exponential. Each decay component can be associated with the rate of quenching exerted on each donor by each of the acceptors that label the other Biotin molecules, depending on the binding site they occupy. The main features that lead to this result are that (i) the transition dipole moments of the up-to-four Atto550 fluorophores that label the complexes are fixed as to both relative positions and mutual orientations; (ii) the fluorophores are identical and the role of donor in each Biotin-Streptavidin complex is randomly attributed to the one that has absorbed the excitation light (homo-FRET). Obviously the high-temporal resolution of the excitation-detection apparatus is necessary to discriminate among the fluorescence decay components. Copyright © 2016 Elsevier B.V. All rights reserved.

Fluorescence resonance energy transfer (FRET-based subcellular visualization of pathogen-induced host receptor signaling

Directory of Open Access Journals (Sweden)

Zimmermann Timo

2009-11-01

Full Text Available Abstract Background Bacteria-triggered signaling events in infected host cells are key elements in shaping the host response to pathogens. Within the eukaryotic cell, signaling complexes are spatially organized. However, the investigation of protein-protein interactions triggered by bacterial infection in the cellular context is technically challenging. Here, we provide a methodological approach to exploit fluorescence resonance energy transfer (FRET to visualize pathogen-initiated signaling events in human cells. Results Live-cell microscopy revealed the transient recruitment of the Src family tyrosine kinase Hck upon bacterial engagement of the receptor carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3. In cells expressing a CEACAM3 variant lacking the cytoplasmic domain, the Src homology 2 (SH2 domain of Hck (Hck-SH2 was not recruited, even though bacteria still bound to the receptor. FRET measurements on the basis of whole cell lysates revealed intimate binding between Hck-SH2 (using enhanced yellow fluorescent protein (YPet-Hck-SH2 and the tyrosine-phosphorylated enhanced cyan fluorescent protein-labeled cytoplasmic domain of wild-type CEACAM3 (CEACAM3 WT-CyPet and a flow cytometry-based FRET approach verified this association in intact cells. Using confocal microscopy and acceptor photobleaching, FRET between Hck-SH2 and CEACAM3 was localized to the sites of bacteria-host cell contact. Conclusion These data demonstrate not only the intimate binding of the SH2 domain of Hck to the tyrosine-phosphorylated cytoplasmic domain of CEACAM3 in intact cells, but furthermore, FRET measurements allow the subcellular localization of this process during bacterial infection. FRET-based assays are valuable tools to resolve bacteria-induced protein-protein interactions in the context of the intact host cell.

Human attention filters for single colors

Science.gov (United States)

Sun, Peng; Chubb, Charles; Wright, Charles E.; Sperling, George

2016-01-01

The visual images in the eyes contain much more information than the brain can process. An important selection mechanism is feature-based attention (FBA). FBA is best described by attention filters that specify precisely the extent to which items containing attended features are selectively processed and the extent to which items that do not contain the attended features are attenuated. The centroid-judgment paradigm enables quick, precise measurements of such human perceptual attention filters, analogous to transmission measurements of photographic color filters. Subjects use a mouse to locate the centroid—the center of gravity—of a briefly displayed cloud of dots and receive precise feedback. A subset of dots is distinguished by some characteristic, such as a different color, and subjects judge the centroid of only the distinguished subset (e.g., dots of a particular color). The analysis efficiently determines the precise weight in the judged centroid of dots of every color in the display (i.e., the attention filter for the particular attended color in that context). We report 32 attention filters for single colors. Attention filters that discriminate one saturated hue from among seven other equiluminant distractor hues are extraordinarily selective, achieving attended/unattended weight ratios >20:1. Attention filters for selecting a color that differs in saturation or lightness from distractors are much less selective than attention filters for hue (given equal discriminability of the colors), and their filter selectivities are proportional to the discriminability distance of neighboring colors, whereas in the same range hue attention-filter selectivity is virtually independent of discriminabilty. PMID:27791040

Mining the Sinorhizobium meliloti transportome to develop FRET biosensors for sugars, dicarboxylates and cyclic polyols.

Directory of Open Access Journals (Sweden)

Alexandre Bourdès

Full Text Available Förster resonance energy transfer (FRET biosensors are powerful tools to detect biologically important ligands in real time. Currently FRET bisosensors are available for twenty-two compounds distributed in eight classes of chemicals (two pentoses, two hexoses, two disaccharides, four amino acids, one nucleobase, two nucleotides, six ions and three phytoestrogens. To expand the number of available FRET biosensors we used the induction profile of the Sinorhizobium meliloti transportome to systematically screen for new FRET biosensors.Two new vectors were developed for cloning genes for solute-binding proteins (SBPs between those encoding FRET partner fluorescent proteins. In addition to a vector with the widely used cyan and yellow fluorescent protein FRET partners, we developed a vector using orange (mOrange2 and red fluorescent protein (mKate2 FRET partners. From the sixty-nine SBPs tested, seven gave a detectable FRET signal change on binding substrate, resulting in biosensors for D-quinic acid, myo-inositol, L-rhamnose, L-fucose, β-diglucosides (cellobiose and gentiobiose, D-galactose and C4-dicarboxylates (malate, succinate, oxaloacetate and fumarate. To our knowledge, we describe the first two FRET biosensor constructs based on SBPs from Tripartite ATP-independent periplasmic (TRAP transport systems.FRET based on orange (mOrange2 and red fluorescent protein (mKate2 partners allows the use of longer wavelength light, enabling deeper penetration of samples at lower energy and increased resolution with reduced back-ground auto-fluorescence. The FRET biosensors described in this paper for four new classes of compounds; (i cyclic polyols, (ii L-deoxy sugars, (iii β-linked disaccharides and (iv C4-dicarboxylates could be developed to study metabolism in vivo.

Single Molecule Conductance of Oligothiophene Derivatives

Science.gov (United States)

Dell, Emma J.

This thesis studies the electronic properties of small organic molecules based on the thiophene motif. If we are to build next-generation devices, advanced materials must be designed which possess requisite electronic functionality. Molecules present attractive candidates for these ad- vanced materials since nanoscale devices are particularly sought after. However, selecting a molecule that is suited to a certain electronic function remains a challenge, and characterization of electronic behavior is therefore critical. Single molecule conductance measurements are a powerful tool to determine properties on the nanoscale and, as such, can be used to investigate novel building blocks that may fulfill the design requirements of next-generation devices. Combining these conductance results with strategic chemical synthesis allows for the development of new families of molecules that show attractive properties for future electronic devices. Since thiophene rings are the fruitflies of organic semiconductors on the bulk scale, they present an intriguing starting point for building functional materials on the nanoscale, and therefore form the structural basis of all molecules studied herein. First, the single-molecule conductance of a family of bithiophene derivatives was measured. A broad distribution in the single-molecule conductance of bithiophene was found compared with that of a biphenyl. This increased breadth in the conductance distribution was shown to be explained by the difference in 5-fold symmetry of thiophene rings as compared to the 6-fold symmetry of benzene rings. The reduced symmetry of thiophene rings results in a restriction on the torsion angle space available to these molecules when bound between two metal electrodes in a junction, causing each molecular junction to sample a different set of conformers in the conductance measurements. By contrast, the rotations of biphenyl are essentially unimpeded by junction binding, allowing each molecular junction

Advances in Spiropyrans/Spirooxazines and Applications Based on Fluorescence Resonance Energy Transfer (FRET with Fluorescent Materials

Directory of Open Access Journals (Sweden)

Hongyan Xia

2017-12-01

Full Text Available Studies on the following were reviewed: (1 the structure of spiropyrans and spirooxazines (two kinds of spiro compounds under external stimuli and (2 the construction and applications of composite systems based on fluorescence resonance energy transfer (FRET with fluorescent materials. When treated with different stimuli (light, acids and bases, solvents, metal ions, temperature, redox potential, and so on, spiropyrans/spirooxazines undergo transformations between the ring-closed form (SP, the ring-opened merocyanine (MC form, and the protonated ring-opened form (MCH. This is due to the breakage of the spiro C–O bond and the protonation of MC, along with a color change. Various novel, multifunctional materials based on photochromic spiropyrans and spirooxazines have been successfully developed because of the vastly differently physiochemical properties posssed by the SP, MC and MCH forms. Among the three different structural forms, the MC form has been studied most extensively. The MC form not only gives complexes with various inorganic particles, biological molecules, and organic chemicals but also acts as the energy acceptor (of energy from fluorescent molecules during energy transfer processes that take place under proper conditions. Furthermore, spiropyran and spirooxazine compounds exhibit reversible physicochemical property changes under proper stimuli; this provides more advantages compared with other photochromic compounds. Additionally, the molecular structures of spiropyrans and spirooxazines can be easily modified and extended, so better compounds can be obtained to expand the scope of already known applications. Described in detail are: (1 the structural properties of spiropyrans and spirooxazines and related photochromic mechanisms; (2 composite systems based on spiropyrans and spirooxazines, and (3 fluorescent materials which have potential applications in sensing, probing, and a variety of optical elements.

Improved Dye Stability in Single-Molecule Fluorescence Experiments

Science.gov (United States)

EcheverrÍa Aitken, Colin; Marshall, R. Andrew; Pugi, Joseph D.

Complex biological systems challenge existing single-molecule methods. In particular, dye stability limits observation time in singlemolecule fluorescence applications. Current approaches to improving dye performance involve the addition of enzymatic oxygen scavenging systems and small molecule additives. We present an enzymatic oxygen scavenging system that improves dye stability in single-molecule experiments. Compared to the currently-employed glucose-oxidase/catalase system, the protocatechuate-3,4-dioxygenase system achieves lower dissolved oxygen concentration and stabilizes single Cy3, Cy5, and Alexa488 fluorophores. Moreover, this system possesses none of the limitations associated with the glucose oxidase/catalase system. We also tested the effects of small molecule additives in this system. Biological reducing agents significantly destabilize the Cy5 fluorophore as a function of reducing potential. In contrast, anti-oxidants stabilize the Cy3 and Alexa488 fluorophores. We recommend use of the protocatechuate-3,4,-dioxygenase system with antioxidant additives, and in the absence of biological reducing agents. This system should have wide application to single-molecule fluorescence experiments.

Correlative FRET: new method improves rigor and reproducibility in determining distances within synaptic nanoscale architecture

Science.gov (United States)

Shinogle-Decker, Heather; Martinez-Rivera, Noraida; O'Brien, John; Powell, Richard D.; Joshi, Vishwas N.; Connell, Samuel; Rosa-Molinar, Eduardo

2018-02-01

A new correlative Förster Resonance Energy Transfer (FRET) microscopy method using FluoroNanogold™, a fluorescent immunoprobe with a covalently attached Nanogold® particle (1.4nm Au), overcomes resolution limitations in determining distances within synaptic nanoscale architecture. FRET by acceptor photobleaching has long been used as a method to increase fluorescence resolution. The transfer of energy from a donor to an acceptor generally occurs between 10-100Å, which is the relative distance between the donor molecule and the acceptor molecule. For the correlative FRET microscopy method using FluoroNanogold™, we immuno-labeled GFP-tagged-HeLa-expressing Connexin 35 (Cx35) with anti-GFP and with anti-Cx35/36 antibodies, and then photo-bleached the Cx before processing the sample for electron microscopic imaging. Preliminary studies reveal the use of Alexa Fluor® 594 FluoroNanogold™ slightly increases FRET distance to 70Å, in contrast to the 62.5Å using AlexaFluor 594®. Preliminary studies also show that using a FluoroNanogold™ probe inhibits photobleaching. After one photobleaching session, Alexa Fluor 594® fluorescence dropped to 19% of its original fluorescence; in contrast, after one photobleaching session, Alexa Fluor 594® FluoroNanogold™ fluorescence dropped to 53% of its original intensity. This result confirms that Alexa Fluor 594® FluoroNanogold™ is a much better donor probe than is Alexa Fluor 594®. The new method (a) creates a double confirmation method in determining structure and orientation of synaptic architecture, (b) allows development of a two-dimensional in vitro model to be used for precise testing of multiple parameters, and (c) increases throughput. Future work will include development of FluoroNanogold™ probes with different sizes of gold for additional correlative microscopy studies.

Cell biochemistry studied by single-molecule imaging.

Science.gov (United States)

Mashanov, G I; Nenasheva, T A; Peckham, M; Molloy, J E

2006-11-01

Over the last decade, there have been remarkable developments in live-cell imaging. We can now readily observe individual protein molecules within living cells and this should contribute to a systems level understanding of biological pathways. Direct observation of single fluorophores enables several types of molecular information to be gathered. Temporal and spatial trajectories enable diffusion constants and binding kinetics to be deduced, while analyses of fluorescence lifetime, intensity, polarization or spectra give chemical and conformational information about molecules in their cellular context. By recording the spatial trajectories of pairs of interacting molecules, formation of larger molecular complexes can be studied. In the future, multicolour and multiparameter imaging of single molecules in live cells will be a powerful analytical tool for systems biology. Here, we discuss measurements of single-molecule mobility and residency at the plasma membrane of live cells. Analysis of diffusional paths at the plasma membrane gives information about its physical properties and measurement of temporal trajectories enables rates of binding and dissociation to be derived. Meanwhile, close scrutiny of individual fluorophore trajectories enables ideas about molecular dimerization and oligomerization related to function to be tested directly.

Probing Intranuclear Environments at the Single-Molecule Level

Science.gov (United States)

Grünwald, David; Martin, Robert M.; Buschmann, Volker; Bazett-Jones, David P.; Leonhardt, Heinrich; Kubitscheck, Ulrich; Cardoso, M. Cristina

2008-01-01

Genome activity and nuclear metabolism clearly depend on accessibility, but it is not known whether and to what extent nuclear structures limit the mobility and access of individual molecules. We used fluorescently labeled streptavidin with a nuclear localization signal as an average-sized, inert protein to probe the nuclear environment. The protein was injected into the cytoplasm of mouse cells, and single molecules were tracked in the nucleus with high-speed fluorescence microscopy. We analyzed and compared the mobility of single streptavidin molecules in structurally and functionally distinct nuclear compartments of living cells. Our results indicated that all nuclear subcompartments were easily and similarly accessible for such an average-sized protein, and even condensed heterochromatin neither excluded single molecules nor impeded their passage. The only significant difference was a higher frequency of transient trappings in heterochromatin, which lasted only tens of milliseconds. The streptavidin molecules, however, did not accumulate in heterochromatin, suggesting comparatively less free volume. Interestingly, the nucleolus seemed to exclude streptavidin, as it did many other nuclear proteins, when visualized by conventional fluorescence microscopy. The tracking of single molecules, nonetheless, showed no evidence for repulsion at the border but relatively unimpeded passage through the nucleolus. These results clearly show that single-molecule tracking can provide novel insights into mobility of proteins in the nucleus that cannot be obtained by conventional fluorescence microscopy. Our results suggest that nuclear processes may not be regulated at the level of physical accessibility but rather by local concentration of reactants and availability of binding sites. PMID:18065482

Multistabilities and symmetry-broken one-color and two-color states in closely coupled single-mode lasers.

Science.gov (United States)

Clerkin, Eoin; O'Brien, Stephen; Amann, Andreas

2014-03-01

We theoretically investigate the dynamics of two mutually coupled, identical single-mode semi-conductor lasers. For small separation and large coupling between the lasers, symmetry-broken one-color states are shown to be stable. In this case the light outputs of the lasers have significantly different intensities while at the same time the lasers are locked to a single common frequency. For intermediate coupling we observe stable symmetry-broken two-color states, where both lasers lase simultaneously at two optical frequencies which are separated by up to 150 GHz. Using a five-dimensional model, we identify the bifurcation structure which is responsible for the appearance of symmetric and symmetry-broken one-color and two-color states. Several of these states give rise to multistabilities and therefore allow for the design of all-optical memory elements on the basis of two coupled single-mode lasers. The switching performance of selected designs of optical memory elements is studied numerically.

Electrophoresis- and FRET-Based Measures of Serpin Polymerization.

Science.gov (United States)

Faull, Sarah V; Brown, Anwen E; Haq, Imran; Irving, James A

2017-01-01

Many serpinopathies, including alpha-1 antitrypsin (A1AT) deficiency, are associated with the formation of unbranched polymer chains of mutant serpins. In vivo, this deficiency is the result of mutations that cause kinetic or thermodynamic destabilization of the molecule. However, polymerization can also be induced in vitro from mutant or wild-type serpins under destabilizing conditions. The characteristics of the resulting polymers are dependent upon induction conditions. Due to their relationship to disease, serpin polymers, mainly those formed from A1AT, have been widely studied. Here, we describe Förster resonance energy transfer (FRET) and gel-based approaches for their characterization.

Mechanisms of fretting-fatigue of titanium alloys

Energy Technology Data Exchange (ETDEWEB)

Antoniou, R A; Radtke, T C [Defence Sci. and Technol. Organ., Melbourne, Vic. (Australia). Aeronautical and Maritime Res. Lab.

1997-09-30

The effect of continuous fretting in air at 20 C on fatigue performance has been studied for Ti-17 and Ti-6Al-4V, high strength titanium alloys used for gas-turbine fan and compressor disks and blades, respectively. The effect of fretting was to reduce the fatigue stress limit from 700 MPa for plain fatigue to 200 MPa for fretting-fatigue. A number of models, supported by metallographic and fractographic evidence, are proposed which explain (i) how the cyclic loading of individual asperities results in crack initiation; (ii) the formation of multiple cracks; (iii) the existence of non-propagating cracks; and (iv) how fretting influences crack propagation once fatigue cracks have formed. (orig.) 46 refs.

Single molecule image formation, reconstruction and processing: introduction.

Science.gov (United States)

Ashok, Amit; Piestun, Rafael; Stallinga, Sjoerd

2016-07-01

The ability to image at the single molecule scale has revolutionized research in molecular biology. This feature issue presents a collection of articles that provides new insights into the fundamental limits of single molecule imaging and reports novel techniques for image formation and analysis.

Single-Molecule Photocurrent at a Metal-Molecule-Semiconductor Junction.

Science.gov (United States)

Vezzoli, Andrea; Brooke, Richard J; Higgins, Simon J; Schwarzacher, Walther; Nichols, Richard J

2017-11-08

We demonstrate here a new concept for a metal-molecule-semiconductor nanodevice employing Au and GaAs contacts that acts as a photodiode. Current-voltage traces for such junctions are recorded using a STM, and the "blinking" or "I(t)" method is used to record electrical behavior at the single-molecule level in the dark and under illumination, with both low and highly doped GaAs samples and with two different types of molecular bridge: nonconjugated pentanedithiol and the more conjugated 1,4-phenylene(dimethanethiol). Junctions with highly doped GaAs show poor rectification in the dark and a low photocurrent, while junctions with low doped GaAs show particularly high rectification ratios in the dark (>10 3 for a 1.5 V bias potential) and a high photocurrent in reverse bias. In low doped GaAs, the greater thickness of the depletion layer not only reduces the reverse bias leakage current, but also increases the volume that contributes to the photocurrent, an effect amplified by the point contact geometry of the junction. Furthermore, since photogenerated holes tunnel to the metal electrode assisted by the HOMO of the molecular bridge, the choice of the latter has a strong influence on both the steady state and transient metal-molecule-semiconductor photodiode response. The control of junction current via photogenerated charge carriers adds new functionality to single-molecule nanodevices.

A new microcavity design for single molecule detection

International Nuclear Information System (INIS)

Steiner, M.; Schleifenbaum, F.; Stupperich, C.; Failla, A.V.; Hartschuh, A.; Meixner, A.J.

2006-01-01

We present a new microcavity design which allows for efficient detection of single molecules by measuring the molecular fluorescence emission coupled into a resonant cavity mode. The Fabry-Perot-type microresonator consists of two silver mirrors separated by a thin polymer film doped with dye molecules in ultralow concenctration. By slightly tilting one of the mirrors different cavity lengths can be selected within the same sample. Locally, on a μm scale, the microcavity still acts as a planar Fabry-Perot resonator. Using scanning confocal fluorescence microscopy, single emitters on resonance with a single mode of the microresonator can be spatially addressed. Our microcavity is demonstrated to be well-suited for investigating the coupling mechanism between single quantum emitters and single modes of the electromagnetic field. The microcavity layout could be integrated in a lab-on-a-microchip design for ultrasensitive microfluidic analytics and can be considered as an important improvement for single photon sources based on single molecules operating at room temperature

Sensing single electrons with single molecules

International Nuclear Information System (INIS)

Plakhotnik, Taras

2007-01-01

We propose a new methodology for probing transport of just one electron, a process of great importance both in nature and in artificial devices. Our idea for locating a single electron is analogues to the conventional GPS where signals from several satellites are used to locate a macro object. Using fluorescent molecules as tiny sensors, it is possible to determine 3D displacement vector of an electron

Molecular electronics with single molecules in solid-state devices

DEFF Research Database (Denmark)

Moth-Poulsen, Kasper; Bjørnholm, Thomas

2009-01-01

The ultimate aim of molecular electronics is to understand and master single-molecule devices. Based on the latest results on electron transport in single molecules in solid-state devices, we focus here on new insights into the influence of metal electrodes on the energy spectrum of the molecule...

Research Update: Molecular electronics: The single-molecule switch and transistor

Directory of Open Access Journals (Sweden)

Kai Sotthewes

2014-01-01

Full Text Available In order to design and realize single-molecule devices it is essential to have a good understanding of the properties of an individual molecule. For electronic applications, the most important property of a molecule is its conductance. Here we show how a single octanethiol molecule can be connected to macroscopic leads and how the transport properties of the molecule can be measured. Based on this knowledge we have realized two single-molecule devices: a molecular switch and a molecular transistor. The switch can be opened and closed at will by carefully adjusting the separation between the electrical contacts and the voltage drop across the contacts. This single-molecular switch operates in a broad temperature range from cryogenic temperatures all the way up to room temperature. Via mechanical gating, i.e., compressing or stretching of the octanethiol molecule, by varying the contact's interspace, we are able to systematically adjust the conductance of the electrode-octanethiol-electrode junction. This two-terminal single-molecule transistor is very robust, but the amplification factor is rather limited.

Correlation between three color coordinates of human teeth

Science.gov (United States)

Lee, Yong-Keun

2014-11-01

The objective was to determine whether there were significant correlations in the three color coordinates within each of two color coordinate systems, such as the Commission Internationale de l'Eclairage (CIE) L*a*b* system, and the lightness, chroma, and hue angle system, of human vital teeth. The color of six maxillary and six mandibular anterior teeth was measured by the Shade Vision System. Pearson correlations between each pair of the color coordinates were determined (α=0.01). The influence of two color coordinates on the other color coordinate was determined with a multiple regression analysis (α=0.01). Based on correlation analyses, all the color coordinate pairs showed significant correlations except for the chroma and hue angle pair. The CIE L* was negatively correlated with the CIE a*, b*, and chroma, but positively correlated with the hue angle. The CIE a* was positively correlated with the CIE b* and chroma. Tooth color coordinates were correlated each other. Lighter teeth were less chromatic both in the CIE a* and b* coordinates. Therefore, it was postulated that the three color coordinates of human teeth were harmonized within certain color attribute ranges, and a lack of correlations in these coordinates might indicate external/internal discolorations and/or anomalies of teeth.

Decoupling of size and shape fluctuations in heteropolymeric sequences reconciles discrepancies in SAXS vs. FRET measurements.

Science.gov (United States)

Fuertes, Gustavo; Banterle, Niccolò; Ruff, Kiersten M; Chowdhury, Aritra; Mercadante, Davide; Koehler, Christine; Kachala, Michael; Estrada Girona, Gemma; Milles, Sigrid; Mishra, Ankur; Onck, Patrick R; Gräter, Frauke; Esteban-Martín, Santiago; Pappu, Rohit V; Svergun, Dmitri I; Lemke, Edward A

2017-08-01

Unfolded states of proteins and native states of intrinsically disordered proteins (IDPs) populate heterogeneous conformational ensembles in solution. The average sizes of these heterogeneous systems, quantified by the radius of gyration ( R G ), can be measured by small-angle X-ray scattering (SAXS). Another parameter, the mean dye-to-dye distance ( R E ) for proteins with fluorescently labeled termini, can be estimated using single-molecule Förster resonance energy transfer (smFRET). A number of studies have reported inconsistencies in inferences drawn from the two sets of measurements for the dimensions of unfolded proteins and IDPs in the absence of chemical denaturants. These differences are typically attributed to the influence of fluorescent labels used in smFRET and to the impact of high concentrations and averaging features of SAXS. By measuring the dimensions of a collection of labeled and unlabeled polypeptides using smFRET and SAXS, we directly assessed the contributions of dyes to the experimental values R G and R E For chemically denatured proteins we obtain mutual consistency in our inferences based on R G and R E , whereas for IDPs under native conditions, we find substantial deviations. Using computations, we show that discrepant inferences are neither due to methodological shortcomings of specific measurements nor due to artifacts of dyes. Instead, our analysis suggests that chemical heterogeneity in heteropolymeric systems leads to a decoupling between R E and R G that is amplified in the absence of denaturants. Therefore, joint assessments of R G and R E combined with measurements of polymer shapes should provide a consistent and complete picture of the underlying ensembles.

Studying DNA Looping by Single-Molecule FRET

OpenAIRE

Le, Tung T.; Kim, Harold D.

2014-01-01

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into nucleosomes. Thermodynamics of dsDNA bending has been studied by a method called cyclization which relies on DNA ligase to covalently join short sticky ends of a dsDNA. However, ligation efficiency can be affected by many factors that are not related to dsDNA looping such as the DNA structure surrounding the joined sticky ends, and ligase can als...

Single molecule microscopy and spectroscopy: concluding remarks.

Science.gov (United States)

van Hulst, Niek F

2015-01-01

Chemistry is all about molecules: control, synthesis, interaction and reaction of molecules. All too easily on a blackboard, one draws molecules, their structures and dynamics, to create an insightful picture. The dream is to see these molecules in reality. This is exactly what "Single Molecule Detection" provides: a look at molecules in action at ambient conditions; a breakthrough technology in chemistry, physics and biology. Within the realms of the Royal Society of Chemistry, the Faraday Discussion on "Single Molecule Microscopy and Spectroscopy" was a very appropriate topic for presentation, deliberation and debate. Undoubtedly, the Faraday Discussions have a splendid reputation in stimulating scientific debates along the traditions set by Michael Faraday. Interestingly, back in the 1830's, Faraday himself pursued an experiment that led to the idea that atoms in a compound were joined by an electrical component. He placed two opposite electrodes in a solution of water containing a dissolved compound, and observed that one of the elements of the compound accumulated on one electrode, while the other was deposited on the opposite electrode. Although Faraday was deeply opposed to atomism, he had to recognize that electrical forces were responsible for the joining of atoms. Probably a direct view on the atoms or molecules in his experiment would have convinced him. As such, Michael Faraday might have liked the gathering at Burlington House in September 2015 (). Surely, with the questioning eyes of his bust on the 1st floor corridor, the non-believer Michael Faraday has incited each passer-by to enter into discussion and search for deeper answers at the level of single molecules. In these concluding remarks, highlights of the presented papers and discussions are summarized, complemented by a conclusion on future perspectives.

Toward Bayesian inference of the spatial distribution of proteins from three-cube Förster resonance energy transfer data

DEFF Research Database (Denmark)

Hooghoudt, Jan Otto; Barroso, Margarida; Waagepetersen, Rasmus Plenge

2017-01-01

Főrster resonance energy transfer (FRET) is a quantum-physical phenomenon where energy may be transferred from one molecule to a neighbour molecule if the molecules are close enough. Using fluorophore molecule marking of proteins in a cell it is possible to measure in microscopic images to what....... In this paper we propose a new likelihood-based approach to statistical inference for FRET microscopic data. The likelihood function is obtained from a detailed modeling of the FRET data generating mechanism conditional on a protein configuration. We next follow a Bayesian approach and introduce a spatial point...

Dual-Shell Fluorescent Nanoparticles for Self-Monitoring of pH-Responsive Molecule-Releasing in a Visualized Way.

Science.gov (United States)

Yang, Lingang; Cui, Chuanfeng; Wang, Lingzhi; Lei, Juying; Zhang, Jinlong

2016-07-27

The rational design and controlled synthesis of a smart device with flexibly tailored response ability is all along desirable for bioapplication but long remains a considerable challenge. Here, a pH-stimulated valve system with a visualized "on-off" mode is constructed through a dual-shell fluorescence resonance energy transfer (FRET) strategy. The dual shells refer to carbon dots and fluorescent molecules embedded polymethacrylic acid (F-PMAA) layers successively coating around a SiO2 core (ca. 120 nm), which play the roles as energy donor and acceptor, respectively. The total thickness of the dual-shell in the solid composite is ca. 10 nm. The priorities of this dual-shell FRET nanovalve stem from three facts: (1) the thin shell allows the formation of efficient FRET system without chemical bonding between energy donor and acceptor; (2) the maximum emission wavelength of CD layer is tunable in the range of 400-600 nm, thus providing a flexible energy donor for a wide variety of energy acceptors; (3) the outer F-PMAA shell with a pH-sensitive swelling-shrinking (on-off) behavior functions as a valve for regulating the FRET process. As such, a sensitive and stable pH ratiometric sensor with a working pH range of 3-6 has been built by simply encapsulating pH-responsive fluorescein isothiocyanate (FITC) into PMAA; a pH-dependent swelling-shrinking shuttle carrier with a finely controllable molecule-release behavior has been further fabricated using rhodamine B isothiocyanate (RBITC) as the energy donor and model guest molecule. Significantly, the controlled releasing process is visually self-monitorable.

Fluorescence Lifetime Readouts of Troponin-C-Based Calcium FRET Sensors: A Quantitative Comparison of CFP and mTFP1 as Donor Fluorophores

Science.gov (United States)

Laine, Romain; Stuckey, Daniel W.; Manning, Hugh; Warren, Sean C.; Kennedy, Gordon; Carling, David

2012-01-01

We have compared the performance of two Troponin-C-based calcium FRET sensors using fluorescence lifetime read-outs. The first sensor, TN-L15, consists of a Troponin-C fragment inserted between CFP and Citrine while the second sensor, called mTFP-TnC-Cit, was realized by replacing CFP in TN-L15 with monomeric Teal Fluorescent Protein (mTFP1). Using cytosol preparations of transiently transfected mammalian cells, we have measured the fluorescence decay profiles of these sensors at controlled concentrations of calcium using time-correlated single photon counting. These data were fitted to discrete exponential decay models using global analysis to determine the FRET efficiency, fraction of donor molecules undergoing FRET and calcium affinity of these sensors. We have also studied the decay profiles of the donor fluorescent proteins alone and determined the sensitivity of the donor lifetime to temperature and emission wavelength. Live-cell fluorescence lifetime imaging (FLIM) of HEK293T cells expressing each of these sensors was also undertaken. We confirmed that donor fluorescence of mTFP-TnC-Cit fits well to a two-component decay model, while the TN-L15 lifetime data was best fitted to a constrained four-component model, which was supported by phasor analysis of the measured lifetime data. If the constrained global fitting is employed, the TN-L15 sensor can provide a larger dynamic range of lifetime readout than the mTFP-TnC-Cit sensor but the CFP donor is significantly more sensitive to changes in temperature and emission wavelength compared to mTFP and, while the mTFP-TnC-Cit solution phase data broadly agreed with measurements in live cells, this was not the case for the TN-L15 sensor. Our titration experiment also indicates that a similar precision in determination of calcium concentration can be achieved with both FRET biosensors when fitting a single exponential donor fluorescence decay model to the fluorescence decay profiles. We therefore suggest that m

Single-molecule force-conductance spectroscopy of hydrogen-bonded complexes

Science.gov (United States)

Pirrotta, Alessandro; De Vico, Luca; Solomon, Gemma C.; Franco, Ignacio

2017-03-01

The emerging ability to study physical properties at the single-molecule limit highlights the disparity between what is observable in an ensemble of molecules and the heterogeneous contributions of its constituent parts. A particularly convenient platform for single-molecule studies are molecular junctions where forces and voltages can be applied to individual molecules, giving access to a series of electromechanical observables that can form the basis of highly discriminating multidimensional single-molecule spectroscopies. Here, we computationally examine the ability of force and conductance to inform about molecular recognition events at the single-molecule limit. For this, we consider the force-conductance characteristics of a prototypical class of hydrogen bonded bimolecular complexes sandwiched between gold electrodes. The complexes consist of derivatives of a barbituric acid and a Hamilton receptor that can form up to six simultaneous hydrogen bonds. The simulations combine classical molecular dynamics of the mechanical deformation of the junction with non-equilibrium Green's function computations of the electronic transport. As shown, in these complexes hydrogen bonds mediate transport either by directly participating as a possible transport pathway or by stabilizing molecular conformations with enhanced conductance properties. Further, we observe that force-conductance correlations can be very sensitive to small changes in the chemical structure of the complexes and provide detailed information about the behavior of single molecules that cannot be gleaned from either measurement alone. In fact, there are regions during the elongation that are only mechanically active, others that are only conductance active, and regions where both force and conductance changes as the complex is mechanically manipulated. The implication is that force and conductance provide complementary information about the evolution of molecules in junctions that can be used to

Semisynthetic protein nanoreactor for single-molecule chemistry

OpenAIRE

Lee, Joongoo; Bayley, Hagan

2015-01-01

The modulation of ionic current flowing through an individual protein pore provides information at the single-molecule level about chemical reactions occurring within the pore. However, chemistry investigated in this way has been largely confined to the reactions of thiolates, presented by the side chains of cysteine residues. The introduction of unnatural amino acids would provide a large variety of reactive side chains with which additional single-molecule chemistry could be investigated. H...

Flexible single molecule simulation of reaction-diffusion processes

International Nuclear Information System (INIS)

Hellander, Stefan; Loetstedt, Per

2011-01-01

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.

Modulation and Control of Charge Transport Through Single-Molecule Junctions.

Science.gov (United States)

Wang, Kun; Xu, Bingqian

2017-02-01

The ability to modulate and control charge transport though single-molecule junction devices is crucial to achieving the ultimate goal of molecular electronics: constructing real-world-applicable electronic components from single molecules. This review aims to highlight the progress made in single-molecule electronics, emphasizing the development of molecular junction electronics in recent years. Among many techniques that attempt to wire a molecule to metallic electrodes, the single-molecule break junction (SMBJ) technique is one of the most reliable and tunable experimental platforms for achieving metal-molecule-metal configurations. It also provides great freedom to tune charge transport through the junction. Soon after the SMBJ technique was introduced, it was extensively used to measure the conductances of individual molecules; however, different conductances were obtained for the same molecule, and it proved difficult to interpret this wide distribution of experimental data. This phenomenon was later found to be mainly due to a lack of precise experimental control and advanced data analysis methods. In recent years, researchers have directed considerable effort into advancing the SMBJ technique by gaining a deeper physical understanding of charge transport through single molecules and thus enhancing its potential applicability in functional molecular-scale electronic devices, such as molecular diodes and molecular transistors. In parallel with that research, novel data analysis methods and approaches that enable the discovery of hidden yet important features in the data are being developed. This review discusses various aspects of molecular junction electronics, from the initial goal of molecular electronics, the development of experimental techniques for creating single-molecule junctions and determining single-molecule conductance, to the characterization of functional current-voltage features and the investigation of physical properties other than charge

Single-Molecule Nanomagnets

Science.gov (United States)

Friedman, Jonathan R.; Sarachik, Myriam P.

2010-04-01

Single-molecule magnets straddle the classical and quantum mechanical worlds, displaying many fascinating phenomena. They may have important technological applications in information storage and quantum computation. We review the physical properties of two prototypical molecular nanomagnets, Mn12-acetate and Fe8: Each behaves as a rigid, spin-10 object and exhibits tunneling between up and down directions. As temperature is lowered, the spin-reversal process evolves from thermal activation to pure quantum tunneling. At low temperatures, magnetic avalanches occur in which the magnetization of an entire sample rapidly reverses. We discuss the important role that symmetry-breaking fields play in driving tunneling and in producing Berry-phase interference. Recent experimental advances indicate that quantum coherence can be maintained on timescales sufficient to allow a meaningful number of quantum computing operations to be performed. Efforts are under way to create monolayers and to address and manipulate individual molecules.

Quantum design rules for single molecule logic gates.

Science.gov (United States)

Renaud, N; Hliwa, M; Joachim, C

2011-08-28

Recent publications have demonstrated how to implement a NOR logic gate with a single molecule using its interaction with two surface atoms as logical inputs [W. Soe et al., ACS Nano, 2011, 5, 1436]. We demonstrate here how this NOR logic gate belongs to the general family of quantum logic gates where the Boolean truth table results from a full control of the quantum trajectory of the electron transfer process through the molecule by very local and classical inputs practiced on the molecule. A new molecule OR gate is proposed for the logical inputs to be also single metal atoms, one per logical input.

Photoinduced nuclear spin conversion of methyl groups of single molecules

International Nuclear Information System (INIS)

Sigl, A.

2007-01-01

A methyl group is an outstanding quantum system due to its special symmetry properties. The threefold rotation around one of its bond is isomorphic to the group of even permutations of the remaining protons, a property which imposes severe quantum restrictions on the system, for instance a strict correlation of rotational states with nuclear spin states. The resulting long lifetimes of the rotational tunneling states of the methyl group can be exploited for applying certain high resolution optical techniques, like hole burning or single molecule spectroscopy to optically switch the methyl group from one tunneling state to another therebye changing the nuclear spin of the protons. One goal of the thesis was to perform this switching in single methyl groups. To this end the methyl group was attached to a chromophoric system, in the present case terrylene, which is well suited for single molecule spectroscopy as well as for hole burning. Experiments were performed with the bare terrylene molecule in a hexadecane lattice which served as a reference system, with alphamethyl terrylene and betamethyl terrylene, both embedded in hexadecane, too. A single molecular probe is a highly sensitive detector for dynamic lattice instabilities. Already the bare terrylene probe showed a wealth of interesting local dynamic effects of the hexadecane lattice which could be well acounted for by the assumption of two nearly degenerate sites with rather different optical and thermal properties, all of which could be determined in a quantitative fashion. As to the methylated terrylene systems, the experiments verified that for betamethyl terrylene it is indeed possible to measure rotational tunneling events in single methyl groups. However, the spectral patterns obtained was much more complicated than expected pointing to the presence of three spectroscopically different methyl groups. In order to achieve a definite assignement, molecular mechanics simulations of the terrylene probes in the

Supramolecular Systems and Chemical Reactions in Single-Molecule Break Junctions.

Science.gov (United States)

Li, Xiaohui; Hu, Duan; Tan, Zhibing; Bai, Jie; Xiao, Zongyuan; Yang, Yang; Shi, Jia; Hong, Wenjing

2017-04-01

The major challenges of molecular electronics are the understanding and manipulation of the electron transport through the single-molecule junction. With the single-molecule break junction techniques, including scanning tunneling microscope break junction technique and mechanically controllable break junction technique, the charge transport through various single-molecule and supramolecular junctions has been studied during the dynamic fabrication and continuous characterization of molecular junctions. This review starts from the charge transport characterization of supramolecular junctions through a variety of noncovalent interactions, such as hydrogen bond, π-π interaction, and electrostatic force. We further review the recent progress in constructing highly conductive molecular junctions via chemical reactions, the response of molecular junctions to external stimuli, as well as the application of break junction techniques in controlling and monitoring chemical reactions in situ. We suggest that beyond the measurement of single molecular conductance, the single-molecule break junction techniques provide a promising access to study molecular assembly and chemical reactions at the single-molecule scale.

Electrochemical proton relay at the single-molecule level

DEFF Research Database (Denmark)

Kuznetsov, A. M.; Medvedev, I. G.; Ulstrup, Jens

2009-01-01

A scheme for the experimental study of single-proton transfer events, based on proton-coupled two-electron transfer between a proton donor and a proton acceptor molecule confined in the tunneling gap between two metal leads in electrolyte solution is suggested. Expressions for the electric current...... are derived and compared with formalism for electron tunneling through redox molecules. The scheme allows studying the kinetics of proton and hydrogen atom transfer as well as kinetic isotope effects at the single-molecule level under electrochemical potential control....

Coherent interaction of single molecules and plasmonic nanowires

Science.gov (United States)

Gerhardt, Ilja; Grotz, Bernhard; Siyushev, Petr; Wrachtrup, Jörg

2017-09-01

Quantum plasmonics opens the option to integrate complex quantum optical circuitry onto chip scale devices. In the past, often external light sources were used and nonclassical light was coupled in and out of plasmonic structures, such as hole arrays or waveguide structures. Another option to launch single plasmonic excitations is the coupling of single emitters in the direct proximity of, e.g., a silver or gold nanostructure. Here, we present our attempts to integrate the research of single emitters with wet-chemically grown silver nanowires. The emitters of choice are single organic dye molecules under cryogenic conditions, which are known to act as high-brightness and extremely narrow-band single photon sources. Another advantage is their high optical nonlinearity, such that they might mediate photon-photon interactions on the nanoscale. We report on the coupling of a single molecule fluorescence emission through the wire over the length of several wavelengths. The transmission of coherently emitted photons is proven by an extinction type experiment. As for influencing the spectral properties of a single emitter, we are able to show a remote change of the line-width of a single terrylene molecule, which is in close proximity to the nanowire.

FRET-mediated pH-responsive dual fluorescent nanoparticles prepared via click chemistry

Science.gov (United States)

Ouadahi, Karima; Sbargoud, Kamal; Allard, Emmanuel; Larpent, Chantal

2012-01-01

Herein, we report an easy preparation of azide-coated polystyrene-based nanoparticles (15 nm in diameter) and their surface functionalization via CuAAC with fluorophores in water. Resultant dual fluorescent nanoparticles coated with dansyl and pH-sensitive fluorescein moieties as the donor/acceptor FRET pair show a ratiometric response to pH upon excitation at a single wavelength.Herein, we report an easy preparation of azide-coated polystyrene-based nanoparticles (15 nm in diameter) and their surface functionalization via CuAAC with fluorophores in water. Resultant dual fluorescent nanoparticles coated with dansyl and pH-sensitive fluorescein moieties as the donor/acceptor FRET pair show a ratiometric response to pH upon excitation at a single wavelength. Electronic supplementary information (ESI) available: Experimental details and figures S1-S16 as mentioned in the text. See DOI: 10.1039/c2nr11413e

The spontaneous formation of single-molecule junctions via terminal alkynes

International Nuclear Information System (INIS)

Pla-Vilanova, Pepita; Aragonès, Albert C; Sanz, Fausto; Darwish, Nadim; Diez-Perez, Ismael; Ciampi, Simone

2015-01-01

Herein, we report the spontaneous formation of single-molecule junctions via terminal alkyne contact groups. Self-assembled monolayers that form spontaneously from diluted solutions of 1, 4-diethynylbenzene (DEB) were used to build single-molecule contacts and assessed using the scanning tunneling microscopy-break junction technique (STM-BJ). The STM-BJ technique in both its dynamic and static approaches was used to characterize the lifetime (stability) and the conductivity of a single-DEB wire. It is demonstrated that single-molecule junctions form spontaneously with terminal alkynes and require no electrochemical control or chemical deprotonation. The alkyne anchoring group was compared against typical contact groups exploited in single-molecule studies, i.e. amine (benzenediamine) and thiol (benzendithiol) contact groups. The alkyne contact showed a conductance magnitude comparable to that observed with amine and thiol groups. The lifetime of the junctions formed from alkynes were only slightly less than that of thiols and greater than that observed for amines. These findings are important as (a) they extend the repertoire of chemical contacts used in single-molecule measurements to 1-alkynes, which are synthetically accessible and stable and (b) alkynes have a remarkable affinity toward silicon surfaces, hence opening the door for the study of single-molecule transport on a semiconducting electronic platform. (fast track communication)

rFRET: A comprehensive, Matlab-based program for analyzing intensity-based ratiometric microscopic FRET experiments.

Science.gov (United States)

Nagy, Peter; Szabó, Ágnes; Váradi, Tímea; Kovács, Tamás; Batta, Gyula; Szöllősi, János

2016-04-01

Fluorescence or Förster resonance energy transfer (FRET) remains one of the most widely used methods for assessing protein clustering and conformation. Although it is a method with solid physical foundations, many applications of FRET fall short of providing quantitative results due to inappropriate calibration and controls. This shortcoming is especially valid for microscopy where currently available tools have limited or no capability at all to display parameter distributions or to perform gating. Since users of multiparameter flow cytometry usually apply these tools, the absence of these features in applications developed for microscopic FRET analysis is a significant limitation. Therefore, we developed a graphical user interface-controlled Matlab application for the evaluation of ratiometric, intensity-based microscopic FRET measurements. The program can calculate all the necessary overspill and spectroscopic correction factors and the FRET efficiency and it displays the results on histograms and dot plots. Gating on plots and mask images can be used to limit the calculation to certain parts of the image. It is an important feature of the program that the calculated parameters can be determined by regression methods, maximum likelihood estimation (MLE) and from summed intensities in addition to pixel-by-pixel evaluation. The confidence interval of calculated parameters can be estimated using parameter simulations if the approximate average number of detected photons is known. The program is not only user-friendly, but it provides rich output, it gives the user freedom to choose from different calculation modes and it gives insight into the reliability and distribution of the calculated parameters. © 2016 International Society for Advancement of Cytometry. © 2016 International Society for Advancement of Cytometry.

Central dogma at the single-molecule level in living cells.

Science.gov (United States)

Li, Gene-Wei; Xie, X Sunney

2011-07-20

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 the central dogma of molecular biology and the demonstration that a stochastic single-molecule event can determine the phenotype of a cell.

Advanced KSNP fuel, plus7 : grid-to-rod fretting wear resistance of the plus7 spacer grids

International Nuclear Information System (INIS)

Kim, Kyu Tae; Kim, Yong Hwan; Jang, Young Ki; Choi, Joon Hyung

2003-01-01

Vibration-induced grid-to-rod fretting wear initiates at a certain critical gap correlated with a critical work rate. A critical gap between grid and rod forms due to in-reactor performance of fuel, thermal relaxation of grid spring and irradiation growth of grid strap, etc. A critical work rate may be generated by three vibration mechanisms proposed in this paper. Three vibration mechanisms have been derived with various fretting wear experience in commercial reactors as well as various out-of-pile hydraulic test results. The first active vibration mechanism is high turbulence-induced excessive fuel rod vibration with the combination of excessive grid-to-rod gap. The second active vibration mechanism is self-excited fuel assembly vibration in a low frequency range caused by hydraulically unbalanced mixing vanes of the spacer grid assembly. The third active vibration mechanism is self-excited spacer grid strap vibration in quite a high frequency range caused by some spacer grid designs. In this study, each vibration mechanism on the grid-to-rod fretting wear damage is discussed. On the other hand, the effects of various grid designs on the fretting wear damage in the commercial reactors are predicted using the long-term fretting wear test results. It is found that the larger grid-to-rod initial contact area generates the less fretting wear damage. Consequently the conformal spring of PLUS7 is superior to typical convex shaped spring with regard to fretting wear resistance since the former generates relatively larger contact area than the latter

Torque Measurement at the Single Molecule Level

Science.gov (United States)

Forth, Scott; Sheinin, Maxim Y.; Inman, James; Wang, Michelle D.

2017-01-01

Methods for exerting and measuring forces on single molecules have revolutionized the study of the physics of biology. However, it is often the case that biological processes involve rotation or torque generation, and these parameters have been more difficult to access experimentally. Recent advances in the single molecule field have led to the development of techniques which add the capability of torque measurement. By combining force, displacement, torque, and rotational data, a more comprehensive description of the mechanics of a biomolecule can be achieved. In this review, we highlight a number of biological processes for which torque plays a key mechanical role. We describe the various techniques that have been developed to directly probe the torque experienced by a single molecule, and detail a variety of measurements made to date using these new technologies. We conclude by discussing a number of open questions and propose systems of study which would be well suited for analysis with torsional measurement techniques. PMID:23541162

Rotation of a single molecule within a supramolecular bearing

DEFF Research Database (Denmark)

Gimzewski, J.K.; Joachim, C.; Schlittler, R.R.

1998-01-01

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...

Molecular electronics with single molecules in solid-state devices.

Science.gov (United States)

Moth-Poulsen, Kasper; Bjørnholm, Thomas

2009-09-01

The ultimate aim of molecular electronics is to understand and master single-molecule devices. Based on the latest results on electron transport in single molecules in solid-state devices, we focus here on new insights into the influence of metal electrodes on the energy spectrum of the molecule, and on how the electron transport properties of the molecule depend on the strength of the electronic coupling between it and the electrodes. A variety of phenomena are observed depending on whether this coupling is weak, intermediate or strong.

Three-flavor color superconductivity

Energy Technology Data Exchange (ETDEWEB)

Malekzadeh, H.

2007-12-15

I investigate some of the inert phases in three-flavor, spin-zero color-superconducting quark matter: the CFL phase (the analogue of the B phase in superfluid {sup 3}He), the A and A{sup *} phases, and the 2SC and sSC phases. I compute the pressure of these phases with and without the neutrality condition. Without the neutrality condition, after the CFL phase the sSC phase is the dominant phase. However, including the neutrality condition, the CFL phase is again the energetically favored phase except for a small region of intermediate densities where the 2SC/A{sup *} phase is favored. It is shown that the 2SC phase is identical to the A{sup *} phase up to a color rotation. In addition, I calculate the self-energies and the spectral densities of longitudinal and transverse gluons at zero temperature in color-superconducting quark matter in the CFL phase. I find a collective excitation, a plasmon, at energies smaller than two times the gap parameter and momenta smaller than about eight times the gap. The dispersion relation of this mode exhibits a minimum at some nonzero value of momentum, indicating a van Hove singularity. (orig.)

Three-flavor color superconductivity

International Nuclear Information System (INIS)

Malekzadeh, H.

2007-12-01

I investigate some of the inert phases in three-flavor, spin-zero color-superconducting quark matter: the CFL phase (the analogue of the B phase in superfluid 3 He), the A and A * phases, and the 2SC and sSC phases. I compute the pressure of these phases with and without the neutrality condition. Without the neutrality condition, after the CFL phase the sSC phase is the dominant phase. However, including the neutrality condition, the CFL phase is again the energetically favored phase except for a small region of intermediate densities where the 2SC/A * phase is favored. It is shown that the 2SC phase is identical to the A * phase up to a color rotation. In addition, I calculate the self-energies and the spectral densities of longitudinal and transverse gluons at zero temperature in color-superconducting quark matter in the CFL phase. I find a collective excitation, a plasmon, at energies smaller than two times the gap parameter and momenta smaller than about eight times the gap. The dispersion relation of this mode exhibits a minimum at some nonzero value of momentum, indicating a van Hove singularity. (orig.)

Music-to-Color Associations of Single-Line Piano Melodies in Non-synesthetes.

Science.gov (United States)

Palmer, Stephen E; Langlois, Thomas A; Schloss, Karen B

2016-01-01

Prior research has shown that non-synesthetes' color associations to classical orchestral music are strongly mediated by emotion. The present study examines similar cross-modal music-to-color associations for much better controlled musical stimuli: 64 single-line piano melodies that were generated from four basic melodies by Mozart, whose global musical parameters were manipulated in tempo(slow/fast), note-density (sparse/dense), mode (major/minor) and pitch-height (low/high). Participants first chose the three colors (from 37) that they judged to be most consistent with (and, later, the three that were most inconsistent with) the music they were hearing. They later rated each melody and each color for the strength of its association along four emotional dimensions: happy/sad, agitated/calm, angry/not-angry and strong/weak. The cross-modal choices showed that faster music in the major mode was associated with lighter, more saturated, yellower (warmer) colors than slower music in the minor mode. These results replicate and extend those of Palmer et al. (2013, Proc. Natl Acad. Sci. 110, 8836-8841) with more precisely controlled musical stimuli. Further results replicated strong evidence for emotional mediation of these cross-modal associations, in that the emotional ratings of the melodies were very highly correlated with the emotional associations of the colors chosen as going best/worst with the melodies (r = 0.92, 0.85, 0.82 and 0.70 for happy/sad, strong/weak,angry/not-angry and agitated/calm, respectively). The results are discussed in terms of common emotional associations forming a cross-modal bridge between highly disparate sensory inputs.

Single-molecule imaging and manipulation of biomolecular machines and systems.

Science.gov (United States)

Iino, Ryota; Iida, Tatsuya; Nakamura, Akihiko; Saita, Ei-Ichiro; You, Huijuan; Sako, Yasushi

2018-02-01

Biological molecular machines support various activities and behaviors of cells, such as energy production, signal transduction, growth, differentiation, and migration. We provide an overview of single-molecule imaging methods involving both small and large probes used to monitor the dynamic motions of molecular machines in vitro (purified proteins) and in living cells, and single-molecule manipulation methods used to measure the forces, mechanical properties and responses of biomolecules. We also introduce several examples of single-molecule analysis, focusing primarily on motor proteins and signal transduction systems. Single-molecule analysis is a powerful approach to unveil the operational mechanisms both of individual molecular machines and of systems consisting of many molecular machines. Quantitative, high-resolution single-molecule analyses of biomolecular systems at the various hierarchies of life will help to answer our fundamental question: "What is life?" This article is part of a Special Issue entitled "Biophysical Exploration of Dynamical Ordering of Biomolecular Systems" edited by Dr. Koichi Kato. Copyright © 2017 Elsevier B.V. All rights reserved.

A plasmonic biosensor with single-molecule sensitivity

NARCIS (Netherlands)

Zijlstra, P.; Paulo, P.M.R.; Yuan, H.; Khatua, S.; Yorulmaz, M.; Orrit, M.

2013-01-01

The plasmon resonance of a single metal nanoparticle induces an enhancement of the local electromagnetic field. We exploit this field enhancement to detect single molecules that are (1) poorly fluorescent or (2) completely non-fluorescent.

Molecular electronics--resonant transport through single molecules.

Science.gov (United States)

Lörtscher, Emanuel; Riel, Heike

2010-01-01

The mechanically controllable break-junction technique (MCBJ) enables us to investigate charge transport through an individually contacted and addressed molecule in ultra-high vacuum (UHV) environment at variable temperature ranging from room temperature down to 4 K. Using a statistical measurement and analysis approach, we acquire current-voltage (I-V) characteristics during the repeated formation, manipulation, and breaking of a molecular junction. At low temperatures, voltages accessing the first molecular orbitals in resonance can be applied, providing spectroscopic information about the junction's energy landscape, in particular about the molecular level alignment in respect to the Fermi energy of the electrodes. Thereby, we can investigate the non-linear transport properties of various types of functional molecules and explore their potential use as functional building blocks for future nano-electronics. An example will be given by the reversible and controllable switching between two distinct conductive states of a single molecule. As a proof-of-principle for functional molecular devices, a single-molecule memory element will be demonstrated.

Vesicle Encapsulation Studies Reveal that Single Molecule Ribozyme Heterogeneities Are Intrinsic

Science.gov (United States)

Okumus, Burak; Wilson, Timothy J.; Lilley, David M. J.; Ha, Taekjip

2004-01-01

Single-molecule measurements have revealed that what were assumed to be identical molecules can differ significantly in their static and dynamic properties. One of the most striking examples is the hairpin ribozyme, which was shown to exhibit two to three orders of magnitude variation in folding kinetics between molecules. Although averaged behavior of single molecules matched the bulk solution data, it was not possible to exclude rigorously the possibility that the variations around the mean values arose from different ways of interacting with the surface environment. To test this, we minimized the molecules' interaction with the surface by encapsulating DNA or RNA molecules inside 100- to 200-nm diameter unilamellar vesicles, following the procedures described by Haran and coworkers. Vesicles were immobilized on a supported lipid bilayer via biotin-streptavidin linkages. We observed no direct binding of DNA or RNA on the supported bilayer even at concentrations exceeding 100 nM, indicating that these molecules do not bind stably on the membrane. Since the vesicle diameter is smaller than the resolution of optical microscopy, the lateral mobility of the molecules is severely constrained, allowing long observation periods. We used fluorescence correlation spectroscopy, nuclease digestion, and external buffer exchange to show that the molecules were indeed encapsulated within the vesicles. When contained within vesicles, the natural form of the hairpin ribozyme exhibited 50-fold variation in both folding and unfolding rates in 0.5 mM Mg2+, which is identical to what was observed from the molecules tethered directly on the surface. This strongly indicates that the observed heterogeneity in dynamic properties does not arise as an artifact of surface attachment, but is intrinsic to the nature of the molecules. PMID:15454471

Stereoelectronic Effect-Induced Conductance Switching in Aromatic Chain Single-Molecule Junctions.

Science.gov (United States)

Xin, Na; Wang, Jinying; Jia, Chuancheng; Liu, Zitong; Zhang, Xisha; Yu, Chenmin; Li, Mingliang; Wang, Shuopei; Gong, Yao; Sun, Hantao; Zhang, Guanxin; Liu, Zhirong; Zhang, Guangyu; Liao, Jianhui; Zhang, Deqing; Guo, Xuefeng

2017-02-08

Biphenyl, as the elementary unit of organic functional materials, has been widely used in electronic and optoelectronic devices. However, over decades little has been fundamentally understood regarding how the intramolecular conformation of biphenyl dynamically affects its transport properties at the single-molecule level. Here, we establish the stereoelectronic effect of biphenyl on its electrical conductance based on the platform of graphene-molecule single-molecule junctions, where a specifically designed hexaphenyl aromatic chain molecule is covalently sandwiched between nanogapped graphene point contacts to create stable single-molecule junctions. Both theoretical and temperature-dependent experimental results consistently demonstrate that phenyl twisting in the aromatic chain molecule produces different microstates with different degrees of conjugation, thus leading to stochastic switching between high- and low-conductance states. These investigations offer new molecular design insights into building functional single-molecule electrical devices.

A single molecule DNA flow stretching microscope for undergraduates

NARCIS (Netherlands)

Williams, Kelly; Grafe, Brendan; Burke, Kathryn M.; Tanner, Nathan; van Oijen, Antoine M.; Loparo, Joseph; Price, Allen C.

2011-01-01

The design of a simple, safe, and inexpensive single molecule flow stretching instrument is presented. The instrument uses a low cost upright microscope coupled to a webcam for imaging single DNA molecules that are tethered in an easy to construct microfluidic flow cell. The system requires no

Electrons, Photons, and Force: Quantitative Single-Molecule Measurements from Physics to Biology

Science.gov (United States)

2011-01-01

Single-molecule measurement techniques have illuminated unprecedented details of chemical behavior, including observations of the motion of a single molecule on a surface, and even the vibration of a single bond within a molecule. Such measurements are critical to our understanding of entities ranging from single atoms to the most complex protein assemblies. We provide an overview of the strikingly diverse classes of measurements that can be used to quantify single-molecule properties, including those of single macromolecules and single molecular assemblies, and discuss the quantitative insights they provide. Examples are drawn from across the single-molecule literature, ranging from ultrahigh vacuum scanning tunneling microscopy studies of adsorbate diffusion on surfaces to fluorescence studies of protein conformational changes in solution. PMID:21338175

Biological Nanopores: Confined Spaces for Electrochemical Single-Molecule Analysis.

Science.gov (United States)

Cao, Chan; Long, Yi-Tao

2018-02-20

Nanopore sensing is developing into a powerful single-molecule approach to investigate the features of biomolecules that are not accessible by studying ensemble systems. When a target molecule is transported through a nanopore, the ions occupying the pore are excluded, resulting in an electrical signal from the intermittent ionic blockade event. By statistical analysis of the amplitudes, duration, frequencies, and shapes of the blockade events, many properties of the target molecule can be obtained in real time at the single-molecule level, including its size, conformation, structure, charge, geometry, and interactions with other molecules. With the development of the use of α-hemolysin to characterize individual polynucleotides, nanopore technology has attracted a wide range of research interest in the fields of biology, physics, chemistry, and nanoscience. As a powerful single-molecule analytical method, nanopore technology has been applied for the detection of various biomolecules, including oligonucleotides, peptides, oligosaccharides, organic molecules, and disease-related proteins. In this Account, we highlight recent developments of biological nanopores in DNA-based sensing and in studying the conformational structures of DNA and RNA. Furthermore, we introduce the application of biological nanopores to investigate the conformations of peptides affected by charge, length, and dipole moment and to study disease-related proteins' structures and aggregation transitions influenced by an inhibitor, a promoter, or an applied voltage. To improve the sensing ability of biological nanopores and further extend their application to a wider range of molecular sensing, we focus on exploring novel biological nanopores, such as aerolysin and Stable Protein 1. Aerolysin exhibits an especially high sensitivity for the detection of single oligonucleotides both in current separation and duration. Finally, to facilitate the use of nanopore measurements and statistical analysis

Measurement of the conductance properties of single organic molecules using gold nanoparticles

Science.gov (United States)

Gordin, Yoav

conduct more than an order of magnitude less than those that are fully conjugated. A distinct feature of the conjugated molecule is the appearance of pronounced peaks in its conductance at certain voltage values. We have shown that these peaks can be gated randomly by the electrostatic environment, but the peak spectrum is reproducible among the different samples of the same molecular species that we studied. To properly study and understand the peak structure we developed the ability to add gate dependent measurements to our system. Unfortunately the backdrop of this was a drastic reduction in the yield of good samples for measurement. We focused on four different conjugated molecules to attempt to understand the effect of the molecular structure on the properties of the peak spectra. We have been able to measure three of these molecules, and obtained SET diamond plots reminiscent of those seen for the single particles. The molecular diamonds have a larger energy gap than that found in single particles, as can be expected from their smaller size. We do not yet have enough data on this issue to make any definite statements on the influence of the molecular structure on the peak structure. Another topic investigated in this work is the physics of the two gold nanoparticles, giving rise to double quantum dot (DQD) phenomena. This physics is observed in dimers that do not exhibit "molecular" (high energy) features, or at low voltages before the appearance of the molecular peaks. We have used these phenomena to fully characterize the properties of our system and understand better the role the molecule plays in transport at low bias (below the voltage of the first peak). I begin this thesis with an introduction to the field of molecular electronics; I briefly review the theoretical approaches and the experimental methods used. I then describe in detail the dimer method, whose development took up a major part of this work, relaying in detail the relevant issues and

Two-Color Single-Photon Photoinitiation and Photoinhibition for Subdiffraction Photolithography

Science.gov (United States)

Scott, Timothy F.; Kowalski, Benjamin A.; Sullivan, Amy C.; Bowman, Christopher N.; McLeod, Robert R.

2009-05-01

Controlling and reducing the developed region initiated by photoexposure is one of the fundamental goals of optical lithography. Here, we demonstrate a two-color irradiation scheme whereby initiating species are generated by single-photon absorption at one wavelength while inhibiting species are generated by single-photon absorption at a second, independent wavelength. Co-irradiation at the second wavelength thus reduces the polymerization rate, delaying gelation of the material and facilitating enhanced spatial control over the polymerization. Appropriate overlapping of the two beams produces structures with both feature sizes and monomer conversions otherwise unobtainable with use of single- or two-photon absorption photopolymerization. Additionally, the generated inhibiting species rapidly recombine when irradiation with the second wavelength ceases, allowing for fast sequential exposures not limited by memory effects in the material and thus enabling fabrication of complex two- or three-dimensional structures.

Probing protein-lipid interactions by FRET between membrane fluorophores

Science.gov (United States)

Trusova, Valeriya M.; Gorbenko, Galyna P.; Deligeorgiev, Todor; Gadjev, Nikolai

2016-09-01

Förster resonance energy transfer (FRET) is a powerful fluorescence technique that has found numerous applications in medicine and biology. One area where FRET proved to be especially informative involves the intermolecular interactions in biological membranes. The present study was focused on developing and verifying a Monte-Carlo approach to analyzing the results of FRET between the membrane-bound fluorophores. This approach was employed to quantify FRET from benzanthrone dye ABM to squaraine dye SQ-1 in the model protein-lipid system containing a polycationic globular protein lysozyme and negatively charged lipid vesicles composed of phosphatidylcholine and phosphatidylglycerol. It was found that acceptor redistribution between the lipid bilayer and protein binding sites resulted in the decrease of FRET efficiency. Quantification of this effect in terms of the proposed methodology yielded both structural and binding parameters of lysozyme-lipid complexes.

Nuclear Fuel Fretting Mechanisms in a Room Temperature Unlubricated Condition

Energy Technology Data Exchange (ETDEWEB)

Lee, Young Ho; Kim, Hyung Kyu [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

2008-10-15

Recently, efforts for evaluating the fretting wear mechanism have been carried out by many researchers in various conditions. In an unlubricated condition, especially, effects of a wear debris and/or its layer on the fretting wear behavior were proposed that the formation of a well-developed glaze layer has a beneficial effect for decreasing a friction coefficient. Otherwise, a wear rate was accelerated by a third-body abrasion. At this time, it is well known that wear debris behaviors are affected by test variables such as a temperature, environment, material characteristics, etc. In a nuclear fuel fretting, however, its contact condition is quite different when compared with general fretting wear studies and could be summarized as the following; first, a fuel rod is supported by spacer grid springs and dimples that were elastically deformable. This results in a unique friction loop and a different fretting mechanism when a fuel rod is vibrated due to a flow-induced vibration (FIV). Next, it is possible that some region of the wear scar area with a specific spring shape condition could be hidden due to different wear debris behavior. So, some of the wear debris layers could be found on the worn surfaces in previous studies even though fretting wear tests were performed in a water lubricated condition. Finally, initial contact condition could be changed both an actual operating condition in power plants (i.e. high temperature and pressurized water (HTHP) under severe irradiation conditions) and the fretting wear tests for evaluating the wear resistant spring in lab conditions (i.e. from room temperature to HTHP without irradiation conditions) due to material degradations and the formation of the wear scar, respectively. In summary, the spring shape effect and the variation of the contact condition with increasing fretting cycle should be evaluated in order to improve the wear resistance of the spacer grid spring. So, in this study, fretting wear tests have been

Synthesis of single-molecule nanocars.

Science.gov (United States)

Vives, Guillaume; Tour, James M

2009-03-17

The drive to miniaturize devices has led to a variety of molecular machines inspired by macroscopic counterparts such as molecular motors, switches, shuttles, turnstiles, barrows, elevators, and nanovehicles. Such nanomachines are designed for controlled mechanical motion and the transport of nanocargo. As researchers miniaturize devices, they can consider two complementary approaches: (1) the "top-down" approach, which reduces the size of macroscopic objects to reach an equivalent microscopic entity using photolithography and related techniques and (2) the "bottom-up" approach, which builds functional microscopic or nanoscopic entities from molecular building blocks. The top-down approach, extensively used by the semiconductor industry, is nearing its scaling limits. On the other hand, the bottom-up approach takes advantage of the self-assembly of smaller molecules into larger networks by exploiting typically weak molecular interactions. But self-assembly alone will not permit complex assembly. Using nanomachines, we hope to eventually consider complex, enzyme-like directed assembly. With that ultimate goal, we are currently exploring the control of nanomachines that would provide a basis for the future bottom-up construction of complex systems. This Account describes the synthesis of a class of molecular machines that resemble macroscopic vehicles. We designed these so-called nanocars for study at the single-molecule level by scanning probe microscopy (SPM). The vehicles have a chassis connected to wheel-terminated axles and convert energy inputs such as heat, electric fields, or light into controlled motion on a surface, ultimately leading to transport of nanocargo. At first, we used C(60) fullerenes as wheels, which allowed the demonstration of a directional rolling mechanism of a nanocar on a gold surface by STM. However, because of the low solubility of the fullerene nanocars and the incompatibility of fullerenes with photochemical processes, we developed new

Automated imaging system for single molecules

Science.gov (United States)

Schwartz, David Charles; Runnheim, Rodney; Forrest, Daniel

2012-09-18

There is provided a high throughput automated single molecule image collection and processing system that requires minimal initial user input. The unique features embodied in the present disclosure allow automated collection and initial processing of optical images of single molecules and their assemblies. Correct focus may be automatically maintained while images are collected. Uneven illumination in fluorescence microscopy is accounted for, and an overall robust imaging operation is provided yielding individual images prepared for further processing in external systems. Embodiments described herein are useful in studies of any macromolecules such as DNA, RNA, peptides and proteins. The automated image collection and processing system and method of same may be implemented and deployed over a computer network, and may be ergonomically optimized to facilitate user interaction.

Making ultracold molecules in a two color pump-dump photoassociation scheme using chirped pulses

OpenAIRE

Koch, Christiane P.; Luc-Koenig, Eliane; Masnou-Seeuws, Françoise

2005-01-01

This theoretical paper investigates the formation of ground state molecules from ultracold cesium atoms in a two-color scheme. Following previous work on photoassociation with chirped picosecond pulses [Luc-Koenig et al., Phys. Rev. A {\\bf 70}, 033414 (2004)], we investigate stabilization by a second (dump) pulse. By appropriately choosing the dump pulse parameters and time delay with respect to the photoassociation pulse, we show that a large number of deeply bound molecules are created in t...

Single-molecule force-conductance spectroscopy of hydrogen-bonded complexes

DEFF Research Database (Denmark)

Pirrotta, Alessandro; De Vico, Luca; Solomon, Gemma C.

2017-01-01

to inform about molecular recognition events at the single-molecule limit. For this, we consider the force-conductance characteristics of a prototypical class of hydrogen bonded bimolecular complexes sandwiched between gold electrodes. The complexes consist of derivatives of a barbituric acid and a Hamilton...... is mechanically manipulated. The implication is that force and conductance provide complementary information about the evolution of molecules in junctions that can be used to interrogate basic structure-transport relations at the single-molecule limit....

Fast temporal fluctuations in single-molecule junctions.

Science.gov (United States)

Ochs, Roif; Secker, Daniel; Elbing, Mark; Mayor, Marcel; Weber, Heiko B

2006-01-01

The noise within the electrical current through single-molecule junctions is studied cryogenic temperature. The organic sample molecules were contacted with the mechanically controlled break-junction technique. The noise spectra refer to a where only few Lorentzian fluctuators occur in the conductance. The frequency dependence shows qualitative variations from sample to sample.

Double ionization of nitrogen molecules in orthogonal two-color femtosecond laser fields

Science.gov (United States)

Song, Qiying; Li, Hui; Wang, Junping; Lu, Peifen; Gong, Xiaochun; Ji, Qinying; Lin, Kang; Zhang, Wenbin; Ma, Junyang; Li, Hanxiao; Zeng, Heping; He, Feng; Wu, Jian

2018-04-01

Double ionization of nitrogen molecules in orthogonally polarized two-color femtosecond laser fields is investigated by varying the relative intensity between the fundamental wave (FW) and its second harmonic (SH) components. The yield ratios of the double ionization channels, i.e., the non-dissociative {{{{N}}}2}2+ and Coulomb exploded (N+, N+), to the singly charged N2 + channel exhibit distinct dependences on the relative strength between the FW and SH fields. As the intensity ratio of SH to FW increases, the yield ratio of (N+, N+)/N2 + gradually increases, while the ratio of {{{{N}}}2}2+/N2 + first descends and then increases constituting a valley shape which is similar to the behavior of Ar2+/Ar+ observed in the same experimental condition. Based on the classical trajectory simulations, we found that the different characteristics of the two doubly ionized channels stem from two mechanisms, i.e., the {{{{N}}}2}2+ is mostly accessed by the (e, 2e) impact ionization while the recollision-induced excitation with subsequent ionization plays an important role in producing the (N+, N+) channel.

Efficient FRET-based fuorescent ratiometric chemosensors for Fe3+ and its application in living cells

International Nuclear Information System (INIS)

Wang, Cuicui; Liu, Yaqi; Cheng, Junye; Song, Jianhua; Zhao, Yufen; Ye, Yong

2015-01-01

A series of novel FRET-based fluorescent ratiometric chemosensors (L 1 –L 6 ) were designed and synthesized. Sensor L 2 showed reversible and the best selective recognition toward Fe 3+ over other metal ions with a detection limit of 0.418 ppm, which can meet the selective requirements for practical application. Experiment results showed that the response behavior of L 2 toward Fe 3+ is pH independent in weak acid condition (pH 4.0–6.0). In addition, sensor L 2 was successfully applied for ratiometric visualization of Fe 3+ in living cells. - Highlights: • The detection limit of a new FRET probe for Fe 3+ was 0.418 ppm. • The probe exhibited high selectivity and sensitivity detection to Fe 3+ with a pH span of 4.0–6.0. • The significant changes in color could be used for naked-eye detection • The fluorescence imaging experiment demonstrated its value of practical application

Intercalating dye as an acceptor in quantum-dot-mediated FRET

International Nuclear Information System (INIS)

Lim, Teck Chuan; Bailey, Vasudev J; Wang, T-H; Ho, Y-P

2008-01-01

Fluorescence resonance energy transfer (FRET) is a popular tool to study intermolecular distances and characterize structural or conformational changes of biological macromolecules. We investigate a novel inorganic/organic FRET pair with quantum dots (QDs) as donors and DNA intercalating dyes, BOBO-3, as acceptors by using DNA as a linker. Typically, FRET efficiency increases with the number of stained DNA linked to a QD. However, with the use of intercalating dyes, we demonstrate that FRET efficiency at a fixed DNA:QD ratio can be further enhanced by increasing the number of dyes stained to a DNA strand through the use of an increased staining dye/bp ratio. We exploit this flexibility in the staining ratio to maintain a high FRET efficiency of >0.90 despite a sixfold decrease in DNA concentration. Having characterized this new QD-mediated FRET system, we test this system in a cellular environment using nanocomplexes generated by encapsulating DNA with commercial non-viral gene carriers. Using this novel FRET pair, we are able to monitor the configuration changes and fate of the DNA nanocomplexes during intracellular delivery, thereby providing an insight into the mechanistic study of gene delivery

The Leakage determination on corrosion fretting machine

International Nuclear Information System (INIS)

Sriyono; Satmoko, Ari; Hafid, Abdul; Febrianto; Prasetio, Joko; Abtokhi; Sumarno, Edy; Handoyo, Ismu; Hidayati, Nur Rahmah; Histori

1998-01-01

Fretting machine is an experimental loop to learn fretting corrosion phenomena wich is caused by loading and vibration. On the steam generator, one of the corrosion process that's occurred, it can be caused by vibration between tubes and bending material. Because of high flow rate inside the tube, the high frequency vibration will appeared so it can make the corrosion on bending material more faster. This process can be simulate by fretting machine. This machine has already damage because of leakage. So it will be repaired by dismantling, radiography testing and redrawing. from the result of radiography, the leakage is caused by cracking on bellows seals of the dynamic main support

Single-molecule electronics: Cooling individual vibrational modes by the tunneling current.

Science.gov (United States)

Lykkebo, Jacob; Romano, Giuseppe; Gagliardi, Alessio; Pecchia, Alessandro; Solomon, Gemma C

2016-03-21

Electronic devices composed of single molecules constitute the ultimate limit in the continued downscaling of electronic components. A key challenge for single-molecule electronics is to control the temperature of these junctions. Controlling heating and cooling effects in individual vibrational modes can, in principle, be utilized to increase stability of single-molecule junctions under bias, to pump energy into particular vibrational modes to perform current-induced reactions, or to increase the resolution in inelastic electron tunneling spectroscopy by controlling the life-times of phonons in a molecule by suppressing absorption and external dissipation processes. Under bias the current and the molecule exchange energy, which typically results in heating of the molecule. However, the opposite process is also possible, where energy is extracted from the molecule by the tunneling current. Designing a molecular "heat sink" where a particular vibrational mode funnels heat out of the molecule and into the leads would be very desirable. It is even possible to imagine how the vibrational energy of the other vibrational modes could be funneled into the "cooling mode," given the right molecular design. Previous efforts to understand heating and cooling mechanisms in single molecule junctions have primarily been concerned with small models, where it is unclear which molecular systems they correspond to. In this paper, our focus is on suppressing heating and obtaining current-induced cooling in certain vibrational modes. Strategies for cooling vibrational modes in single-molecule junctions are presented, together with atomistic calculations based on those strategies. Cooling and reduced heating are observed for two different cooling schemes in calculations of atomistic single-molecule junctions.

Multiplexed single-molecule force spectroscopy using a centrifuge.

Science.gov (United States)

Yang, Darren; Ward, Andrew; Halvorsen, Ken; Wong, Wesley P

2016-03-17

We present a miniature centrifuge force microscope (CFM) that repurposes a benchtop centrifuge for high-throughput single-molecule experiments with high-resolution particle tracking, a large force range, temperature control and simple push-button operation. Incorporating DNA nanoswitches to enable repeated interrogation by force of single molecular pairs, we demonstrate increased throughput, reliability and the ability to characterize population heterogeneity. We perform spatiotemporally multiplexed experiments to collect 1,863 bond rupture statistics from 538 traceable molecular pairs in a single experiment, and show that 2 populations of DNA zippers can be distinguished using per-molecule statistics to reduce noise.

Detection of protease activity by fluorescent protein FRET sensors: from computer simulation to live cells

Science.gov (United States)

Goryashchenko, Alexander S.; Khrenova, Maria G.; Savitsky, Alexander P.

2018-04-01

Förster resonance energy transfer (FRET) sensors are widely used for the detection of protease activity in vitro and in vivo. Usually they consist of a FRET pair connected with a polypeptide linker containing a specific cleavage site for the relevant protease. Use of the fluorescent proteins as components of the FRET pair allows genetic encoding of such sensors and solves the problem of their delivery into live cells and animals. There are several ways to improve the properties of such sensors, mainly to increase FRET efficiency and therefore the dynamic range. One of the ways to achieve this is to use a non-fluorescent chromoprotein as an acceptor. Molecular dynamic simulations may assist in the construction of linker structures connecting donor and acceptor molecules. Estimation of the orientation factor κ 2 can be obtained by methods based on quantum theory and combined quantum mechanics/molecular mechanics approaches. The linker can be structured by hydrophobic interactions, bringing it into a closed conformation that shortens the distance between donor and acceptor and, consequently, increases FRET efficiency. We analyzed the effects of different linker structures on the detection of caspase-3 activity using a non-fluorescent acceptor. Also we have constructed the Tb3+- TagRFP sensor in which a complex of the terbium ion and terbium-binding peptide is used as a donor. This allowed us to use the unique property of lanthanide ions—fluorescence lifetime up to milliseconds—to perform measurements with time delay and exclude the nanosecond-order fluorescence. Using our systems as a starting point, by changing the recognition site in the linker it is possible to perform imaging of different protease activity in vitro or in vivo.

Toward The Reconstitution of the Maturation of Okazaki Fragments Multiprotein Complex in Human At The Single Molecule Level

KAUST Repository

Joudeh, Luay

2017-04-01

The maturation of Okazaki fragments on the lagging strand in eukaryotes is mediated by a highly coordinated multistep process involving several proteins that ensure the accurate and efficient replication of genomic DNA. Human proliferating cell nuclear antigen (PCNA) that slides on double-stranded DNA is the key player that coordinates the access of various proteins to the different intermediary steps in this process. In this study, I am focusing on characterizing how PCNA recruits and stimulates the structure specific flap endonuclease 1 (FEN1) to process the aberrant double flap (DF) structures that are produced during maturation of Okazaki fragments. FEN1 distorts the DF structures into a bent conformer to place the scissile phosphate into the active site for cleavage. The product is a nick substrate that can be sealed by DNA ligase I whose recruitment is also mediated by its interaction with PCNA. Using single-molecule Förster resonance energy transfer (smFRET) measurements that simultaneously monitored bending and cleavage of various DF substrates by FEN1 alone or in the presence of PCNA, we found that FEN1 and PCNA bends cognate and non-cognate substrates but display remarkable selectivity to stabilize the bent conformer in cognate substrate while promoting the dissociation of non-cognate substrates. This mechanism provides efficiency and accuracy for FEN1 and PCNA to cleave the correct substrate while avoiding the deleterious cleavage of incorrect substrates. This work provides a true molecular level understanding of the key step during the maturation of Okazaki fragment and contributes towards the reconstitution of its entire activity at the single molecule level.

Isolated single-molecule magnets on native gold.

Science.gov (United States)

Zobbi, Laura; Mannini, Matteo; Pacchioni, Mirko; Chastanet, Guillaume; Bonacchi, Daniele; Zanardi, Chiara; Biagi, Roberto; Del Pennino, Umberto; Gatteschi, Dante; Cornia, Andrea; Sessoli, Roberta

2005-03-28

The incorporation of thioether groups in the structure of a Mn12 single-molecule magnet, [Mn12(O12)(L)16(H2O)4] with L = 4-(methylthio)benzoate, is a successful route to the deposition of well-separated clusters on native gold surfaces and to the addressing of individual molecules by scanning tunnelling microscopy.

Atomic-Scale Control of Electron Transport through Single Molecules

DEFF Research Database (Denmark)

Wang, Y. F.; Kroger, J.; Berndt, R.

2010-01-01

Tin-phthalocyanine molecules adsorbed on Ag(111) were contacted with the tip of a cryogenic scanning tunneling microscope. Orders-of-magnitude variations of the single-molecule junction conductance were achieved by controllably dehydrogenating the molecule and by modifying the atomic structure...

Silicon photon-counting avalanche diodes for single-molecule fluorescence spectroscopy

Science.gov (United States)

Michalet, Xavier; Ingargiola, Antonino; Colyer, Ryan A.; Scalia, Giuseppe; Weiss, Shimon; Maccagnani, Piera; Gulinatti, Angelo; Rech, Ivan; Ghioni, Massimo

2014-01-01

Solution-based single-molecule fluorescence spectroscopy is a powerful experimental tool with applications in cell biology, biochemistry and biophysics. The basic feature of this technique is to excite and collect light from a very small volume and work in a low concentration regime resulting in rare burst-like events corresponding to the transit of a single molecule. Detecting photon bursts is a challenging task: the small number of emitted photons in each burst calls for high detector sensitivity. Bursts are very brief, requiring detectors with fast response time and capable of sustaining high count rates. Finally, many bursts need to be accumulated to achieve proper statistical accuracy, resulting in long measurement time unless parallelization strategies are implemented to speed up data acquisition. In this paper we will show that silicon single-photon avalanche diodes (SPADs) best meet the needs of single-molecule detection. We will review the key SPAD parameters and highlight the issues to be addressed in their design, fabrication and operation. After surveying the state-of-the-art SPAD technologies, we will describe our recent progress towards increasing the throughput of single-molecule fluorescence spectroscopy in solution using parallel arrays of SPADs. The potential of this approach is illustrated with single-molecule Förster resonance energy transfer measurements. PMID:25309114

Single-molecule tracking of small GTPase Rac1 uncovers spatial regulation of membrane translocation and mechanism for polarized signaling

Science.gov (United States)

Das, Sulagna; Yin, Taofei; Yang, Qingfen; Zhang, Jingqiao; Wu, Yi I.; Yu, Ji

2015-01-01

Polarized Rac1 signaling is a hallmark of many cellular functions, including cell adhesion, motility, and cell division. The two steps of Rac1 activation are its translocation to the plasma membrane and the exchange of nucleotide from GDP to GTP. It is, however, unclear whether these two processes are regulated independent of each other and what their respective roles are in polarization of Rac1 signaling. We designed a single-particle tracking (SPT) method to quantitatively analyze the kinetics of Rac1 membrane translocation in living cells. We found that the rate of Rac1 translocation was significantly elevated in protrusions during cell spreading on collagen. Furthermore, combining FRET sensor imaging with SPT measurements in the same cell, the recruitment of Rac1 was found to be polarized to an extent similar to that of the nucleotide exchange process. Statistical analysis of single-molecule trajectories and optogenetic manipulation of membrane lipids revealed that Rac1 membrane translocation precedes nucleotide exchange, and is governed primarily by interactions with phospholipids, particularly PI(3,4,5)P3, instead of protein factors. Overall, the study highlights the significance of membrane translocation in spatial Rac1 signaling, which is in addition to the traditional view focusing primarily on GEF distribution and exchange reaction. PMID:25561548

Single-Molecule Analysis for RISC Assembly and Target Cleavage.

Science.gov (United States)

Sasaki, Hiroshi M; Tadakuma, Hisashi; Tomari, Yukihide

2018-01-01

RNA-induced silencing complex (RISC) is a small RNA-protein complex that mediates silencing of complementary target RNAs. Biochemistry has been successfully used to characterize the molecular mechanism of RISC assembly and function for nearly two decades. However, further dissection of intermediate states during the reactions has been warranted to fill in the gaps in our understanding of RNA silencing mechanisms. Single-molecule analysis with total internal reflection fluorescence (TIRF) microscopy is a powerful imaging-based approach to interrogate complex formation and dynamics at the individual molecule level with high sensitivity. Combining this technique with our recently established in vitro reconstitution system of fly Ago2-RISC, we have developed a single-molecule observation system for RISC assembly. In this chapter, we summarize the detailed protocol for single-molecule analysis of chaperone-assisted assembly of fly Ago2-RISC as well as its target cleavage reaction.

Evaluation of the Kinetic Property of Single-Molecule Junctions by Tunneling Current Measurements.

Science.gov (United States)

Harashima, Takanori; Hasegawa, Yusuke; Kiguchi, Manabu; Nishino, Tomoaki

2018-01-01

We investigated the formation and breaking of single-molecule junctions of two kinds of dithiol molecules by time-resolved tunneling current measurements in a metal nanogap. The resulting current trajectory was statistically analyzed to determine the single-molecule conductance and, more importantly, to reveal the kinetic property of the single-molecular junction. These results suggested that combining a measurement of the single-molecule conductance and statistical analysis is a promising method to uncover the kinetic properties of the single-molecule junction.

Terbium to Quantum Dot FRET Bioconjugates for Clinical Diagnostics: Influence of Human Plasma on Optical and Assembly Properties

Directory of Open Access Journals (Sweden)

Niko Hildebrandt

2011-10-01

Full Text Available Förster resonance energy transfer (FRET from luminescent terbium complexes (LTC as donors to semiconductor quantum dots (QDs as acceptors allows extraordinary large FRET efficiencies due to the long Förster distances afforded. Moreover, time-gated detection permits an efficient suppression of autofluorescent background leading to sub-picomolar detection limits even within multiplexed detection formats. These characteristics make FRET-systems with LTC and QDs excellent candidates for clinical diagnostics. So far, such proofs of principle for highly sensitive multiplexed biosensing have only been performed under optimized buffer conditions and interactions between real-life clinical media such as human serum or plasma and LTC-QD-FRET-systems have not yet been taken into account. Here we present an extensive spectroscopic analysis of absorption, excitation and emission spectra along with the luminescence decay times of both the single components as well as the assembled FRET-systems in TRIS-buffer, TRIS-buffer with 2% bovine serum albumin, and fresh human plasma. Moreover, we evaluated homogeneous LTC-QD FRET assays in QD conjugates assembled with either the well-known, specific biotin-streptavidin biological interaction or, alternatively, the metal-affinity coordination of histidine to zinc. In the case of conjugates assembled with biotin-streptavidin no significant interference with the optical and binding properties occurs whereas the histidine-zinc system appears to be affected by human plasma.

Single molecules and single nanoparticles as windows to the nanoscale

Science.gov (United States)

Caldarola, Martín; Orrit, Michel

2018-05-01

Since the first optical detection of single molecules, they have been used as nanometersized optical sensors to explore the physical properties of materials and light-matter interaction at the nanoscale. Understanding nanoscale properties of materials is fundamental for the development of new technology that requires precise control of atoms and molecules when the quantum nature of matter cannot be ignored. In the following lines, we illustrate this journey into nanoscience with some experiments from our group.

Alternative types of molecule-decorated atomic chains in Au–CO–Au single-molecule junctions

Directory of Open Access Journals (Sweden)

Zoltán Balogh

2015-06-01

Full Text Available We investigate the formation and evolution of Au–CO single-molecule break junctions. The conductance histogram exhibits two distinct molecular configurations, which are further investigated by a combined statistical analysis. According to conditional histogram and correlation analysis these molecular configurations show strong anticorrelations with each other and with pure Au monoatomic junctions and atomic chains. We identify molecular precursor configurations with somewhat higher conductance, which are formed prior to single-molecule junctions. According to detailed length analysis two distinct types of molecule-affected chain-formation processes are observed, and we compare these results to former theoretical calculations considering bridge- and atop-type molecular configurations where the latter has reduced conductance due to destructive Fano interference.

Protecting AREVA ATRIUM™ BWR fuel from debris fretting failure

International Nuclear Information System (INIS)

Cole, Steven E.; Garner, Norman L.; Lippert, Hans-Joachim; Graebert, Rüdiger; Mollard, Pierre; Hahn, Gregory C.

2014-01-01

Historically, debris fretting has been the leading cause of fuel rod failure in BWR fuel assemblies, costing the industry millions of dollars in lost generation and negatively impacting the working area of plant site personnel. In this paper the focus will be on recent BWR fuel product innovation designed to eliminate debris related failures. Experience feedback from more than three decades of operation history with non-line-of-sight FUELGUARD™ lower tie plate debris filters will be presented. The development and relative effectiveness of successive generations of filtration technology will be discussed. It will be shown that modern, state of the art debris filters are an effective defense against debris fretting failure. Protective measures extend beyond inlet nozzle debris filters. The comprehensive debris resistance features built into AREVA’s newest fuel design, the ATRIUM™ 11, reduce the overall risk of debris entrapment as well as providing a degree of protection from debris that may fall down on the fuel assembly from above, e.g., during refueling operations. The positive recent experience in a debris sensitive plant will be discussed showing that the combination of advanced fuel technology and a robust foreign material exclusion program at the reactor site can eliminate the debris fretting failure mechanism. (author)

A Color-Opponency Based Biological Model for Color Constancy

Directory of Open Access Journals (Sweden)

Yongjie Li

2011-05-01

Full Text Available Color constancy is the ability of the human visual system to adaptively correct color-biased scenes under different illuminants. Most of the existing color constancy models are nonphysiologically plausible. Among the limited biological models, the great majority is Retinex and its variations, and only two or three models directly simulate the feature of color-opponency, but only of the very earliest stages of visual pathway, i.e., the single-opponent mechanisms involved at the levels of retinal ganglion cells and lateral geniculate nucleus (LGN neurons. Considering the extensive physiological evidences supporting that both the single-opponent cells in retina and LGN and the double-opponent neurons in primary visual cortex (V1 are the building blocks for color constancy, in this study we construct a color-opponency based color constancy model by simulating the opponent fashions of both the single-opponent and double-opponent cells in a forward manner. As for the spatial structure of the receptive fields (RF, both the classical RF (CRF center and the nonclassical RF (nCRF surround are taken into account for all the cells. The proposed model was tested on several typical image databases commonly used for performance evaluation of color constancy methods, and exciting results were achieved.

Single-molecule analysis of DNA replication in Xenopus egg extracts

NARCIS (Netherlands)

Yardimci, Hasan; Loveland, Anna B.; van Oijen, Antoine M.; Walter, Johannes C.; Mechali, Marcel

The recent advent in single-molecule imaging and manipulation methods has made a significant impact on the understanding of molecular mechanisms underlying many essential cellular processes. Single-molecule techniques such as electron microscopy and DNA fiber assays have been employed to study the

Single Molecule Spectroscopy on Photosynthetic Pigment-Protein Complexes

CERN Document Server

Jelezko, F; Schuler, S; Thews, E; Tietz, C; Wechsler, A; Wrachtrup, J

2001-01-01

Single molecule spectroscopy was applied to unravel the energy transfer pathway in photosynthetic pigment-protein complexes. Detailed analysis of excitation and fluorescence emission spectra has been made for peripheral plant antenna LHC II and Photosystem I from cyanobacterium Synechococcus elongatus. Optical transitions of individual pigments were resolved under nonselective excitation of antenna chlorophylls. High-resolution fluorescence spectroscopy of individual plant antenna LHC II indicates that at low temperatures, the excitation energy is localized on the red-most Chl a pool absorbing at 680 nm. More than one pigment molecule is responsible for the fluorescence emission of the LHC II trimer. The spectral lines of single Chl a molecules absorbing at 675 nm are broadened because of the Foerster energy transfer towards the red-most pigments. Low-temperature spectroscopy on single PS I trimers indicates that two subgroups of pigments, which are present in the red antenna pool, differ by the strength of t...

Single-Molecule Interfacial Electron Transfer

Energy Technology Data Exchange (ETDEWEB)

Ho, Wilson [Univ. of California, Irvine, CA (United States)

2018-02-03

Interfacial electron transfer (ET) plays an important role in many chemical and biological processes. Specifically, interfacial ET in TiO₂-based systems is important to solar energy technology, catalysis, and environmental remediation technology. However, the microscopic mechanism of interfacial ET is not well understood with regard to atomic surface structure, molecular structure, bonding, orientation, and motion. In this project, we used two complementary methodologies; single-molecule fluorescence spectroscopy, and scanning-tunneling microscopy and spectroscopy (STM and STS) to address this scientific need. The goal of this project was to integrate these techniques and measure the molecular dependence of ET between adsorbed molecules and TiO₂ semiconductor surfaces and the ET induced reactions such as the splitting of water. The scanning probe techniques, STM and STS, are capable of providing the highest spatial resolution but not easily time-resolved data. Single-molecule fluorescence spectroscopy is capable of good time resolution but requires further development to match the spatial resolution of the STM. The integrated approach involving Peter Lu at Bowling Green State University (BGSU) and Wilson Ho at the University of California, Irvine (UC Irvine) produced methods for time and spatially resolved chemical imaging of interfacial electron transfer dynamics and photocatalytic reactions. An integral aspect of the joint research was a significant exchange of graduate students to work at the two institutions. This project bridged complementary approaches to investigate a set of common problems by working with the same molecules on a variety of solid surfaces, but using appropriate techniques to probe under ambient (BGSU) and ultrahigh vacuum (UCI) conditions. The molecular level understanding of the fundamental interfacial electron transfer processes obtained in this joint project will be important for developing efficient light harvesting

Single-molecule magnetism in three related {Co(III)2Dy(III)2}-acetylacetonate complexes with multiple relaxation mechanisms.

Science.gov (United States)

Langley, Stuart K; Chilton, Nicholas F; Moubaraki, Boujemaa; Murray, Keith S

2013-06-17

Three new heterometallic complexes with formulas of [Dy(III)2Co(III)2(OMe)2(teaH)2(acac)4(NO3)2] (1), [Dy(III)2Co(III)2(OH)2(teaH)2(acac)4(NO3)2]·4H2O (2), and [Dy(III)2Co(III)2(OMe)2(mdea)2(acac)4(NO3)2] (3) were characterized by single-crystal X-ray diffraction and by dc and ac magnetic susceptibility measurements. All three complexes have an identical "butterfly"-type metallic core that consists of two Dy(III) ions occupying the "body" position and two diamagnetic low-spin Co(III) ions occupying the outer "wing-tips". Each complex displays single-molecule magnet (SMM) behavior in zero applied magnetic field, with thermally activated anisotropy barriers of 27, 28, and 38 K above 7.5 K for 1-3, respectively, as well as observing a temperature-independent mechanism of relaxation below 5 K for 1 and 2 and at 3 K for 3, indicating fast quantum tunneling of magnetization (QTM). A second, faster thermally activated relaxation mechanism may also be active under a zero applied dc field as derived from the Cole-Cole data. Interestingly, these complexes demonstrate further relaxation modes that are strongly dependent upon the application of a static dc magnetic field. Dilution experiments that were performed on 1, in the {Y(III)2Co(III)2} diamagnetic analog, show that the slow magnetic relaxation is of a single-ion origin, but it was found that the neighboring ion also plays an important role in the overall relaxation dynamics.

Metal-Controlled Magnetoresistance at Room Temperature in Single-Molecule Devices.

Science.gov (United States)

Aragonès, Albert C; Aravena, Daniel; Valverde-Muñoz, Francisco J; Real, José Antonio; Sanz, Fausto; Díez-Pérez, Ismael; Ruiz, Eliseo

2017-04-26

The appropriate choice of the transition metal complex and metal surface electronic structure opens the possibility to control the spin of the charge carriers through the resulting hybrid molecule/metal spinterface in a single-molecule electrical contact at room temperature. The single-molecule conductance of a Au/molecule/Ni junction can be switched by flipping the magnetization direction of the ferromagnetic electrode. The requirements of the molecule include not just the presence of unpaired electrons: the electronic configuration of the metal center has to provide occupied or empty orbitals that strongly interact with the junction metal electrodes and that are close in energy to their Fermi levels for one of the electronic spins only. The key ingredient for the metal surface is to provide an efficient spin texture induced by the spin-orbit coupling in the topological surface states that results in an efficient spin-dependent interaction with the orbitals of the molecule. The strong magnetoresistance effect found in this kind of single-molecule wire opens a new approach for the design of room-temperature nanoscale devices based on spin-polarized currents controlled at molecular level.

Heterobifunctional crosslinkers for tethering single ligand molecules to scanning probes

International Nuclear Information System (INIS)

Riener, Christian K.; Kienberger, Ferry; Hahn, Christoph D.; Buchinger, Gerhard M.; Egwim, Innocent O.C.; Haselgruebler, Thomas; Ebner, Andreas; Romanin, Christoph; Klampfl, Christian; Lackner, Bernd; Prinz, Heino; Blaas, Dieter; Hinterdorfer, Peter; Gruber, Hermann J.

2003-01-01

Single molecule recognition force microscopy (SMRFM) is a versatile atomic force microscopy (AFM) method to probe specific interactions of cognitive molecules on the single molecule level. It allows insights to be gained into interaction potentials and kinetic barriers and is capable of mapping interaction sites with nm positional accuracy. These applications require a ligand to be attached to the AFM tip, preferably by a distensible poly(ethylene glycol) (PEG) chain between the measuring tip and the ligand molecule. The PEG chain greatly facilitates specific binding of the ligand to immobile receptor sites on the sample surface. The present study contributes to tip-PEG-ligand tethering in three ways: (i) a convenient synthetic route was found to prepare NH 2 -PEG-COOH which is the key intermediate for long heterobifunctional crosslinkers; (ii) a variety of heterobifunctional PEG derivatives for tip-PEG-ligand linking were prepared from NH 2 -PEG-COOH; (iii) in particular, a new PEG crosslinker with one thiol-reactive end and one terminal nitrilotriacetic acid (NTA) group was synthesized and successfully used to tether His 6 -tagged protein molecules to AFM tips via noncovalent NTA-Ni 2+ -His 6 bridges. The new crosslinker was applied to link a recombinant His 6 -tagged fragment of the very-low density lipoprotein receptor to the AFM tip whereupon specific docking to the capsid of human rhinovirus particles was observed by force microscopy. In a parallel study, the specific interaction of the small GTPase Ran with the nuclear import receptor importin β1 was studied in detail by SMRFM, using the new crosslinker to link His 6 -tagged Ran to the measuring tip [Nat. Struct. Biol. (2003), 10, 553-557

Single-Molecule Chemistry with Surface- and Tip-Enhanced Raman Spectroscopy.

Science.gov (United States)

Zrimsek, Alyssa B; Chiang, Naihao; Mattei, Michael; Zaleski, Stephanie; McAnally, Michael O; Chapman, Craig T; Henry, Anne-Isabelle; Schatz, George C; Van Duyne, Richard P

2017-06-14

Single-molecule (SM) surface-enhanced Raman spectroscopy (SERS) and tip-enhanced Raman spectroscopy (TERS) have emerged as analytical techniques for characterizing molecular systems in nanoscale environments. SERS and TERS use plasmonically enhanced Raman scattering to characterize the chemical information on single molecules. Additionally, TERS can image single molecules with subnanometer spatial resolution. In this review, we cover the development and history of SERS and TERS, including the concept of SERS hot spots and the plasmonic nanostructures necessary for SM detection, the past and current methodologies for verifying SMSERS, and investigations into understanding the signal heterogeneities observed with SMSERS. Moving on to TERS, we cover tip fabrication and the physical origins of the subnanometer spatial resolution. Then, we highlight recent advances of SMSERS and TERS in fields such as electrochemistry, catalysis, and SM electronics, which all benefit from the vibrational characterization of single molecules. SMSERS and TERS provide new insights on molecular behavior that would otherwise be obscured in an ensemble-averaged measurement.

Design improvement for fretting-wear reduction of HANARO fuel assembly

Energy Technology Data Exchange (ETDEWEB)

Cho, Yeong Garp; Chae, H. T.; Ryu, J. S.; Kim, H. R

2000-06-01

In the course of the visual inspection of the fuel assemblies un-loaded from the reactor core in December 1996, it was observed that many of fuel assemblies had mechanical damages on some components. The major damage was the freting-wear on spacer plates and endplates due to the flow induced vibration of the fuel assembly in the flow tube. Since the reactor is activated and the system modification for complete removal of the driving factors of the vibration of fuel assemblies is practically very difficult, the focus has been on the design change of the fuel assemblies. Consequently, various design changes were proposed to strengthen the wear resistance of the components based on the evaluation of the visual inspection results. The validity of the proposals was verified through the performance tests for the modified components, and the vibration test and endurance test for the fuel assemblies using the single-channel test rig(SCTR) in AECL.The subsequent design changes were additionally proposed based on the visual inspections for the fuel assemblies that had been fabricated according to the first design change and loaded in the core. As the effects of the first design change, the fretting-wear of spacer plates was remarkably reduced and the period until fretting-wear damage was extended by 60% for the first modified 36-rod fuel assembly. It is too early to say the endurance life time for the first modified 18-rod fuel assembly because of insufficient statistical data of only two bundles damaged, but the fretting-wear at the bottom endplate slot was reduced to about 50%. The second modified fuel assemblies, that were not loaded into the core yet, are expected to meet the design requirements for the core residence time due to strengthening the weak parts from the fretting-wear point of view. This report describes design changes and tests for fuel assemblies of HANARO to reduce the fretting-wear, and estimates the effects of design improvement quantitatively compared

Design improvement for fretting-wear reduction of HANARO fuel assembly

International Nuclear Information System (INIS)

Cho, Yeong Garp; Chae, H. T.; Ryu, J. S.; Kim, H. R.

2000-06-01

In the course of the visual inspection of the fuel assemblies un-loaded from the reactor core in December 1996, it was observed that many of fuel assemblies had mechanical damages on some components. The major damage was the freting-wear on spacer plates and endplates due to the flow induced vibration of the fuel assembly in the flow tube. Since the reactor is activated and the system modification for complete removal of the driving factors of the vibration of fuel assemblies is practically very difficult, the focus has been on the design change of the fuel assemblies. Consequently, various design changes were proposed to strengthen the wear resistance of the components based on the evaluation of the visual inspection results. The validity of the proposals was verified through the performance tests for the modified components, and the vibration test and endurance test for the fuel assemblies using the single-channel test rig(SCTR) in AECL.The subsequent design changes were additionally proposed based on the visual inspections for the fuel assemblies that had been fabricated according to the first design change and loaded in the core. As the effects of the first design change, the fretting-wear of spacer plates was remarkably reduced and the period until fretting-wear damage was extended by 60% for the first modified 36-rod fuel assembly. It is too early to say the endurance life time for the first modified 18-rod fuel assembly because of insufficient statistical data of only two bundles damaged, but the fretting-wear at the bottom endplate slot was reduced to about 50%. The second modified fuel assemblies, that were not loaded into the core yet, are expected to meet the design requirements for the core residence time due to strengthening the weak parts from the fretting-wear point of view. This report describes design changes and tests for fuel assemblies of HANARO to reduce the fretting-wear, and estimates the effects of design improvement quantitatively compared

Nonlinear and Nonsymmetric Single-Molecule Electronic Properties Towards Molecular Information Processing.

Science.gov (United States)

Tamaki, Takashi; Ogawa, Takuji

2017-09-05

This review highlights molecular design for nonlinear and nonsymmetric single-molecule electronic properties such as rectification, negative differential resistance, and switching, which are important components of future single-molecule information processing devices. Perspectives on integrated "molecular circuits" are also provided. Nonlinear and nonsymmetric single-molecule electronics can be designed by utilizing (1) asymmetric molecular cores, (2) asymmetric anchoring groups, (3) an asymmetric junction environment, and (4) asymmetric electrode materials. This review mainly focuses on the design of molecular cores.

Estimating single molecule conductance from spontaneous evolution of a molecular contact

Science.gov (United States)

Gil, M.; Malinowski, T.; Iazykov, M.; Klein, H. R.

2018-03-01

We present an original method to estimate the conductivity of a single molecule anchored to nanometric-sized metallic electrodes, using a Mechanically Controlled Break Junction operated at room temperature in the liquid. We record the conductance through the metal/molecules/metal nanocontact while keeping the metallic electrodes at a fixed distance. Taking advantage of thermal diffusion and electromigration, we let the contact naturally explore the more stable configurations around a chosen conductance value. The conductance of a single molecule is estimated from a statistical analysis of raw conductance and conductance standard deviation data for molecular contacts containing up to 14 molecules. The single molecule conductance values are interpreted as time-averaged conductance of an ensemble of conformers at thermal equilibrium.

Single-Molecule Light-Sheet Imaging of Suspended T Cells.

Science.gov (United States)

Ponjavic, Aleks; McColl, James; Carr, Alexander R; Santos, Ana Mafalda; Kulenkampff, Klara; Lippert, Anna; Davis, Simon J; Klenerman, David; Lee, Steven F

2018-05-08

Adaptive immune responses are initiated by triggering of the T cell receptor. Single-molecule imaging based on total internal reflection fluorescence microscopy at coverslip/basal cell interfaces is commonly used to study this process. These experiments have suggested, unexpectedly, that the diffusional behavior and organization of signaling proteins and receptors may be constrained before activation. However, it is unclear to what extent the molecular behavior and cell state is affected by the imaging conditions, i.e., by the presence of a supporting surface. In this study, we implemented single-molecule light-sheet microscopy, which enables single receptors to be directly visualized at any plane in a cell to study protein dynamics and organization in live, resting T cells. The light sheet enabled the acquisition of high-quality single-molecule fluorescence images that were comparable to those of total internal reflection fluorescence microscopy. By comparing the apical and basal surfaces of surface-contacting T cells using single-molecule light-sheet microscopy, we found that most coated-glass surfaces and supported lipid bilayers profoundly affected the diffusion of membrane proteins (T cell receptor and CD45) and that all the surfaces induced calcium influx to various degrees. Our results suggest that, when studying resting T cells, surfaces are best avoided, which we achieve here by suspending cells in agarose. Copyright © 2018. Published by Elsevier Inc.

Single-cell multiple gene expression analysis based on single-molecule-detection microarray assay for multi-DNA determination

Energy Technology Data Exchange (ETDEWEB)

Li, Lu [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China); Wang, Xianwei [School of Life Sciences, Shandong University, Jinan 250100 (China); Zhang, Xiaoli [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China); Wang, Jinxing [School of Life Sciences, Shandong University, Jinan 250100 (China); Jin, Wenrui, E-mail: jwr@sdu.edu.cn [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China)

2015-01-07

Highlights: • A single-molecule-detection (SMD) microarray for 10 samples is fabricated. • The based-SMD microarray assay (SMA) can determine 8 DNAs for each sample. • The limit of detection of SMA is as low as 1.3 × 10{sup −16} mol L{sup −1}. • The SMA can be applied in single-cell multiple gene expression analysis. - Abstract: We report a novel ultra-sensitive and high-selective single-molecule-detection microarray assay (SMA) for multiple DNA determination. In the SMA, a capture DNA (DNAc) microarray consisting of 10 subarrays with 9 spots for each subarray is fabricated on a silanized glass coverslip as the substrate. On the subarrays, the spot-to-spot spacing is 500 μm and each spot has a diameter of ∼300 μm. The sequence of the DNAcs on the 9 spots of a subarray is different, to determine 8 types of target DNAs (DNAts). Thus, 8 types of DNAts are captured to their complementary DNAcs at 8 spots of a subarray, respectively, and then labeled with quantum dots (QDs) attached to 8 types of detection DNAs (DNAds) with different sequences. The ninth spot is used to detect the blank value. In order to determine the same 8 types of DNAts in 10 samples, the 10 DNAc-modified subarrays on the microarray are identical. Fluorescence single-molecule images of the QD-labeled DNAts on each spot of the subarray are acquired using a home-made single-molecule microarray reader. The amounts of the DNAts are quantified by counting the bright dots from the QDs. For a microarray, 8 types of DNAts in 10 samples can be quantified in parallel. The limit of detection of the SMA for DNA determination is as low as 1.3 × 10{sup −16} mol L{sup −1}. The SMA for multi-DNA determination can also be applied in single-cell multiple gene expression analysis through quantification of complementary DNAs (cDNAs) corresponding to multiple messenger RNAs (mRNAs) in single cells. To do so, total RNA in single cells is extracted and reversely transcribed into their cDNAs. Three

a Study on the Fretting Fatigue Life of Zircaloy Alloys

Science.gov (United States)

Kwon, Jae-Do; Park, Dae-Kyu; Woo, Seung-Wan; Chai, Young-Suck

Studies on the strength and fatigue life of machines and structures have been conducted in accordance with the development of modern industries. In particular, fine and repetitive cyclic damage occurring in contact regions has been known to have an impact on fretting fatigue fractures. The main component of zircaloy alloy is Zr, and it possesses good mechanical characteristics at high temperatures. This alloy is used in the fuel rod material of nuclear power plants because of its excellent resistance. In this paper, the effect of the fretting damage on the fatigue behavior of the zircaloy alloy is studied. Further, various types of mechanical tests such as tension and plain fatigue tests are performed. Fretting fatigue tests are performed with a flat-flat contact configuration using a bridge-type contact pad and plate-type specimen. Through these experiments, it is found that the fretting fatigue strength decreases by about 80% as compared to the plain fatigue strength. Oblique cracks are observed in the initial stage of the fretting fatigue, in which damaged areas are found. These results can be used as the basic data for the structural integrity evaluation of corrosion-resisting alloys considering the fretting damages.

Polyfluorophore Excimers and Exciplexes as FRET Donors in DNA

Science.gov (United States)

Teo, Yin Nah; Kool, Eric T.

2009-01-01

We describe studies aimed at testing whether oligomeric exciplex- and excimer fluorophores conjugated to DNA have the potential to act as donors for energy transfer by the Förster mechanism. Oligodeoxyfluorosides (ODFs) are composed of stacked, electronically interacting fluorophores replacing the bases on a DNA scaffold. The monomer chromophores in the twenty tetramer-length ODFs studied here include pyrene (Y), benzopyrene (B), perylene (E), dimethylaminostilbene (D), and a nonfluorescent spacer (S); these are conjugated in varied combinations at the 3’ end of a 14mer DNA probe sequence. In the absence of an acceptor chromophore, many of the ODF-DNAs show broad, unstructured long-wavelength emission peaks characteristic of excimer and exciplex excited states, similar to what has been observed for unconjugated ODFs. Although such delocalized excited states have been widely studied, we know of no prior report of their use in FRET. We tested the ability of the twenty ODFs to donate energy to Cy5 and TAMRA dyes conjugated to a complementary strand of DNA, with these acceptors oriented either at the near or far end of the ODF-conjugated probes. Results showed that a number of the ODF fluorophores exhibited relatively efficient energy transfer characteristic of the Förster mechanism, as judged by drops in donor emission quantum yield and fluorescence lifetime, accompanied by increases in intensity of acceptor emission bands. Excimer/exciplex bands in the donors were selectively quenched while shorter-wavelength monomer emission stayed relatively constant, consistent with the notion that the delocalized excited states, rather than individual fluorophores, are the donors. Interestingly, only specific sequences of ODFs were able to act as donors, while others did not, even though their emission wavelengths were similar. The new FRET donors possess large Stokes shifts, which can be beneficial for multiple applications. In addition, all ODFs can be excited at a single

Understanding and modeling Förster-type resonance energy transfer (FRET)

CERN Document Server

Hernández Martínez, Pedro Ludwig; Demir, Hilmi Volkan

2017-01-01

This Brief presents a complete study of the generalized theory of Förster-type energy transfer in nanostructures with mixed dimensionality. Here the aim is to obtain a generalized theory of FRET including a comprehensive set of analytical equations for all combinations and configurations of nanostructures and deriving generic expressions for the dimensionality involved. In this brief, the modification of FRET mechanism with respect to the nanostructure serving as the donor vs. the acceptor will be included, focusing on the rate’s distance dependency and the role of the effective dielectric function in FRET, which will be a unique, useful source for those who study and model FRET.

CONSIDERATIONS REGARDING THE FRETTING PHENOMENON USING LEAF SPRINGS

Directory of Open Access Journals (Sweden)

Stefan GHIMIȘI

2015-05-01

Full Text Available The fretting phenomenon represents particulary and complex form of wear who is; generaly, and/or weary of fretting who is produced on the load contact in a relative oscialatory movement lay small amplitude.A simultaneoustly applied tangential force and normal into contact appears a adhesion force

Electrical properties and mechanical stability of anchoring groups for single-molecule electronics

Directory of Open Access Journals (Sweden)

Riccardo Frisenda

2015-07-01

Full Text Available We report on an experimental investigation of transport through single molecules, trapped between two gold nano-electrodes fabricated with the mechanically controlled break junction (MCBJ technique. The four molecules studied share the same core structure, namely oligo(phenylene ethynylene (OPE3, while having different aurophilic anchoring groups: thiol (SAc, methyl sulfide (SMe, pyridyl (Py and amine (NH2. The focus of this paper is on the combined characterization of the electrical and mechanical properties determined by the anchoring groups. From conductance histograms we find that thiol anchored molecules provide the highest conductance; a single-level model fit to current–voltage characteristics suggests that SAc groups exhibit a higher electronic coupling to the electrodes, together with better level alignment than the other three groups. An analysis of the mechanical stability, recording the lifetime in a self-breaking method, shows that Py and SAc yield the most stable junctions while SMe form short-lived junctions. Density functional theory combined with non-equlibrium Green’s function calculations help in elucidating the experimental findings.

Optical determination and magnetic manipulation of a single nitrogen-vacancy color center in diamond nanocrystal

International Nuclear Information System (INIS)

Diep Lai, Ngoc; Zheng, Dingwei; Treussart, François; Roch, Jean-François

2010-01-01

The controlled and coherent manipulation of individual quantum systems is fundamental for the development of quantum information processing. The nitrogen-vacancy (NV) color center in diamond is a promising system since its photoluminescence is perfectly stable at room temperature and its electron spin can be optically read out at the individual level. We review here the experiments currently realized in our laboratory concerning the use of a single NV color center as the single photon source and the coherent magnetic manipulation of the electron spin associated with a single NV color center. Furthermore, we demonstrate a nanoscopy experiment based on the saturation absorption effect, which allows to optically pin-point a single NV color center at sub-λ resolution. This offers the possibility to independently address two or multiple magnetically coupled single NV color centers, which is a necessary step towards the realization of a diamond-based quantum computer

Deep learning for single-molecule science

Science.gov (United States)

Albrecht, Tim; Slabaugh, Gregory; Alonso, Eduardo; Al-Arif, SM Masudur R.

2017-10-01

Exploring and making predictions based on single-molecule data can be challenging, not only due to the sheer size of the datasets, but also because a priori knowledge about the signal characteristics is typically limited and poor signal-to-noise ratio. For example, hypothesis-driven data exploration, informed by an expectation of the signal characteristics, can lead to interpretation bias or loss of information. Equally, even when the different data categories are known, e.g., the four bases in DNA sequencing, it is often difficult to know how to make best use of the available information content. The latest developments in machine learning (ML), so-called deep learning (DL) offer interesting, new avenues to address such challenges. In some applications, such as speech and image recognition, DL has been able to outperform conventional ML strategies and even human performance. However, to date DL has not been applied much in single-molecule science, presumably in part because relatively little is known about the ‘internal workings’ of such DL tools within single-molecule science as a field. In this Tutorial, we make an attempt to illustrate in a step-by-step guide how one of those, a convolutional neural network (CNN), may be used for base calling in DNA sequencing applications. We compare it with a SVM as a more conventional ML method, and discuss some of the strengths and weaknesses of the approach. In particular, a ‘deep’ neural network has many features of a ‘black box’, which has important implications on how we look at and interpret data.

Chemo-mechanical pushing of proteins along single-stranded DNA.

Science.gov (United States)

Sokoloski, Joshua E; Kozlov, Alexander G; Galletto, Roberto; Lohman, Timothy M

2016-05-31

Single-stranded (ss)DNA binding (SSB) proteins bind with high affinity to ssDNA generated during DNA replication, recombination, and repair; however, these SSBs must eventually be displaced from or reorganized along the ssDNA. One potential mechanism for reorganization is for an ssDNA translocase (ATP-dependent motor) to push the SSB along ssDNA. Here we use single molecule total internal reflection fluorescence microscopy to detect such pushing events. When Cy5-labeled Escherichia coli (Ec) SSB is bound to surface-immobilized 3'-Cy3-labeled ssDNA, a fluctuating FRET signal is observed, consistent with random diffusion of SSB along the ssDNA. Addition of Saccharomyces cerevisiae Pif1, a 5' to 3' ssDNA translocase, results in the appearance of isolated, irregularly spaced saw-tooth FRET spikes only in the presence of ATP. These FRET spikes result from translocase-induced directional (5' to 3') pushing of the SSB toward the 3' ssDNA end, followed by displacement of the SSB from the DNA end. Similar ATP-dependent pushing events, but in the opposite (3' to 5') direction, are observed with EcRep and EcUvrD (both 3' to 5' ssDNA translocases). Simulations indicate that these events reflect active pushing by the translocase. The ability of translocases to chemo-mechanically push heterologous SSB proteins along ssDNA provides a potential mechanism for reorganization and clearance of tightly bound SSBs from ssDNA.

Controlling the formation process and atomic structures of single pyrazine molecular junction by tuning the strength of the metal-molecule interaction.

Science.gov (United States)

Kaneko, Satoshi; Takahashi, Ryoji; Fujii, Shintaro; Nishino, Tomoaki; Kiguchi, Manabu

2017-04-12

The formation process and atomic structures were investigated for single pyrazine molecular junctions sandwiched by three different Au, Ag, and Cu electrodes using a mechanically controllable break junction technique in ultrahigh vacuum conditions at 300 K. We demonstrated that the formation process of the single-molecule junction crucially depended on the choice of the metal electrodes. While single-molecule junction showing two distinct conductance states were found for the Au electrodes, only the single conductance state was evident for the Ag electrodes, and there was no junction formation for the Cu electrodes. These results suggested that metal-molecule interaction dominates the formation process and probability of the single-molecule junction. In addition to the metal-molecule interaction, temperature affected the formation process of the single-molecule junction. The single pyrazine molecular junction formed between Au electrodes exhibited significant temperature dependence where the junction-formation probability was about 8% at 300 K, while there was no junction-formation at 100 K. Instead of the junction formation, an Au atomic wire was formed at the low temperature. This study provides insight into the tuning of the junction-forming process for single-molecule junctions, which is needed to construct device structures on a single molecule scale.

Vibrationally coupled electron transport through single-molecule junctions

Energy Technology Data Exchange (ETDEWEB)

Haertle, Rainer

2012-04-26

Single-molecule junctions are among the smallest electric circuits. They consist of a molecule that is bound to a left and a right electrode. With such a molecular nanocontact, the flow of electrical currents through a single molecule can be studied and controlled. Experiments on single-molecule junctions show that a single molecule carries electrical currents that can even be in the microampere regime. Thereby, a number of transport phenomena have been observed, such as, for example, diode- or transistor-like behavior, negative differential resistance and conductance switching. An objective of this field, which is commonly referred to as molecular electronics, is to relate these transport phenomena to the properties of the molecule in the contact. To this end, theoretical model calculations are employed, which facilitate an understanding of the underlying transport processes and mechanisms. Thereby, one has to take into account that molecules are flexible structures, which respond to a change of their charge state by a profound reorganization of their geometrical structure or may even dissociate. It is thus important to understand the interrelation between the vibrational degrees of freedom of a singlemolecule junction and the electrical current flowing through the contact. In this thesis, we investigate vibrational effects in electron transport through singlemolecule junctions. For these studies, we calculate and analyze transport characteristics of both generic and first-principles based model systems of a molecular contact. To this end, we employ a master equation and a nonequilibrium Green's function approach. Both methods are suitable to describe this nonequilibrium transport problem and treat the interactions of the tunneling electrons on the molecular bridge non-perturbatively. This is particularly important with respect to the vibrational degrees of freedom, which may strongly interact with the tunneling electrons. We show in detail that the resulting

Prediction of pressure tube fretting-wear damage due to fuel vibration

International Nuclear Information System (INIS)

Yetisir, M.; Fisher, N.J.

1997-01-01

Fretting marks between fuel bundle bearing pads and pressure tubes have been observed at the inlet end of some Darlington Nuclear Generating Station (NGS) and Bruce NGS fuel channels. The excitation mechanisms that lead to fretting are not fully understood. In this paper, the possibility of bearing pad-to-pressure tube fretting due to turbulence-induced motion of the fuel element is investigated. Numerical simulations indicate that this mechanism by itself is not likely to cause the level of fretting experienced in Darlington and Bruce NGSs. (orig.)

Repurposing a Benchtop Centrifuge for High-Throughput Single-Molecule Force Spectroscopy.

Science.gov (United States)

Yang, Darren; Wong, Wesley P

2018-01-01

We present high-throughput single-molecule manipulation using a benchtop centrifuge, overcoming limitations common in other single-molecule approaches such as high cost, low throughput, technical difficulty, and strict infrastructure requirements. An inexpensive and compact Centrifuge Force Microscope (CFM) adapted to a commercial centrifuge enables use by nonspecialists, and integration with DNA nanoswitches facilitates both reliable measurements and repeated molecular interrogation. Here, we provide detailed protocols for constructing the CFM, creating DNA nanoswitch samples, and carrying out single-molecule force measurements.

Full-Color LCD Microdisplay System Based on OLED Backlight Unit and Field-Sequential Color Driving Method

Directory of Open Access Journals (Sweden)

Sungho Woo

2011-01-01

Full Text Available We developed a single-panel LCD microdisplay system using a field-sequential color (FSC driving method and an organic light-emitting diode (OLED as a backlight unit (BLU. The 0.76′′ OLED BLU with red, green, and blue (RGB colors was fabricated by a conventional UV photolithography patterning process and by vacuum deposition of small molecule organic layers. The field-sequential driving frequency was set to 255 Hz to allow each of the RGB colors to be generated without color mixing at the given display frame rate. A prototype FSC LCD microdisplay system consisting of a 0.7′′ LCD microdisplay panel and the 0.76′′ OLED BLU successfully exhibited color display and moving picture images using the FSC driving method.

Implications of molecular heterogeneity for the cooperativity of biological macromolecules.

Science.gov (United States)

Solomatin, Sergey V; Greenfeld, Max; Herschlag, Daniel

2011-06-01

Cooperativity, a universal property of biological macromolecules, is typically characterized by a Hill slope, which can provide fundamental information about binding sites and interactions. We demonstrate, through simulations and single-molecule FRET (smFRET) experiments, that molecular heterogeneity lowers bulk cooperativity from the intrinsic value for the individual molecules. As heterogeneity is common in smFRET experiments, appreciation of its influence on fundamental measures of cooperativity is critical for deriving accurate molecular models.

Mutation-Specific Mechanisms of Hyperactivation of Noonan Syndrome SOS Molecules Detected with Single-molecule Imaging in Living Cells.

Science.gov (United States)

Nakamura, Yuki; Umeki, Nobuhisa; Abe, Mitsuhiro; Sako, Yasushi

2017-10-26

Noonan syndrome (NS) is a congenital hereditary disorder associated with developmental and cardiac defects. Some patients with NS carry mutations in SOS, a guanine nucleotide exchange factor (GEF) for the small GTPase RAS. NS mutations have been identified not only in the GEF domain, but also in various domains of SOS, suggesting that multiple mechanisms disrupt SOS function. In this study, we examined three NS mutations in different domains of SOS to clarify the abnormality in its translocation to the plasma membrane, where SOS activates RAS. The association and dissociation kinetics between SOS tagged with a fluorescent protein and the living cell surface were observed in single molecules. All three mutants showed increased affinity for the plasma membrane, inducing excessive RAS signalling. However, the mechanisms by which their affinity was increased were specific to each mutant. Conformational disorder in the resting state, increased probability of a conformational change on the plasma membrane, and an increased association rate constant with the membrane receptor are the suggested mechanisms. These different properties cause the specific phenotypes of the mutants, which should be rescuable with different therapeutic strategies. Therefore, single-molecule kinetic analyses of living cells are useful for the pathological analysis of genetic diseases.

Real-time single-molecule imaging of quantum interference.

Science.gov (United States)

Juffmann, Thomas; Milic, Adriana; Müllneritsch, Michael; Asenbaum, Peter; Tsukernik, Alexander; Tüxen, Jens; Mayor, Marcel; Cheshnovsky, Ori; Arndt, Markus

2012-03-25

The observation of interference patterns in double-slit experiments with massive particles is generally regarded as the ultimate demonstration of the quantum nature of these objects. Such matter-wave interference has been observed for electrons, neutrons, atoms and molecules and, in contrast to classical physics, quantum interference can be observed when single particles arrive at the detector one by one. The build-up of such patterns in experiments with electrons has been described as the "most beautiful experiment in physics". Here, we show how a combination of nanofabrication and nano-imaging allows us to record the full two-dimensional build-up of quantum interference patterns in real time for phthalocyanine molecules and for derivatives of phthalocyanine molecules, which have masses of 514 AMU and 1,298 AMU respectively. A laser-controlled micro-evaporation source was used to produce a beam of molecules with the required intensity and coherence, and the gratings were machined in 10-nm-thick silicon nitride membranes to reduce the effect of van der Waals forces. Wide-field fluorescence microscopy detected the position of each molecule with an accuracy of 10 nm and revealed the build-up of a deterministic ensemble interference pattern from single molecules that arrived stochastically at the detector. In addition to providing this particularly clear demonstration of wave-particle duality, our approach could also be used to study larger molecules and explore the boundary between quantum and classical physics.

Single-molecule chemical reactions on DNA origami

DEFF Research Database (Denmark)

Voigt, Niels Vinther; Tørring, Thomas; Rotaru, Alexandru

2010-01-01

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......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...

Development of new photon-counting detectors for single-molecule fluorescence microscopy

Science.gov (United States)

Michalet, X.; Colyer, R. A.; Scalia, G.; Ingargiola, A.; Lin, R.; Millaud, J. E.; Weiss, S.; Siegmund, Oswald H. W.; Tremsin, Anton S.; Vallerga, John V.; Cheng, A.; Levi, M.; Aharoni, D.; Arisaka, K.; Villa, F.; Guerrieri, F.; Panzeri, F.; Rech, I.; Gulinatti, A.; Zappa, F.; Ghioni, M.; Cova, S.

2013-01-01

Two optical configurations are commonly used in single-molecule fluorescence microscopy: point-like excitation and detection to study freely diffusing molecules, and wide field illumination and detection to study surface immobilized or slowly diffusing molecules. Both approaches have common features, but also differ in significant aspects. In particular, they use different detectors, which share some requirements but also have major technical differences. Currently, two types of detectors best fulfil the needs of each approach: single-photon-counting avalanche diodes (SPADs) for point-like detection, and electron-multiplying charge-coupled devices (EMCCDs) for wide field detection. However, there is room for improvements in both cases. The first configuration suffers from low throughput owing to the analysis of data from a single location. The second, on the other hand, is limited to relatively low frame rates and loses the benefit of single-photon-counting approaches. During the past few years, new developments in point-like and wide field detectors have started addressing some of these issues. Here, we describe our recent progresses towards increasing the throughput of single-molecule fluorescence spectroscopy in solution using parallel arrays of SPADs. We also discuss our development of large area photon-counting cameras achieving subnanosecond resolution for fluorescence lifetime imaging applications at the single-molecule level. PMID:23267185

Molecular spintronics using single-molecule magnets

Science.gov (United States)

Bogani, Lapo; Wernsdorfer, Wolfgang

2008-03-01

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 charges in electronic devices containing one or more molecules. We discuss the advantages over more conventional materials, and the potential applications in information storage and processing. We also outline current challenges in the field, and propose convenient schemes to overcome them.

Modulation of intermolecular interactions in single-molecule magnets

Science.gov (United States)

Heroux, Katie Jeanne

Polynuclear manganese clusters exhibiting interesting magnetic and quantum properties have been an area of intense research since the discovery of the first single-molecule magnet (SMM) in 1993. These molecules, below their blocking temperature, function as single-domain magnetic particles which exhibit classical macroscale magnetic properties as well as quantum mechanical phenomena such as quantum tunnelling of magnetization (QTM) and quantum phase interference. The union of classical and quantum behavior in these nanomaterials makes SMMs ideal candidates for high-density information storage and quantum computing. However, environmental coupling factors (nuclear spins, phonons, neighboring molecules) must be minimized if such applications are ever to be fully realized. The focus of this work is making small structural changes in well-known manganese SMMs in order to drastically enhance the overall magnetic and quantum properties of the system. Well-isolated molecules of high crystalline quality should lead to well-defined energetic and spectral properties as well. An advantage of SMMs over bulk magnetic materials is that they can be chemically altered from a "bottom-up" approach providing a synthetic tool for tuning magnetic properties. This systematic approach is utilized in the work presented herein by incorporating bulky ligands and/or counterions to "isolate" the magnetic core of [Mn4] dicubane SMMs. Reducing intermolecular interactions in the crystal lattice (neighboring molecules, solvate molecules, dipolar interactions) is an important step toward developing viable quantum computing devices. Detailed bulk magnetic studies as well as single crystal magnetization hysteresis and high-frequency EPR studies on these sterically-isolated complexes show enhanced, and sometimes even unexpected, quantum dynamics. The importance of intra- and intermolecular interactions remains a common theme throughout this work, extending to other SMMs of various topology including

Towards single molecule biosensors using super-resolution fluorescence microscopy.

Science.gov (United States)

Lu, Xun; Nicovich, Philip R; Gaus, Katharina; Gooding, J Justin

2017-07-15

Conventional immunosensors require many binding events to give a single transducer output which represents the concentration of the analyte in the sample. Because of the requirements to selectively detect species in complex samples, immunosensing interfaces must allow immobilisation of antibodies while repelling nonspecific adsorption of other species. These requirements lead to quite sophisticated interfacial design, often with molecular level control, but we have no tools to characterise how well these interfaces work at the molecular level. The work reported herein is an initial feasibility study to show that antibody-antigen binding events can be monitored at the single molecule level using single molecule localisation microscopy (SMLM). The steps to achieve this first requires showing that indium tin oxide surfaces can be used for SMLM, then that these surfaces can be modified with self-assembled monolayers using organophosphonic acid derivatives, that the amount of antigens and antibodies on the surface can be controlled and monitored at the single molecule level and finally antibody binding to antigen modified surfaces can be monitored. The results show the amount of antibody that binds to an antigen modified surface is dependent on both the concentration of antigen on the surface and the concentration of antibody in solution. This study demonstrates the potential of SMLM for characterising biosensing interfaces and as the transducer in a massively parallel, wide field, single molecule detection scheme for quantitative analysis. Copyright © 2016 Elsevier B.V. All rights reserved.

Electrochemical Single-Molecule Transistors with Optimized Gate Coupling

DEFF Research Database (Denmark)

Osorio, Henrry M.; Catarelli, Samantha; Cea, Pilar

2015-01-01

Electrochemical gating at the single molecule level of viologen molecular bridges in ionic liquids is examined. Contrary to previous data recorded in aqueous electrolytes, a clear and sharp peak in the single molecule conductance versus electrochemical potential data is obtained in ionic liquids....... These data are rationalized in terms of a two-step electrochemical model for charge transport across the redox bridge. In this model the gate coupling in the ionic liquid is found to be fully effective with a modeled gate coupling parameter, ξ, of unity. This compares to a much lower gate coupling parameter...

Single-molecule fluorescence microscopy review: shedding new light on old problems.

Science.gov (United States)

Shashkova, Sviatlana; Leake, Mark C

2017-08-31

Fluorescence microscopy is an invaluable tool in the biosciences, a genuine workhorse technique offering exceptional contrast in conjunction with high specificity of labelling with relatively minimal perturbation to biological samples compared with many competing biophysical techniques. Improvements in detector and dye technologies coupled to advances in image analysis methods have fuelled recent development towards single-molecule fluorescence microscopy, which can utilize light microscopy tools to enable the faithful detection and analysis of single fluorescent molecules used as reporter tags in biological samples. For example, the discovery of GFP, initiating the so-called 'green revolution', has pushed experimental tools in the biosciences to a completely new level of functional imaging of living samples, culminating in single fluorescent protein molecule detection. Today, fluorescence microscopy is an indispensable tool in single-molecule investigations, providing a high signal-to-noise ratio for visualization while still retaining the key features in the physiological context of native biological systems. In this review, we discuss some of the recent discoveries in the life sciences which have been enabled using single-molecule fluorescence microscopy, paying particular attention to the so-called 'super-resolution' fluorescence microscopy techniques in live cells, which are at the cutting-edge of these methods. In particular, how these tools can reveal new insights into long-standing puzzles in biology: old problems, which have been impossible to tackle using other more traditional tools until the emergence of new single-molecule fluorescence microscopy techniques. © 2017 The Author(s).

Investigation of photobleaching and saturation of single molecules by fluorophore recrossing events

Energy Technology Data Exchange (ETDEWEB)

Burrows, Sean M.; Reif, Randall D. [Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409-1061 (United States); Pappas, Dimitri [Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409-1061 (United States)], E-mail: d.pappas@ttu.edu

2007-08-15

A method for investigation of photobleaching and saturation of single molecules by fluorophore recrossing events in a laser beam is described. The diffraction-limited probe volumes encountered in single-molecule detection (SMD) produce high excitation irradiance, which can decrease available signal. The single molecules of several dyes were detected and the data was used to extract interpeak times above a defined threshold value. The interpeak times revealed the number of fluorophore recrossing events. The number of molecules detected that were within 2 ms of each other represented a molecular recrossing for this work. Calcein, fluorescein and R-phycoerythrin were analyzed and the saturation irradiance and photobleaching effects were determined as a function of irradiance. This approach is simple and it serves as a method of optimizing experimental conditions for single-molecule detection.

Total Internal Reflection Fluorescence Microscopy Imaging-Guided Confocal Single-Molecule Fluorescence Spectroscopy

OpenAIRE

Zheng, Desheng; Kaldaras, Leonora; Lu, H. Peter

2013-01-01

We have developed an integrated spectroscopy system combining total internal reflection fluorescence microscopy imaging with confocal single-molecule fluorescence spectroscopy for two-dimensional interfaces. This spectroscopy approach is capable of both multiple molecules simultaneously sampling and in situ confocal fluorescence dynamics analyses of individual molecules of interest. We have demonstrated the calibration with fluorescent microspheres, and carried out single-molecule spectroscop...

Enhanced numerical analysis of three-color HgCdTe detectors

Science.gov (United States)

Jóźwikowski, K.; Rogalski, A.

2007-04-01

The performance of three-color HgCdTe photovoltaic heterostructure detector is examined theoretically. In comparison with two-color detectors with two back-to-back junctions, three-color structure contain an absorber of intermediate wavelength placed between two junctions, and electronic barriers are used to isolate this intermediate region. This structure was first proposed by British workers. Enhanced original computer programs are applied to solve the system of non-linear continuity equations for carriers and Poisson equations. In addition, the numerical analysis includes the dependence of absorption coefficient on Burstein effect as well as interference effects in heterostructure with metallic electrical contacts. Three detector structures with different localizations of separating barriers are analyzed. The calculations results are presented in the form of spatial distributions of bandgap energy and quantum efficiency. It is shown that the performance of the detector is critically dependent on the barrier's doping level and position in relation to the junction. This behavior is serious disadvantage of the considered three color detector. A small shift of the barrier location and doping level causes serious changes in spectral responsivity.

Steam generator fretting-wear damage: A summary of recent findings

International Nuclear Information System (INIS)

Guerout, F.M.; Fisher, N.J.

1999-01-01

Flow-induced vibration of steam generator (SG) tubes may sometimes result in fretting-wear damage at the tube-to-support locations. Fretting-wear damage predictions are largely based on experimental data obtained at representative test conditions. Fretting-wear of SG materials has been studied at the Chalk River Laboratories for two decades. Tests are conducted in fretting-wear test machines that simulate SG environmental conditions and tube-to-support dynamic interactions. A new high-temperature force and displacement measuring system was developed to monitor tube-to-support interaction (i.e., work-rate) at operating conditions. This improvement in experimental fretting-wear technology was used to perform a comprehensive study of the effect of various environment and design parameters on SG tube wear damage. This paper summarizes the results of tests performed over the past 4 yr to study the effect of temperature, water chemistry, support geometry, and tube material on fretting-wear. The results show a significant effect of temperature on tube wear damage. Therefore, fretting-wear tests must be performed at operating temperatures in order to be relevant. No significant effect of the type of water treatment on tube wear damage was observed. For predominantly impacting motion, the wear of SG tubes in contact with 410 stainless steel is similar regardless of whether Alloy 690 or Alloy 800 is used as tubing material or whether lattice bars or broached hole supports are used. Based on results presented in this paper, an average wear coefficient value is recommended that is used for the prediction of SG tube wear depth versus time

High-speed three-frame image recording system using colored flash units and low-cost video equipment

Science.gov (United States)

Racca, Roberto G.; Scotten, Larry N.

1995-05-01

This article describes a method that allows the digital recording of sequences of three black and white images at rates of several thousand frames per second using a system consisting of an ordinary CCD camcorder, three flash units with color filters, a PC-based frame grabber board and some additional electronics. The maximum framing rate is determined by the duration of the flashtube emission, and for common photographic flash units lasting about 20 microsecond(s) it can exceed 10,000 frames per second in actual use. The subject under study is strobe- illuminated using a red, a green and a blue flash unit controlled by a special sequencer, and the three images are captured by a color CCD camera on a single video field. Color is used as the distinguishing parameter that allows the overlaid exposures to be resolved. The video output for that particular field will contain three individual scenes, one for each primary color component, which potentially can be resolved with no crosstalk between them. The output is electronically decoded into the primary color channels, frame grabbed and stored into digital memory, yielding three time-resolved images of the subject. A synchronization pulse provided by the flash sequencer triggers the frame grabbing so that the correct video field is acquired. A scheme involving the use of videotape as intermediate storage allows the frame grabbing to be performed using a monochrome video digitizer. Ideally each flash- illuminated scene would be confined to one color channel, but in practice various factors, both optical and electronic, affect color separation. Correction equations have been derived that counteract these effects in the digitized images and minimize 'ghosting' between frames. Once the appropriate coefficients have been established through a calibration procedure that needs to be performed only once for a given configuration of the equipment, the correction process is carried out transparently in software every time a

DNA analysis by single molecule stretching in nanofluidic biochips

DEFF Research Database (Denmark)

Abad, E.; Juarros, A.; Retolaza, A.

2011-01-01

Imprint Lithography (NIL) technology combined with a conventional anodic bonding of the silicon base and Pyrex cover. Using this chip, we have performed single molecule imaging on a bench-top fluorescent microscope system. Lambda phage DNA was used as a model sample to characterize the chip. Single molecules of λ-DNA......Stretching single DNA molecules by confinement in nanofluidic channels has attracted a great interest during the last few years as a DNA analysis tool. We have designed and fabricated a sealed micro/nanofluidic device for DNA stretching applications, based on the use of the high throughput Nano...... stained with the fluorescent dye YOYO-1 were stretched in the nanochannel array and the experimental results were analysed to determine the extension factor of the DNA in the chip and the geometrical average of the nanochannel inner diameter. The determination of the extension ratio of the chip provides...

Fretting Corrosion Behavior of Experimental Ti-20Cr Compared to Titanium.

Science.gov (United States)

Sawada, Tomofumi; Schille, Christine; Almadani, Atif; Geis-Gerstorfer, Jürgen

2017-02-17

Experimental cast titanium alloys containing 20 mass% chromium (Ti-20Cr) show preferable mechanical properties and a good corrosion resistance. This study evaluated the fretting corrosion behavior of Ti-20Cr. Ti-20Cr ( n = 4) and commercially pure titanium (CP-Ti, n = 6) disk specimens were used. The fretting corrosion test was performed by electrochemical corrosion at 0.3 V in 0.9% saline solution and mechanical damage using 10 scratching cycles with three different scratching speeds (10-40 mm/s) at 10 N. After testing, the activation peak, repassivation time and surface morphology of each specimen were analyzed. The differences between the results were tested by parametric tests (α = 0.05). The average activation peaks were significantly higher in CP-Ti than in Ti-20Cr ( p Ti. Slight differences in the repassivation time were observed between the materials at every scratching speed; faster scratching speeds showed shorter repassivation times in both materials ( p Ti showed severe damage and significantly higher wear depth than Ti-20Cr ( p < 0.05). In conclusion, adding chromium to titanium reduced surface damage and improved the fretting corrosion resistance.

Maxwell's color statistics: from reduction of visible errors to reduction to invisible molecules.

Science.gov (United States)

Cat, Jordi

2014-12-01

This paper presents a cross-disciplinary and multi-disciplinary account of Maxwell's introduction of statistical models of molecules for the composition of gases. The account focuses on Maxwell's deployment of statistical models of data in his contemporaneous color researches as established in Cambridge mathematical physics, especially by Maxwell's seniors and mentors. The paper also argues that the cross-disciplinary, or cross-domain, transfer of resources from the natural and social sciences took place in both directions and relied on the complex intra-disciplinary, or intra-domain, dynamics of Maxwell's researches in natural sciences, in color theory, physical astronomy, electromagnetism and dynamical theory of gases, as well as involving a variety of types of communicating and mediating media, from material objects to concepts, techniques and institutions.

Prediction of pressure tube fretting-wear damage due to fuel vibration

Energy Technology Data Exchange (ETDEWEB)

Yetisir, M; Fisher, N J [Atomic Energy of Canada Ltd., Chalk River, ON (Canada)

1996-12-31

Fretting marks between fuel bundle bearing pads and pressure tubes have been observed at the inlet end of some Darlington NGS (nuclear generating station) and Bruce NGS fuel channels. The excitation mechanisms that lead to fretting are not fully understood. In this paper, the possibility of bearing pad-to-pressure tube fretting due to turbulence-induced motion of the fuel element is investigated. Numerical simulations indicate that this mechanism by itself is not likely to cause the level of fretting experienced in Darlington and Bruce NGS`s (nuclear generating stations). (author). 12 refs., 2 tabs., 11 figs.

DNA-Based Single-Molecule Electronics: From Concept to Function

Science.gov (United States)

2018-01-01

Beyond being the repository of genetic information, DNA is playing an increasingly important role as a building block for molecular electronics. Its inherent structural and molecular recognition properties render it a leading candidate for molecular electronics applications. The structural stability, diversity and programmability of DNA provide overwhelming freedom for the design and fabrication of molecular-scale devices. In the past two decades DNA has therefore attracted inordinate amounts of attention in molecular electronics. This review gives a brief survey of recent experimental progress in DNA-based single-molecule electronics with special focus on single-molecule conductance and I–V characteristics of individual DNA molecules. Existing challenges and exciting future opportunities are also discussed. PMID:29342091

DNA-Based Single-Molecule Electronics: From Concept to Function.

Science.gov (United States)

Wang, Kun

2018-01-17

Beyond being the repository of genetic information, DNA is playing an increasingly important role as a building block for molecular electronics. Its inherent structural and molecular recognition properties render it a leading candidate for molecular electronics applications. The structural stability, diversity and programmability of DNA provide overwhelming freedom for the design and fabrication of molecular-scale devices. In the past two decades DNA has therefore attracted inordinate amounts of attention in molecular electronics. This review gives a brief survey of recent experimental progress in DNA-based single-molecule electronics with special focus on single-molecule conductance and I-V characteristics of individual DNA molecules. Existing challenges and exciting future opportunities are also discussed.

Development of a Fluorescence Resonance Energy Transfer (FRET)-Based DNA Biosensor for Detection of Synthetic Oligonucleotide of Ganoderma boninense.

Science.gov (United States)

Mohd Bakhori, Noremylia; Yusof, Nor Azah; Abdullah, Abdul Halim; Hussein, Mohd Zobir

2013-12-01

An optical DNA biosensor based on fluorescence resonance energy transfer (FRET) utilizing synthesized quantum dot (QD) has been developed for the detection of specific-sequence of DNA for Ganoderma boninense, an oil palm pathogen. Modified QD that contained carboxylic groups was conjugated with a single-stranded DNA probe (ssDNA) via amide-linkage. Hybridization of the target DNA with conjugated QD-ssDNA and reporter probe labeled with Cy5 allows for the detection of related synthetic DNA sequence of Ganoderma boninense gene based on FRET signals. Detection of FRET emission before and after hybridization was confirmed through the capability of the system to produce FRET at 680 nm for hybridized sandwich with complementary target DNA. No FRET emission was observed for non-complementary system. Hybridization time, temperature and effect of different concentration of target DNA were studied in order to optimize the developed system. The developed biosensor has shown high sensitivity with detection limit of 3.55 × 10(-9) M. TEM results show that the particle size of QD varies in the range between 5 to 8 nm after ligand modification and conjugation with ssDNA. This approach is capable of providing a simple, rapid and sensitive method for detection of related synthetic DNA sequence of Ganoderma boninense.

Development of a Fluorescence Resonance Energy Transfer (FRET-Based DNA Biosensor for Detection of Synthetic Oligonucleotide of Ganoderma boninense

Directory of Open Access Journals (Sweden)

Noremylia Mohd Bakhori

2013-12-01

Full Text Available An optical DNA biosensor based on fluorescence resonance energy transfer (FRET utilizing synthesized quantum dot (QD has been developed for the detection of specific-sequence of DNA for Ganoderma boninense, an oil palm pathogen. Modified QD that contained carboxylic groups was conjugated with a single-stranded DNA probe (ssDNA via amide-linkage. Hybridization of the target DNA with conjugated QD-ssDNA and reporter probe labeled with Cy5 allows for the detection of related synthetic DNA sequence of Ganoderma boninense gene based on FRET signals. Detection of FRET emission before and after hybridization was confirmed through the capability of the system to produce FRET at 680 nm for hybridized sandwich with complementary target DNA. No FRET emission was observed for non-complementary system. Hybridization time, temperature and effect of different concentration of target DNA were studied in order to optimize the developed system. The developed biosensor has shown high sensitivity with detection limit of 3.55 × 10−9 M. TEM results show that the particle size of QD varies in the range between 5 to 8 nm after ligand modification and conjugation with ssDNA. This approach is capable of providing a simple, rapid and sensitive method for detection of related synthetic DNA sequence of Ganoderma boninense.

DNA origami as biocompatible surface to match single-molecule and ensemble experiments

Science.gov (United States)

Gietl, Andreas; Holzmeister, Phil; Grohmann, Dina; Tinnefeld, Philip

2012-01-01

Single-molecule experiments on immobilized molecules allow unique insights into the dynamics of molecular machines and enzymes as well as their interactions. The immobilization, however, can invoke perturbation to the activity of biomolecules causing incongruities between single molecule and ensemble measurements. Here we introduce the recently developed DNA origami as a platform to transfer ensemble assays to the immobilized single molecule level without changing the nano-environment of the biomolecules. The idea is a stepwise transfer of common functional assays first to the surface of a DNA origami, which can be checked at the ensemble level, and then to the microscope glass slide for single-molecule inquiry using the DNA origami as a transfer platform. We studied the structural flexibility of a DNA Holliday junction and the TATA-binding protein (TBP)-induced bending of DNA both on freely diffusing molecules and attached to the origami structure by fluorescence resonance energy transfer. This resulted in highly congruent data sets demonstrating that the DNA origami does not influence the functionality of the biomolecule. Single-molecule data collected from surface-immobilized biomolecule-loaded DNA origami are in very good agreement with data from solution measurements supporting the fact that the DNA origami can be used as biocompatible surface in many fluorescence-based measurements. PMID:22523083

Tumor specific lung cancer diagnostics with multiplexed FRET immunoassays

Science.gov (United States)

Geißler, D.; Hill, D.; Löhmannsröben, H.-G.; Thomas, E.; Lavigne, A.; Darbouret, B.; Bois, E.; Charbonnière, L. J.; Ziessel, R. F.; Hildebrandt, N.

2010-02-01

An optical multiplexed homogeneous (liquid phase) immunoassay based on FRET from a terbium complex to eight different fluorescent dyes is presented. We achieved highly sensitive parallel detection of four different lung cancer specific tumor markers (CEA, NSE, SCC and CYFRA21-1) within a single assay and show a proof-of-principle for 5- fold multiplexing. The method is well suited for fast and low-cost miniaturized point-of-care testing as well as for highthroughput screening in a broad range of in-vitro diagnostic applications.

Single-molecule conductivity of non-redox and redox molecules at pure and gold-mined Au(111)-electrode surfaces

DEFF Research Database (Denmark)

Zhang, Jingdong; Chi, Qijin; Ulstrup, Jens

The structure, two-dimensional organization, and function of molecules immobilized on solid surfaces can be addressed in a degree of detail that has reached the level of the single-molecule. In this context redox molecules are “smart” molecules adding sophisticated electronic function. Redox meta...

Single Molecule 3D Orientation in Time and Space: A 6D Dynamic Study on Fluorescently Labeled Lipid Membranes

DEFF Research Database (Denmark)

Börner, Richard; Ehrlich, Nicky; Hohlbein, Johannes

2016-01-01

Interactions between single molecules profoundly depend on their mutual three-dimensional orientation. Recently, we demonstrated a technique that allows for orientation determination of single dipole emitters using a polarization-resolved distribution of fluorescence into several detection channels...... interesting in non-isotropic environments such as lipid membranes, which are of great importance in biology. We used giant unilamellar vesicles (GUVs) labeled with fluorescent dyes down to a single molecule concentration as a model system for both, assessing the robustness of the orientation determination...

Color optimization of single emissive white OLEDs via energy transfer between RGB fluorescent dopants

Energy Technology Data Exchange (ETDEWEB)

Kim, Nam Ho; Kim, You-Hyun; Yoon, Ju-An; Lee, Sang Youn [Department of Green Energy and Semiconductor Engineering, Hoseo University, Asan (Korea, Republic of); Ryu, Dae Hyun [Department of Information Technology, Hansei University, Gunpo (Korea, Republic of); Wood, Richard [Department of Engineering Physics, McMaster University, Hamilton, Ontario, Canada L8S 4L7 (Canada); Moon, C.-B. [Department of Green Energy and Semiconductor Engineering, Hoseo University, Asan (Korea, Republic of); Kim, Woo Young, E-mail: wykim@hoseo.edu [Department of Green Energy and Semiconductor Engineering, Hoseo University, Asan (Korea, Republic of); Department of Engineering Physics, McMaster University, Hamilton, Ontario, Canada L8S 4L7 (Canada)

2013-11-15

The electroluminescent characteristics of white organic light-emitting diodes (WOLEDs) were investigated including single emitting layer (SEL) with an ADN host and dopants; BCzVBi, C545T, and DCJTB for blue, green and red emission, respectively. The structure of the high efficiency WOLED device was; ITO/NPB(700 Å)/ADN: BCzVBi-7%:C545T-0.05%:DCJTB-0.1%(300 Å)/Bphen(300 Å)/Liq(20 Å)/Al(1200 Å) for mixing three primary colors. Luminous efficiency was 9.08 cd/A at 3.5 V and Commission Intenationale de L’eclairage (CIE{sub x,y}) coordinates of white emission was measured as (0.320, 0.338) at 8 V while simulated CIE{sub x,y} coordinates were (0.336, 0.324) via estimation from each dopant's PL spectrum. -- Highlights: • This paper observes single-emissive-layered white OLED using fluorescent dopants. • Electrical and optical properties are analyzed. • Color stability of white OLED is confirmed for new planar light source.

Color optimization of single emissive white OLEDs via energy transfer between RGB fluorescent dopants

International Nuclear Information System (INIS)

Kim, Nam Ho; Kim, You-Hyun; Yoon, Ju-An; Lee, Sang Youn; Ryu, Dae Hyun; Wood, Richard; Moon, C.-B.; Kim, Woo Young

2013-01-01

The electroluminescent characteristics of white organic light-emitting diodes (WOLEDs) were investigated including single emitting layer (SEL) with an ADN host and dopants; BCzVBi, C545T, and DCJTB for blue, green and red emission, respectively. The structure of the high efficiency WOLED device was; ITO/NPB(700 Å)/ADN: BCzVBi-7%:C545T-0.05%:DCJTB-0.1%(300 Å)/Bphen(300 Å)/Liq(20 Å)/Al(1200 Å) for mixing three primary colors. Luminous efficiency was 9.08 cd/A at 3.5 V and Commission Intenationale de L’eclairage (CIE x,y ) coordinates of white emission was measured as (0.320, 0.338) at 8 V while simulated CIE x,y coordinates were (0.336, 0.324) via estimation from each dopant's PL spectrum. -- Highlights: • This paper observes single-emissive-layered white OLED using fluorescent dopants. • Electrical and optical properties are analyzed. • Color stability of white OLED is confirmed for new planar light source

Excitonic Behavior of Rhodamine Dimers: A Single-Molecule Study

NARCIS (Netherlands)

Hernando Campos, J.; van der Schaaf, Martijn; van Dijk, E.M.H.P.; Sauer, Markus; Garcia Parajo, M.F.; van Hulst, N.F.

2003-01-01

The optical behavior of a dimer of tetramethylrhodamine-5-isothiocyanate has been investigated by means of single-molecule measurements. Bulk absorption and fluorescence spectra show the existence of two populations of the dimer molecule that exhibit distinct excitonic interactions (strong and weak

A natural-color mapping for single-band night-time image based on FPGA

Science.gov (United States)

Wang, Yilun; Qian, Yunsheng

2018-01-01

A natural-color mapping for single-band night-time image method based on FPGA can transmit the color of the reference image to single-band night-time image, which is consistent with human visual habits and can help observers identify the target. This paper introduces the processing of the natural-color mapping algorithm based on FPGA. Firstly, the image can be transformed based on histogram equalization, and the intensity features and standard deviation features of reference image are stored in SRAM. Then, the real-time digital images' intensity features and standard deviation features are calculated by FPGA. At last, FPGA completes the color mapping through matching pixels between images using the features in luminance channel.

Single OR molecule and OR atomic circuit logic gates interconnected on a Si(100)H surface

International Nuclear Information System (INIS)

Ample, F; Joachim, C; Duchemin, I; Hliwa, M

2011-01-01

Electron transport calculations were carried out for three terminal OR logic gates constructed either with a single molecule or with a surface dangling bond circuit interconnected on a Si(100)H surface. The corresponding multi-electrode multi-channel scattering matrix (where the central three terminal junction OR gate is the scattering center) was calculated, taking into account the electronic structure of the supporting Si(100)H surface, the metallic interconnection nano-pads, the surface atomic wires and the molecule. Well interconnected, an optimized OR molecule can only run at a maximum of 10 nA output current intensity for a 0.5 V bias voltage. For the same voltage and with no molecule in the circuit, the output current of an OR surface atomic scale circuit can reach 4 μA.

Revisitation of FRET methods to measure intraprotein distances in Human Serum Albumin

Energy Technology Data Exchange (ETDEWEB)

Santini, S.; Bizzarri, A.R.; Cannistraro, S., E-mail: cannistr@unitus.it

2016-11-15

We revisited the FRET methods to measure the intraprotein distance between Trp-214 (used as donor) of Human Serum Albumin and its Cys-34, labelled with 1.5-Iaedans (used as acceptor). Variation of Trp fluorescence emission in terms of both intensity and lifetime, as well the enhancement of the acceptor fluorescence emission upon Trp excitation, have been monitored. A careful statistical analysis of the fluorescence results from ten independently prepared samples, combined with suitable spectral corrections, provided reproducible distances estimations by each one of the three methods. Even if monitoring of the donor lifetime variation in the presence of the acceptor reproduces at the best the crystallographic data, by allowing even sub-nanometre distance variations to be appreciated, we suggest that a comparative analysis of all the three methods, applied with statistical significance, should be preferred to achieve a better reliability of the FRET technique.

Voltage-Driven Conformational Switching with Distinct Raman Signature in a Single-Molecule Junction.

Science.gov (United States)

Bi, Hai; Palma, Carlos-Andres; Gong, Yuxiang; Hasch, Peter; Elbing, Mark; Mayor, Marcel; Reichert, Joachim; Barth, Johannes V

2018-04-11

Precisely controlling well-defined, stable single-molecule junctions represents a pillar of single-molecule electronics. Early attempts to establish computing with molecular switching arrays were partly challenged by limitations in the direct chemical characterization of metal-molecule-metal junctions. While cryogenic scanning probe studies have advanced the mechanistic understanding of current- and voltage-induced conformational switching, metal-molecule-metal conformations are still largely inferred from indirect evidence. Hence, the development of robust, chemically sensitive techniques is instrumental for advancement in the field. Here we probe the conformation of a two-state molecular switch with vibrational spectroscopy, while simultaneously operating it by means of the applied voltage. Our study emphasizes measurements of single-molecule Raman spectra in a room-temperature stable single-molecule switch presenting a signal modulation of nearly 2 orders of magnitude.

Evaluation of the Electronic Structure of Single-Molecule Junctions Based on Current-Voltage and Thermopower Measurements: Application to C60 Single-Molecule Junction.

Science.gov (United States)

Komoto, Yuki; Isshiki, Yuji; Fujii, Shintaro; Nishino, Tomoaki; Kiguchi, Manabu

2017-02-16

The electronic structure of molecular junctions has a significant impact on their transport properties. Despite the decisive role of the electronic structure, a complete characterization of the electronic structure remains a challenge. This is because there is no straightforward way of measuring electron spectroscopy for an individual molecule trapped in a nanoscale gap between two metal electrodes. Herein, a comprehensive approach to obtain a detailed description of the electronic structure in single-molecule junctions based on the analysis of current-voltage (I-V) and thermoelectric characteristics is described. It is shown that the electronic structure of the prototypical C 60 single-molecule junction can be resolved by analyzing complementary results of the I-V and thermoelectric measurement. This combined approach confirmed that the C 60 single-molecule junction was highly conductive with molecular electronic conductances of 0.033 and 0.003 G 0 and a molecular Seebeck coefficient of -12 μV K -1 . In addition, we revealed that charge transport was mediated by a LUMO whose energy level was located 0.5≈0.6 eV above the Fermi level of the Au electrode. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Single-molecule study of full-length NaChBac by planar lipid bilayer recording.

Directory of Open Access Journals (Sweden)

Andrew Jo

Full Text Available Planar lipid bilayer device, alternatively known as BLM, is a powerful tool to study functional properties of conducting membrane proteins such as ion channels and porins. In this work, we used BLM to study the prokaryotic voltage-gated sodium channel (Nav NaChBac in a well-defined membrane environment. Navs are an essential component for the generation and propagation of electric signals in excitable cells. The successes in the biochemical, biophysical and crystallographic studies on prokaryotic Navs in recent years has greatly promoted the understanding of the molecular mechanism that underlies these proteins and their eukaryotic counterparts. In this work, we investigated the single-molecule conductance and ionic selectivity behavior of NaChBac. Purified NaChBac protein was first reconstituted into lipid vesicles, which is subsequently incorporated into planar lipid bilayer by fusion. At single-molecule level, we were able to observe three distinct long-lived conductance sub-states of NaChBac. Change in the membrane potential switches on the channel mainly by increasing its opening probability. In addition, we found that individual NaChBac has similar permeability for Na+, K+, and Ca2+. The single-molecule behavior of the full-length protein is essentially highly stochastic. Our results show that planar lipid bilayer device can be used to study purified ion channels at single-molecule level in an artificial environment, and such studies can reveal new protein properties that are otherwise not observable in in vivo ensemble studies.

Single molecule microscopy in 3D cell cultures and tissues.

Science.gov (United States)

Lauer, Florian M; Kaemmerer, Elke; Meckel, Tobias

2014-12-15

From the onset of the first microscopic visualization of single fluorescent molecules in living cells at the beginning of this century, to the present, almost routine application of single molecule microscopy, the method has well-proven its ability to contribute unmatched detailed insight into the heterogeneous and dynamic molecular world life is composed of. Except for investigations on bacteria and yeast, almost the entire story of success is based on studies on adherent mammalian 2D cell cultures. However, despite this continuous progress, the technique was not able to keep pace with the move of the cell biology community to adapt 3D cell culture models for basic research, regenerative medicine, or drug development and screening. In this review, we will summarize the progress, which only recently allowed for the application of single molecule microscopy to 3D cell systems and give an overview of the technical advances that led to it. While initially posing a challenge, we finally conclude that relevant 3D cell models will become an integral part of the on-going success of single molecule microscopy. Copyright © 2014 Elsevier B.V. All rights reserved.

A single molecule switch based on two Pd nanocrystals linked

Indian Academy of Sciences (India)

Conducting molecule; nanocrystals; scanning tunneling microscopy; negative differential resistance. Abstract. Tunneling spectroscopy measurements have been carried out on a single molecule device formed by two Pd ... Current Issue : Vol.

Uncovering Aberrant Mutant PKA Function with Flow Cytometric FRET

Directory of Open Access Journals (Sweden)

Shin-Rong Lee

2016-03-01

Full Text Available Biology has been revolutionized by tools that allow the detection and characterization of protein-protein interactions (PPIs. Förster resonance energy transfer (FRET-based methods have become particularly attractive as they allow quantitative studies of PPIs within the convenient and relevant context of living cells. We describe here an approach that allows the rapid construction of live-cell FRET-based binding curves using a commercially available flow cytometer. We illustrate a simple method for absolutely calibrating the cytometer, validating our binding assay against the gold standard isothermal calorimetry (ITC, and using flow cytometric FRET to uncover the structural and functional effects of the Cushing-syndrome-causing mutation (L206R on PKA’s catalytic subunit. We discover that this mutation not only differentially affects PKAcat’s binding to its multiple partners but also impacts its rate of catalysis. These findings improve our mechanistic understanding of this disease-causing mutation, while illustrating the simplicity, general applicability, and power of flow cytometric FRET.

A comparison of donor-acceptor pairs for genetically encoded FRET sensors: application to the Epac cAMP sensor as an example.

Directory of Open Access Journals (Sweden)

Gerard N M van der Krogt

Full Text Available We recently reported on CFP-Epac-YFP, an Epac-based single polypeptide FRET reporter to resolve cAMP levels in living cells. In this study, we compared and optimized the fluorescent protein donor/acceptor pairs for use in biosensors such as CFP-Epac-YFP. Our strategy was to prepare a wide range of constructs consisting of different donor and acceptor fluorescent proteins separated by a short linker. Constructs were expressed in HEK293 cells and tested for FRET and other relevant properties. The most promising pairs were subsequently used in an attempt to improve the FRET span of the Epac-based cAMP sensor. The results show significant albeit not perfect correlation between performance in the spacer construct and in the Epac sensor. Finally, this strategy enabled us to identify improved sensors both for detection by sensitized emission and by fluorescent lifetime imaging. The present overview should be helpful in guiding development of future FRET sensors.

Quantum-Sequencing: Fast electronic single DNA molecule sequencing

Science.gov (United States)

Casamada Ribot, Josep; Chatterjee, Anushree; Nagpal, Prashant

2014-03-01

A major goal of third-generation sequencing technologies is to develop a fast, reliable, enzyme-free, high-throughput and cost-effective, single-molecule sequencing method. Here, we present the first demonstration of unique ``electronic fingerprint'' of all nucleotides (A, G, T, C), with single-molecule DNA sequencing, using Quantum-tunneling Sequencing (Q-Seq) at room temperature. We show that the electronic state of the nucleobases shift depending on the pH, with most distinct states identified at acidic pH. We also demonstrate identification of single nucleotide modifications (methylation here). Using these unique electronic fingerprints (or tunneling data), we report a partial sequence of beta lactamase (bla) gene, which encodes resistance to beta-lactam antibiotics, with over 95% success rate. These results highlight the potential of Q-Seq as a robust technique for next-generation sequencing.

Colored Chaos

Science.gov (United States)

2004-01-01

[figure removed for brevity, see original site] Released 7 May 2004 This daytime visible color image was collected on May 30, 2002 during the Southern Fall season in Atlantis Chaos. The THEMIS VIS camera is capable of capturing color images of the martian surface using its five different color filters. In this mode of operation, the spatial resolution and coverage of the image must be reduced to accommodate the additional data volume produced from the use of multiple filters. To make a color image, three of the five filter images (each in grayscale) are selected. Each is contrast enhanced and then converted to a red, green, or blue intensity image. These three images are then combined to produce a full color, single image. Because the THEMIS color filters don't span the full range of colors seen by the human eye, a color THEMIS image does not represent true color. Also, because each single-filter image is contrast enhanced before inclusion in the three-color image, the apparent color variation of the scene is exaggerated. Nevertheless, the color variation that does appear is representative of some change in color, however subtle, in the actual scene. Note that the long edges of THEMIS color images typically contain color artifacts that do not represent surface variation. Image information: VIS instrument. Latitude -34.5, Longitude 183.6 East (176.4 West). 38 meter/pixel resolution. Note: this THEMIS visual image has not been radiometrically nor geometrically calibrated for this preliminary release. An empirical correction has been performed to remove instrumental effects. A linear shift has been applied in the cross-track and down-track direction to approximate spacecraft and planetary motion. Fully calibrated and geometrically projected images will be released through the Planetary Data System in accordance with Project policies at a later time. NASA's Jet Propulsion Laboratory manages the 2001 Mars Odyssey mission for NASA's Office of Space Science, Washington, D

New Antifouling Platform Characterized by Single-Molecule Imaging

Science.gov (United States)

2015-01-01

Antifouling surfaces have been widely studied for their importance in medical devices and industry. Antifouling surfaces mostly achieved by methoxy-poly(ethylene glycol) (mPEG) have shown biomolecular adsorption less than 1 ng/cm2 which was measured by surface analytical tools such as surface plasmon resonance (SPR) spectroscopy, quartz crystal microbalance (QCM), or optical waveguide lightmode (OWL) spectroscopy. Herein, we utilize a single-molecule imaging technique (i.e., an ultimate resolution) to study antifouling properties of functionalized surfaces. We found that about 600 immunoglobulin G (IgG) molecules are adsorbed. This result corresponds to ∼5 pg/cm2 adsorption, which is far below amount for the detection limit of the conventional tools. Furthermore, we developed a new antifouling platform that exhibits improved antifouling performance that shows only 78 IgG molecules adsorbed (∼0.5 pg/cm2). The antifouling platform consists of forming 1 nm TiO2 thin layer, on which peptidomimetic antifouling polymer (PMAP) is robustly anchored. The unprecedented antifouling performance can potentially revolutionize a variety of research fields such as single-molecule imaging, medical devices, biosensors, and others. PMID:24503420

New antifouling platform characterized by single-molecule imaging.

Science.gov (United States)

Ryu, Ji Young; Song, In Taek; Lau, K H Aaron; Messersmith, Phillip B; Yoon, Tae-Young; Lee, Haeshin

2014-03-12

Antifouling surfaces have been widely studied for their importance in medical devices and industry. Antifouling surfaces mostly achieved by methoxy-poly(ethylene glycol) (mPEG) have shown biomolecular adsorption less than 1 ng/cm(2) which was measured by surface analytical tools such as surface plasmon resonance (SPR) spectroscopy, quartz crystal microbalance (QCM), or optical waveguide lightmode (OWL) spectroscopy. Herein, we utilize a single-molecule imaging technique (i.e., an ultimate resolution) to study antifouling properties of functionalized surfaces. We found that about 600 immunoglobulin G (IgG) molecules are adsorbed. This result corresponds to ∼5 pg/cm(2) adsorption, which is far below amount for the detection limit of the conventional tools. Furthermore, we developed a new antifouling platform that exhibits improved antifouling performance that shows only 78 IgG molecules adsorbed (∼0.5 pg/cm(2)). The antifouling platform consists of forming 1 nm TiO2 thin layer, on which peptidomimetic antifouling polymer (PMAP) is robustly anchored. The unprecedented antifouling performance can potentially revolutionize a variety of research fields such as single-molecule imaging, medical devices, biosensors, and others.

Single molecule experiments challenge the strict wave-particle dualism of light.

Science.gov (United States)

Greulich, Karl Otto

2010-01-21

Single molecule techniques improve our understanding of the photon and light. If the single photon double slit experiment is performed at the "single photon limit" of a multi-atom light source, faint light pulses with more than one photon hamper the interpretation. Single molecules, quantum dots or defect centres in crystals should be used as light source. "Single photon detectors" do not meet their promise-only "photon number resolving single photon detectors" do so. Particularly, the accumulation time argument, the only safe basis for the postulate of a strictly particle like photon, has so far not yet been verified.

Zero-phonon-line emission of single molecules for applications in quantum information processing

Science.gov (United States)

Kiraz, Alper; Ehrl, M.; Mustecaplioglu, O. E.; Hellerer, T.; Brauchle, C.; Zumbusch, A.

2005-07-01

A single photon source which generates transform limited single photons is highly desirable for applications in quantum optics. Transform limited emission guarantees the indistinguishability of the emitted single photons. This, in turn brings groundbreaking applications in linear optics quantum information processing within an experimental reach. Recently, self-assembled InAs quantum dots and trapped atoms have successfully been demonstrated as such sources for highly indistinguishable single photons. Here, we demonstrate that nearly transform limited zero-phonon-line (ZPL) emission from single molecules can be obtained by using vibronic excitation. Furthermore we report the results of coincidence detection experiments at the output of a Michelson-type interferometer. These experiments reveal Hong-Ou-Mandel correlations as a proof of the indistinguishability of the single photons emitted consecutively from a single molecule. Therefore, single molecules constitute an attractive alternative to single InAs quantum dots and trapped atoms for applications in linear optics quantum information processing. Experiments were performed with a home-built confocal microscope keeping the sample in a superfluid liquid Helium bath at 1.4K. We investigated terrylenediimide (TDI) molecules highly diluted in hexadecane (Shpol'skii matrix). A continuous wave single mode dye laser was used for excitation of vibronic transitions of individual molecules. From the integral fluorescence, the ZPL of single molecules was selected with a spectrally narrow interference filter. The ZPL emission was then sent to a scanning Fabry-Perot interferometer for linewidth measurements or a Michelson-type interferometer for coincidence detection.

Detection of kinetic change points in piece-wise linear single molecule motion

Science.gov (United States)

Hill, Flynn R.; van Oijen, Antoine M.; Duderstadt, Karl E.

2018-03-01

Single-molecule approaches present a powerful way to obtain detailed kinetic information at the molecular level. However, the identification of small rate changes is often hindered by the considerable noise present in such single-molecule kinetic data. We present a general method to detect such kinetic change points in trajectories of motion of processive single molecules having Gaussian noise, with a minimum number of parameters and without the need of an assumed kinetic model beyond piece-wise linearity of motion. Kinetic change points are detected using a likelihood ratio test in which the probability of no change is compared to the probability of a change occurring, given the experimental noise. A predetermined confidence interval minimizes the occurrence of false detections. Applying the method recursively to all sub-regions of a single molecule trajectory ensures that all kinetic change points are located. The algorithm presented allows rigorous and quantitative determination of kinetic change points in noisy single molecule observations without the need for filtering or binning, which reduce temporal resolution and obscure dynamics. The statistical framework for the approach and implementation details are discussed. The detection power of the algorithm is assessed using simulations with both single kinetic changes and multiple kinetic changes that typically arise in observations of single-molecule DNA-replication reactions. Implementations of the algorithm are provided in ImageJ plugin format written in Java and in the Julia language for numeric computing, with accompanying Jupyter Notebooks to allow reproduction of the analysis presented here.

Single-shot color fringe projection for three-dimensional shape measurement of objects with discontinuities.

Science.gov (United States)

Dai, Meiling; Yang, Fujun; He, Xiaoyuan

2012-04-20

A simple but effective fringe projection profilometry is proposed to measure 3D shape by using one snapshot color sinusoidal fringe pattern. One color fringe pattern encoded with a sinusoidal fringe (as red component) and one uniform intensity pattern (as blue component) is projected by a digital video projector, and the deformed fringe pattern is recorded by a color CCD camera. The captured color fringe pattern is separated into its RGB components and division operation is applied to red and blue channels to reduce the variable reflection intensity. Shape information of the tested object is decoded by applying an arcsine algorithm on the normalized fringe pattern with subpixel resolution. In the case of fringe discontinuities caused by height steps, or spatially isolated surfaces, the separated blue component is binarized and used for correcting the phase demodulation. A simple and robust method is also introduced to compensate for nonlinear intensity response of the digital video projector. The experimental results demonstrate the validity of the proposed method.

Pulsed IR Heating Studies of Single-Molecule DNA Duplex Dissociation Kinetics and Thermodynamics

Science.gov (United States)

Holmstrom, Erik D.; Dupuis, Nicholas F.; Nesbitt, David J.

2014-01-01

Single-molecule fluorescence spectroscopy is a powerful technique that makes it possible to observe the conformational dynamics associated with biomolecular processes. The addition of precise temperature control to these experiments can yield valuable thermodynamic information about equilibrium and kinetic rate constants. To accomplish this, we have developed a microscopy technique based on infrared laser overtone/combination band absorption to heat small (≈10−11 liter) volumes of water. Detailed experimental characterization of this technique reveals three major advantages over conventional stage heating methods: 1), a larger range of steady-state temperatures (20–100°C); 2), substantially superior spatial (≤20 μm) control; and 3), substantially superior temporal (≈1 ms) control. The flexibility and breadth of this spatial and temporally resolved laser-heating approach is demonstrated in single-molecule fluorescence assays designed to probe the dissociation of a 21 bp DNA duplex. These studies are used to support a kinetic model based on nucleic acid end fraying that describes dissociation for both short (10 bp) DNA duplexes. These measurements have been extended to explore temperature-dependent kinetics for the 21 bp construct, which permit determination of single-molecule activation enthalpies and entropies for DNA duplex dissociation. PMID:24411254

Multiplex single-molecule interaction profiling of DNA-barcoded proteins.

Science.gov (United States)

Gu, Liangcai; Li, Chao; Aach, John; Hill, David E; Vidal, Marc; Church, George M

2014-11-27

In contrast with advances in massively parallel DNA sequencing, high-throughput protein analyses are often limited by ensemble measurements, individual analyte purification and hence compromised quality and cost-effectiveness. Single-molecule protein detection using optical methods is limited by the number of spectrally non-overlapping chromophores. Here we introduce a single-molecular-interaction sequencing (SMI-seq) technology for parallel protein interaction profiling leveraging single-molecule advantages. DNA barcodes are attached to proteins collectively via ribosome display or individually via enzymatic conjugation. Barcoded proteins are assayed en masse in aqueous solution and subsequently immobilized in a polyacrylamide thin film to construct a random single-molecule array, where barcoding DNAs are amplified into in situ polymerase colonies (polonies) and analysed by DNA sequencing. This method allows precise quantification of various proteins with a theoretical maximum array density of over one million polonies per square millimetre. Furthermore, protein interactions can be measured on the basis of the statistics of colocalized polonies arising from barcoding DNAs of interacting proteins. Two demanding applications, G-protein coupled receptor and antibody-binding profiling, are demonstrated. SMI-seq enables 'library versus library' screening in a one-pot assay, simultaneously interrogating molecular binding affinity and specificity.

A Study on Fretting Behavior in Room Temperature for Inconel Alloy 690

Science.gov (United States)

Kwon, Jae Do; Chai, Young Suck; Bae, Yong Tak; Choi, Sung Jong

The initial crack under fretting condition occurs at lower stress amplitude and lower cycles of cyclic loading than that under plain fatigue condition. The fretting damage, for example, can be observed in fossil and nuclear power plant, aircraft, automobile and petroleum chemical plants etc. INCONEL alloy 690 is a high-chromium nickel alloy having excellent resistance to many corrosive aqueous media and high-temperature atmospheres. This alloy is used extensively in the industries of nuclear power, chemicals, heat-treatment and electronics. In this paper, the effect of fretting damage on fatigue behavior for INCONEL alloy 690 was studied. Also, various kinds of tests on mechanical properties such as hardness, tension and plain fatigue tests are performed. Fretting fatigue tests were carried out with flat-flat contact configuration using a bridge type contact pad and plate type specimen. Through these experiments, it is found that the fretting fatigue strength decreased about 43% compared to the plain fatigue strength. In fretting fatigue, the wear debris is observed on the contact surface, and the oblique micro-cracks are initiated at an earlier stage. These results can be used as the basic data in a structural integrity evaluation of heat and corrosion resistant alloy considering fretting damages.

Surface single-molecule dynamics controlled by entropy at low temperatures

Science.gov (United States)

Gehrig, J. C.; Penedo, M.; Parschau, M.; Schwenk, J.; Marioni, M. A.; Hudson, E. W.; Hug, H. J.

2017-02-01

Configuration transitions of individual molecules and atoms on surfaces are traditionally described using an Arrhenius equation with energy barrier and pre-exponential factor (attempt rate) parameters. Characteristic parameters can vary even for identical systems, and pre-exponential factors sometimes differ by orders of magnitude. Using low-temperature scanning tunnelling microscopy (STM) to measure an individual dibutyl sulfide molecule on Au(111), we show that the differences arise when the relative position of tip apex and molecule changes by a fraction of the molecule size. Altering the tip position on that scale modifies the transition's barrier and attempt rate in a highly correlated fashion, which results in a single-molecular enthalpy-entropy compensation. Conversely, appropriately positioning the STM tip allows selecting the operating point on the compensation line and modifying the transition rates. The results highlight the need to consider entropy in transition rates of single molecules, even at low temperatures.

Fluorescence resonance energy transfer between conjugated molecules infiltrated in three-dimensional opal photonic crystals

International Nuclear Information System (INIS)

Zou, Lu; Sui, Ning; Wang, Ying-Hui; Qian, Cheng; Ma, Yu-Guang; Zhang, Han-Zhuang

2015-01-01

Fluorescence resonance energy transfer (FRET) from Coumarin 6 (C-6) to Sulforhodamine B (S-B) infiltrated into opal PMMA (poly-methyl-methacrylate) photonic crystals (PCs) has been studied in detail. The intrinsic mesh micro-porous structure of opal PCs could increase the luminescent efficiency through inhibiting the intermolecular interaction. Meanwhile, its structure of periodically varying refractive indices could also modify the FRET through affecting the luminescence characteristics of energy donor or energy acceptor. The results demonstrate that the FRET efficiency between conjugated dyes was easily modified by opal PCs. - Highlights: • We investigate the fluorescence resonance energy transfer between two kinds of dyes. • These two kinds of dyes are infiltrated in PMMA opal photonic crystals. • The structure of opal PCs could improve the luminescent characteristics. • The structure of opal PCs could improve the energy transfer characteristics

Single-Molecule Electronics with Cross- Conjugated Molecules: Quantum Interference, IETS and Non-Equilibrium "Temperatures"

DEFF Research Database (Denmark)

Jørgensen, Jacob Lykkebo

Abstract The idea of using single-molecules as components in electronic devices is fas- cinating. For this idea to come into fruition, a number of technical and theo- retical challenges must be overcome. In this PhD thesis, the electron-phonon interaction is studied for a special class of molecules......, which is characterised by destructive quantum interference. The molecules are cross-conjugated, which means that the two parts of the molecules are conjugated to a third part, but not to each other. This gives rise to an anti-resonance in the trans- mission. In the low bias and low temperature regime......-conjugated molecules. We nd that the vibrational modes that would be expected to dominate, following the propensity, rules are very weak. Instead, other modes are found to be the dominant ones. We study this phenomenon for a number of cross-conjugated molecules, and link these ndings to the anti...

Enthalpy-Driven RNA Folding: Single-Molecule Thermodynamics of Tetraloop–Receptor Tertiary Interaction†

Science.gov (United States)

Fiore, Julie L.; Kraemer, Benedikt; Koberling, Felix; Edmann, Rainer; Nesbitt, David J.

2010-01-01

RNA folding thermodynamics are crucial for structure prediction, which requires characterization of both enthalpic and entropic contributions of tertiary motifs to conformational stability. We explore the temperature dependence of RNA folding due to the ubiquitous GAAA tetraloop–receptor docking interaction, exploiting immobilized and freely diffusing single-molecule fluorescence resonance energy transfer (smFRET) methods. The equilibrium constant for intramolecular docking is obtained as a function of temperature (T = 21–47 °C), from which a van’t Hoff analysis yields the enthalpy (ΔH°) and entropy (ΔS°) of docking. Tetraloop–receptor docking is significantly exothermic and entropically unfavorable in 1 mM MgCl2 and 100 mM NaCl, with excellent agreement between immobilized (ΔH° = −17.4 ± 1.6 kcal/mol, and ΔS° = −56.2 ± 5.4 cal mol−1 K−1) and freely diffusing (ΔH° = −17.2 ± 1.6 kcal/mol, and ΔS° = −55.9 ± 5.2 cal mol−1 K−1) species. Kinetic heterogeneity in the tetraloop–receptor construct is unaffected over the temperature range investigated, indicating a large energy barrier for interconversion between the actively docking and nondocking subpopulations. Formation of the tetraloop–receptor interaction can account for ~60% of the ΔH° and ΔS° of P4–P6 domain folding in the Tetrahymena ribozyme, suggesting that it may act as a thermodynamic clamp for the domain. Comparison of the isolated tetraloop–receptor and other tertiary folding thermodynamics supports a theme that enthalpy- versus entropy-driven folding is determined by the number of hydrogen bonding and base stacking interactions. PMID:19186984

Evidence for a single hydrogen molecule connected by an atomic chain

DEFF Research Database (Denmark)

Kiguchi, M.; Stadler, Robert; Bækgaard, Iben Sig Buur

2007-01-01

Stable, single-molecule conducting-bridge configurations are typically identified from peak structures in a conductance histogram. In previous work on Pt with H-2 at cryogenic temperatures it has been shown that a peak near 1G(0) identifies a single-molecule Pt-H-2-Pt bridge. The histogram shows...

Single-Photon Source for Quantum Information Based on Single Dye Molecule Fluorescence in Liquid Crystal Host

International Nuclear Information System (INIS)

Lukishova, S.G.; Knox, R.P.; Freivald, P.; McNamara, A.; Boyd, R.W.; Stroud, Jr. C.R.; Schmid, A.W.; Marshall, K.L.

2006-01-01

This paper describes a new application for liquid crystals: quantum information technology. A deterministically polarized single-photon source that efficiently produces photons exhibiting antibunching is a pivotal hardware element in absolutely secure quantum communication. Planar-aligned nematic liquid crystal hosts deterministically align the single dye molecules which produce deterministically polarized single (antibunched) photons. In addition, 1-D photonic bandgap cholesteric liquid crystals will increase single-photon source efficiency. The experiments and challenges in the observation of deterministically polarized fluorescence from single dye molecules in planar-aligned glassy nematic-liquid-crystal oligomer as well as photon antibunching in glassy cholesteric oligomer are described for the first time

Surface Passivation for Single-molecule Protein Studies

Science.gov (United States)

Chandradoss, Stanley D.; Haagsma, Anna C.; Lee, Young Kwang; Hwang, Jae-Ho; Nam, Jwa-Min; Joo, Chirlmin

2014-01-01

Single-molecule fluorescence spectroscopy has proven to be instrumental in understanding a wide range of biological phenomena at the nanoscale. Important examples of what this technique can yield to biological sciences are the mechanistic insights on protein-protein and protein-nucleic acid interactions. When interactions of proteins are probed at the single-molecule level, the proteins or their substrates are often immobilized on a glass surface, which allows for a long-term observation. This immobilization scheme may introduce unwanted surface artifacts. Therefore, it is essential to passivate the glass surface to make it inert. Surface coating using polyethylene glycol (PEG) stands out for its high performance in preventing proteins from non-specifically interacting with a glass surface. However, the polymer coating procedure is difficult, due to the complication arising from a series of surface treatments and the stringent requirement that a surface needs to be free of any fluorescent molecules at the end of the procedure. Here, we provide a robust protocol with step-by-step instructions. It covers surface cleaning including piranha etching, surface functionalization with amine groups, and finally PEG coating. To obtain a high density of a PEG layer, we introduce a new strategy of treating the surface with PEG molecules over two rounds, which remarkably improves the quality of passivation. We provide representative results as well as practical advice for each critical step so that anyone can achieve the high quality surface passivation. PMID:24797261

Fuel bundle to pressure tube fretting in Bruce and Darlington

Energy Technology Data Exchange (ETDEWEB)

Norsworthy, A G; Ditschun, A [Atomic Energy of Canada Ltd., Mississauga, ON (Canada)

1996-12-31

As the fuel channel elongates due to creep, the fuel string moves relative to the inlet until the fuel pads at the inboard end eventually separate from the spacer sleeve, and the fuel resides on the burnish mark of the pressure tube. The bundle is then supported in a fashion which contributes to increased levels of vibration. Those pads which (due to geometric variation) have contact loads with the pressure tube within a certain range, vibrate, and cause significant fretting on the burnish mark, and further along at the midplane of the bundle. Inspection of the pressure tubes in Bruce A, Bruce B, and Darlington has revealed fret damage up to 0.55 mm at the burnish mark and slightly lower than this at the inlet bundle midplane. To date, all fret marks have been dealt with successfully without the need for tube replacement, but a program of work has been initiated to understand the mechanism and reduce the fretting. Such understanding is necessary to guide future design changes to the fuel bundle, to guide future inspection programs, to guide maintenance programs, and for longer term strategic planning. This paper discusses how the understanding of fretting has evolved and outlines a current hypothesis for the mechanism of fretting. The role of bundle geometry, excitation forces, and reactor conditions are reviewed, along with options under consideration to mitigate damage. (author). 4 refs., 2 tabs., 13 figs.

Fuel bundle to pressure tube fretting in Bruce and Darlington

International Nuclear Information System (INIS)

Norsworthy, A.G.; Ditschun, A.

1995-01-01

As the fuel channel elongates due to creep, the fuel string moves relative to the inlet until the fuel pads at the inboard end eventually separate from the spacer sleeve, and the fuel resides on the burnish mark of the pressure tube. The bundle is then supported in a fashion which contributes to increased levels of vibration. Those pads which (due to geometric variation) have contact loads with the pressure tube within a certain range, vibrate, and cause significant fretting on the burnish mark, and further along at the midplane of the bundle. Inspection of the pressure tubes in Bruce A, Bruce B, and Darlington has revealed fret damage up to 0.55 mm at the burnish mark and slightly lower than this at the inlet bundle midplane. To date, all fret marks have been dealt with successfully without the need for tube replacement, but a program of work has been initiated to understand the mechanism and reduce the fretting. Such understanding is necessary to guide future design changes to the fuel bundle, to guide future inspection programs, to guide maintenance programs, and for longer term strategic planning. This paper discusses how the understanding of fretting has evolved and outlines a current hypothesis for the mechanism of fretting. The role of bundle geometry, excitation forces, and reactor conditions are reviewed, along with options under consideration to mitigate damage. (author). 4 refs., 2 tabs., 13 figs

Single Lens Dual-Aperture 3D Imaging System: Color Modeling

Science.gov (United States)

Bae, Sam Y.; Korniski, Ronald; Ream, Allen; Fritz, Eric; Shearn, Michael

2012-01-01

In an effort to miniaturize a 3D imaging system, we created two viewpoints in a single objective lens camera. This was accomplished by placing a pair of Complementary Multi-band Bandpass Filters (CMBFs) in the aperture area. Two key characteristics about the CMBFs are that the passbands are staggered so only one viewpoint is opened at a time when a light band matched to that passband is illuminated, and the passbands are positioned throughout the visible spectrum, so each viewpoint can render color by taking RGB spectral images. Each viewpoint takes a different spectral image from the other viewpoint hence yielding a different color image relative to the other. This color mismatch in the two viewpoints could lead to color rivalry, where the human vision system fails to resolve two different colors. The difference will be closer if the number of passbands in a CMBF increases. (However, the number of passbands is constrained by cost and fabrication technique.) In this paper, simulation predicting the color mismatch is reported.

Standard test method for damage to contacting solid surfaces under fretting conditions

CERN Document Server

American Society for Testing and Materials. Philadelphia

2010-01-01

1.1 This test method covers the studying or ranking the susceptibility of candidate materials to fretting corrosion or fretting wear for the purposes of material selection for applications where fretting corrosion or fretting wear can limit serviceability. 1.2 This test method uses a tribological bench test apparatus with a mechanism or device that will produce the necessary relative motion between a contacting hemispherical rider and a flat counterface. The rider is pressed against the flat counterface with a loading mass. The test method is intended for use in room temperature air, but future editions could include fretting in the presence of lubricants or other environments. 1.3 The purpose of this test method is to rub two solid surfaces together under controlled fretting conditions and to quantify the damage to both surfaces in units of volume loss for the test method. 1.4 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard. 1.5...

The Effect of Modulation Ratio of Cu/Ni Multilayer Films on the Fretting Damage Behaviour of Ti-811 Titanium Alloy.

Science.gov (United States)

Zhang, Xiaohua; Liu, Daoxin; Li, Xiaoying; Dong, Hanshan; Xi, Yuntao

2017-05-26

To improve the fretting damage (fretting wear and fretting fatigue) resistance of Ti-811 titanium alloy, three Cu/Ni multilayer films with the same modulation period thickness (200 nm) and different modulation ratios (3:1, 1:1, 1:3) were deposited on the surface of the alloy via ion-assisted magnetron sputtering deposition (IAD). The bonding strength, micro-hardness, and toughness of the films were evaluated, and the effect of the modulation ratio on the room-temperature fretting wear (FW) and fretting fatigue (FF) resistance of the alloy was determined. The results indicated that the IAD technique can be successfully used to prepare Cu/Ni multilayer films, with high bonding strength, low-friction, and good toughness, which yield improved room-temperature FF and FW resistance of the alloy. For the same modulation period (200 nm), the micro-hardness, friction, and FW resistance of the coated alloy increased, decreased, and improved, respectively, with increasing modulation ratio of the Ni-to-Cu layer thickness. However, the FF resistance of the coated alloy increased non-monotonically with the increasing modulation ratio. Among the three Cu/Ni multilayer films, those with a modulation ratio of 1:1 can confer the highest FF resistance to the Ti-811 alloy, owing mainly to their unique combination of good toughness, high strength, and low-friction.

The [Fe(III)[Fe(III)(L1)2]3] star-type single-molecule magnet.

Science.gov (United States)

Saalfrank, Rolf W; Scheurer, Andreas; Bernt, Ingo; Heinemann, Frank W; Postnikov, Andrei V; Schünemann, Volker; Trautwein, Alfred X; Alam, Mohammad S; Rupp, Holger; Müller, Paul

2006-06-21

Star-shaped complex [Fe(III)[Fe(III)(L1)2]3] (3) was synthesized starting from N-methyldiethanolamine H2L1 (1) and ferric chloride in the presence of sodium hydride. For 3, two different high-spin iron(III) ion sites were confirmed by Mössbauer spectroscopy at 77 K. Single-crystal X-ray structure determination revealed that 3 crystallizes with four molecules of chloroform, but, with only three molecules of dichloromethane. The unit cell of 3.4CHCl3 contains the enantiomers (delta)-[(S,S)(R,R)(R,R)] and (lambda)-[(R,R)(S,S)(S,S)], whereas in case of 3.3CH2Cl2 four independent molecules, forming pairs of the enantiomers [lambda-(R,R)(R,R)(R,R)]-3 and [lambda-(S,S)(S,S)(S,S)]-3, were observed in the unit cell. According to SQUID measurements, the antiferromagnetic intramolecular coupling of the iron(III) ions in 3 results in a S = 10/2 ground state multiplet. The anisotropy is of the easy-axis type. EPR measurements enabled an accurate determination of the ligand-field splitting parameters. The ferric star 3 is a single-molecule magnet (SMM) and shows hysteretic magnetization characteristics below a blocking temperature of about 1.2 K. However, weak intermolecular couplings, mediated in a chainlike fashion via solvent molecules, have a strong influence on the magnetic properties. Scanning tunneling microscopy (STM) and scanning tunneling spectroscopy (STS) were used to determine the structural and electronic properties of star-type tetranuclear iron(III) complex 3. The molecules were deposited onto highly ordered pyrolytic graphite (HOPG). Small, regular molecule clusters, two-dimensional monolayers as well as separated single molecules were observed. In our STS measurements we found a rather large contrast at the expected locations of the metal centers of the molecules. This direct addressing of the metal centers was confirmed by DFT calculations.

A Single-Molecule Barcoding System using Nanoslits for DNA Analysis

Science.gov (United States)

Jo, Kyubong; Schramm, Timothy M.; Schwartz, David C.

Single DNA molecule approaches are playing an increasingly central role in the analytical genomic sciences because single molecule techniques intrinsically provide individualized measurements of selected molecules, free from the constraints of bulk techniques, which blindly average noise and mask the presence of minor analyte components. Accordingly, a principal challenge that must be addressed by all single molecule approaches aimed at genome analysis is how to immobilize and manipulate DNA molecules for measurements that foster construction of large, biologically relevant data sets. For meeting this challenge, this chapter discusses an integrated approach for microfabricated and nanofabricated devices for the manipulation of elongated DNA molecules within nanoscale geometries. Ideally, large DNA coils stretch via nanoconfinement when channel dimensions are within tens of nanometers. Importantly, stretched, often immobilized, DNA molecules spanning hundreds of kilobase pairs are required by all analytical platforms working with large genomic substrates because imaging techniques acquire sequence information from molecules that normally exist in free solution as unrevealing random coils resembling floppy balls of yarn. However, nanoscale devices fabricated with sufficiently small dimensions fostering molecular stretching make these devices impractical because of the requirement of exotic fabrication technologies, costly materials, and poor operational efficiencies. In this chapter, such problems are addressed by discussion of a new approach to DNA presentation and analysis that establishes scaleable nanoconfinement conditions through reduction of ionic strength; stiffening DNA molecules thus enabling their arraying for analysis using easily fabricated devices that can also be mass produced. This new approach to DNA nanoconfinement is complemented by the development of a novel labeling scheme for reliable marking of individual molecules with fluorochrome labels

Fretting wear of steam generator tubes: high-temperature tests on AECL rig

International Nuclear Information System (INIS)

Guerout, F.; Zbinden, M.

1993-07-01

The R and DD has undertaken the study of fretting-wear of Alloy 600 S.G. tubes which occurs by contact with migrating items. The test series was performed in Canada at AECL Research (Atomic Energy of Canada Limited) as part of an exchange program. Four types of configuration were envisaged: a tube-to-drilled hole support contact which provides reference results and three types of tube-to-support contacts which simulate the tube fretting-wear induced by a welding rod, a threaded rod and a knife-edge rod support. This programme is completed by the study of the contact between a S.G. tube and a neighbouring S.G. tube which has been broken after plugging. (authors). 1 tab., 3 refs

Functionalization of Probe Tips and Supports for Single-Molecule Recognition Force Microscopy

Science.gov (United States)

Ebner, Andreas; Wildling, Linda; Zhu, Rong; Rankl, Christian; Haselgrübler, Thomas; Hinterdorfer, Peter; Gruber, Hermann J.

The measuring tip of a force microscope can be converted into a monomolecular sensor if one or few "ligand" molecules are attached to the apex of the tip while maintaining ligand function. Functionalized tips are used to study fine details of receptor-ligand interaction by force spectroscopy or to map cognate "receptor" molecules on the sample surface. The receptor (or target) molecules can be present on the surface of a biological specimen; alternatively, soluble target molecules must be immobilized on ultraflat supports. This review describes the methods of tip functionalization, as well as target molecule immobilization. Silicon nitride tips, silicon chips, and mica have usually been functionalized in three steps: (1) aminofunctionalization, (2) crosslinker attachment, and (3) ligand/receptor coupling, whereby numerous crosslinkers are available to couple widely different ligand molecules. Gold-covered tips and/or supports have usually been coated with a self-assembled monolayer, on top of which the ligand/receptor molecule has been coupled either directly or via a crosslinker molecule. Apart from these general strategies, many simplified methods have been used for tip and/or support functionalization, even single-step methods such as adsorption or chemisorption being very efficient under suitable circumstances. All methods are described with the same explicitness and critical parameters are discussed. In conclusion, this review should help to find suitable methods for specific problems of tip and support functionalization.

Single Molecule Experiments Challenge the Strict Wave-Particle Dualism of Light

Directory of Open Access Journals (Sweden)

Karl Otto Greulich

2010-01-01

Full Text Available Single molecule techniques improve our understanding of the photon and light. If the single photon double slit experiment is performed at the “single photon limit” of a multi-atom light source, faint light pulses with more than one photon hamper the interpretation. Single molecules, quantum dots or defect centres in crystals should be used as light source. “Single photon detectors” do not meet their promise―only “photon number resolving single photon detectors” do so. Particularly, the accumulation time argument, the only safe basis for the postulate of a strictly particle like photon, has so far not yet been verified.

Single molecule DNA detection with an atomic vapor notch filter

Energy Technology Data Exchange (ETDEWEB)

Uhland, Denis; Rendler, Torsten; Widmann, Matthias; Lee, Sang-Yun [University of Stuttgart and Stuttgart Research Center of Photonic Engineering (SCoPE) and IQST, 3rd Physics Institute, Stuttgart (Germany); Wrachtrup, Joerg; Gerhardt, Ilja [University of Stuttgart and Stuttgart Research Center of Photonic Engineering (SCoPE) and IQST, 3rd Physics Institute, Stuttgart (Germany); Max Planck Institute for Solid State Research, Stuttgart (Germany)

2015-12-01

The detection of single molecules has facilitated many advances in life- and material-science. Commonly the fluorescence of dye molecules is detected, which are attached to a non-fluorescent structure under study. For fluorescence microscopy one desires to maximize the detection efficiency together with an efficient suppression of undesired laser leakage. Here we present the use of the narrow-band filtering properties of hot atomic sodium vapor to selectively filter the excitation light from the red-shifted fluorescence of dye labeled single-stranded DNA molecules. A statistical analysis proves an enhancement in detection efficiency of more than 15% in a confocal and in a wide-field configuration. (orig.)

Wave propagation in coated cylinders with reference to fretting fatigue

Indian Academy of Sciences (India)

is to study stress wave propagation in cylinders with reference to high frequency fretting. ... The motivation for studying of fretting fatigue at higher frequency is to investigate the ... Hence focus in this work is given to thin rods and cylinders. The.

Single-Molecule Sensing with Nanopore Confinement: from Chemical Reactions to Biological Interactions.

Science.gov (United States)

Lin, Yao; Ying, Yi-Lun; Gao, Rui; Long, Yi-Tao

2018-03-25

The nanopore can generate an electrochemical confinement for single-molecule sensing which help understand the fundamental chemical principle in nanoscale dimensions. By observing the generated ionic current, individual bond-making and bond-breaking steps, single biomolecule dynamic conformational changes and electron transfer processes that occur within pore can be monitored with high temporal and current resolution. These single-molecule studies in nanopore confinement are revealing information about the fundamental chemical and biological processes that cannot be extracted from ensemble measurements. In this concept, we introduce and discuss the electrochemical confinement effects on single-molecule covalent reactions, conformational dynamics of individual molecules and host-guest interactions in protein nanopores. Then, we extend the concept of nanopore confinement effects to confine electrochemical redox reactions in solid-state nanopores for developing new sensing mechanisms. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Single-molecule diffusion and conformational dynamics by spatial integration of temporal fluctuations

KAUST Repository

Serag, Maged F.

2014-10-06

Single-molecule localization and tracking has been used to translate spatiotemporal information of individual molecules to map their diffusion behaviours. However, accurate analysis of diffusion behaviours and including other parameters, such as the conformation and size of molecules, remain as limitations to the method. Here, we report a method that addresses the limitations of existing single-molecular localization methods. The method is based on temporal tracking of the cumulative area occupied by molecules. These temporal fluctuations are tied to molecular size, rates of diffusion and conformational changes. By analysing fluorescent nanospheres and double-stranded DNA molecules of different lengths and topological forms, we demonstrate that our cumulative-area method surpasses the conventional single-molecule localization method in terms of the accuracy of determined diffusion coefficients. Furthermore, the cumulative-area method provides conformational relaxation times of structurally flexible chains along with diffusion coefficients, which together are relevant to work in a wide spectrum of scientific fields.

Single-molecule diffusion and conformational dynamics by spatial integration of temporal fluctuations

KAUST Repository

Serag, Maged F.; Abadi, Maram; Habuchi, Satoshi

2014-01-01

Single-molecule localization and tracking has been used to translate spatiotemporal information of individual molecules to map their diffusion behaviours. However, accurate analysis of diffusion behaviours and including other parameters, such as the conformation and size of molecules, remain as limitations to the method. Here, we report a method that addresses the limitations of existing single-molecular localization methods. The method is based on temporal tracking of the cumulative area occupied by molecules. These temporal fluctuations are tied to molecular size, rates of diffusion and conformational changes. By analysing fluorescent nanospheres and double-stranded DNA molecules of different lengths and topological forms, we demonstrate that our cumulative-area method surpasses the conventional single-molecule localization method in terms of the accuracy of determined diffusion coefficients. Furthermore, the cumulative-area method provides conformational relaxation times of structurally flexible chains along with diffusion coefficients, which together are relevant to work in a wide spectrum of scientific fields.

Reflection color filters of the three primary colors with wide viewing angles using common-thickness silicon subwavelength gratings.

Science.gov (United States)

Kanamori, Yoshiaki; Ozaki, Toshikazu; Hane, Kazuhiro

2014-10-20

We fabricated reflection color filters of the three primary colors with wide viewing angles using silicon two-dimensional subwavelength gratings on the same quartz substrate. The grating periods were 400, 340, and 300 nm for red, green, and blue filters, respectively. All of the color filters had the same grating thickness of 100 nm, which enabled simple fabrication of a color filter array. Reflected colors from the red, green, and blue filters under s-polarized white-light irradiation appeared in the respective colors at incident angles from 0 to 50°. By rigorous coupled-wave analysis, the dimensions of each color filter were designed, and the calculated reflectivity was compared with the measured reflectivity.

Evaluation of surface characteristics under fretting of electrical contacts: Removal behaviour of hot dipped tin coating

International Nuclear Information System (INIS)

Park, Young Woo; Ramesh Bapu, G.N.K.; Lee, Kang Yong

2009-01-01

The fretting corrosion behaviour of hot dipped tin coating is investigated at low fretting cycles at ±25 μm displacement amplitude, 0.5N normal load, 3 Hz frequency, 45-50% relative humidity, and 25 ± 1 deg. C temperature. The typical characteristics of the change in contact resistance with fretting cycles are explained. The fretted surface is examined using laser scanning microscope, scanning electron microscope and energy dispersive X-ray analysis to assess the surface profile, extent of fretting damage, extent of oxidation and elemental distribution across the contact zone. The interdependence of extent of wear and oxidation increases the complexity of the fretting corrosion behaviour of tin coating. The variation of contact resistance clearly revealed the fretting of tin coating from 50 to 1200 cycles and the fretting of the substrate above 1200 cycles. The observed low and stable contact resistance region and the fluctuating resistance region at various fretting cycles are explained and substantiated with Scanning electron microscopy (SEM), laser scanning microscope (LSM) and energy dispersive analysis of X-rays (EDAX) analysis results of the fretted surface.

Machine learning approach for single molecule localisation microscopy.

Science.gov (United States)

Colabrese, Silvia; Castello, Marco; Vicidomini, Giuseppe; Del Bue, Alessio

2018-04-01

Single molecule localisation (SML) microscopy is a fundamental tool for biological discoveries; it provides sub-diffraction spatial resolution images by detecting and localizing "all" the fluorescent molecules labeling the structure of interest. For this reason, the effective resolution of SML microscopy strictly depends on the algorithm used to detect and localize the single molecules from the series of microscopy frames. To adapt to the different imaging conditions that can occur in a SML experiment, all current localisation algorithms request, from the microscopy users, the choice of different parameters. This choice is not always easy and their wrong selection can lead to poor performance. Here we overcome this weakness with the use of machine learning. We propose a parameter-free pipeline for SML learning based on support vector machine (SVM). This strategy requires a short supervised training that consists in selecting by the user few fluorescent molecules (∼ 10-20) from the frames under analysis. The algorithm has been extensively tested on both synthetic and real acquisitions. Results are qualitatively and quantitatively consistent with the state of the art in SML microscopy and demonstrate that the introduction of machine learning can lead to a new class of algorithms competitive and conceived from the user point of view.

Light-Induced Switching of Tunable Single-Molecule Junctions

KAUST Repository

Sendler, Torsten; Luka-Guth, Katharina; Wieser, Matthias; Lokamani; Wolf, Jannic Sebastian; Helm, Manfred; Gemming, Sibylle; Kerbusch, Jochen; Scheer, Elke; Huhn, Thomas; Erbe, Artur

2015-01-01

A major goal of molecular electronics is the development and implementation of devices such as single-molecular switches. Here, measurements are presented that show the controlled in situ switching of diarylethene molecules from their nonconductive to conductive state in contact to gold nanoelectrodes via controlled light irradiation. Both the conductance and the quantum yield for switching of these molecules are within a range making the molecules suitable for actual devices. The conductance of the molecular junctions in the opened and closed states is characterized and the molecular level E 0, which dominates the current transport in the closed state, and its level broadening Γ are identified. The obtained results show a clear light-induced ring forming isomerization of the single-molecule junctions. Electron withdrawing side-groups lead to a reduction of conductance, but do not influence the efficiency of the switching mechanism. Quantum chemical calculations of the light-induced switching processes correlate these observations with the fundamentally different low-lying electronic states of the opened and closed forms and their comparably small modification by electron-withdrawing substituents. This full characterization of a molecular switch operated in a molecular junction is an important step toward the development of real molecular electronics devices.

Light-Induced Switching of Tunable Single-Molecule Junctions

KAUST Repository

Sendler, Torsten

2015-04-16

A major goal of molecular electronics is the development and implementation of devices such as single-molecular switches. Here, measurements are presented that show the controlled in situ switching of diarylethene molecules from their nonconductive to conductive state in contact to gold nanoelectrodes via controlled light irradiation. Both the conductance and the quantum yield for switching of these molecules are within a range making the molecules suitable for actual devices. The conductance of the molecular junctions in the opened and closed states is characterized and the molecular level E 0, which dominates the current transport in the closed state, and its level broadening Γ are identified. The obtained results show a clear light-induced ring forming isomerization of the single-molecule junctions. Electron withdrawing side-groups lead to a reduction of conductance, but do not influence the efficiency of the switching mechanism. Quantum chemical calculations of the light-induced switching processes correlate these observations with the fundamentally different low-lying electronic states of the opened and closed forms and their comparably small modification by electron-withdrawing substituents. This full characterization of a molecular switch operated in a molecular junction is an important step toward the development of real molecular electronics devices.

Linker-dependent Junction Formation Probability in Single-Molecule Junctions

Energy Technology Data Exchange (ETDEWEB)

Yoo, Pil Sun; Kim, Taekyeong [HankukUniversity of Foreign Studies, Yongin (Korea, Republic of)

2015-01-15

We compare the junction formation probabilities of single-molecule junctions with different linker molecules by using a scanning tunneling microscope-based break-junction technique. We found that the junction formation probability varies as SH > SMe > NH2 for the benzene backbone molecule with different types of anchoring groups, through quantitative statistical analysis. These results are attributed to different bonding forces according to the linker groups formed with Au atoms in the electrodes, which is consistent with previous works. Our work allows a better understanding of the contact chemistry in the metal.molecule junction for future molecular electronic devices.

Structural and electronic properties of single molecules and organic layers on surfaces

NARCIS (Netherlands)

Sotthewes, Kai

2016-01-01

Single molecules and organic layers on well-defined solid surfaces have attracted tremendous attention owing to their interesting physical and chemical properties. The ultimate utility of single molecules or self-assembled monolayers (SAMs) for potential applications is critically dependent on the

Electrochemistry and bioelectrochemistry towards the single-molecule level: Theoretical notions and systems

DEFF Research Database (Denmark)

Zhang, Jingdong; Chi, Qijin; Albrecht, Tim

2005-01-01

Surface structures controlled at the nanometer and single-molecule levels, with functions crucially determined by interfacial electron transfer (ET) are broadly reported in recent years, with different kinds of electrochemically controlled nanoscale/single molecule systems. One is the broad class...

Manifestation of spin selection rules on the quantum tunneling of magnetization in a single-molecule magnet.

Science.gov (United States)

Henderson, J J; Koo, C; Feng, P L; del Barco, E; Hill, S; Tupitsyn, I S; Stamp, P C E; Hendrickson, D N

2009-07-03

We present low temperature magnetometry measurements on a new Mn3 single-molecule magnet in which the quantum tunneling of magnetization (QTM) displays clear evidence for quantum mechanical selection rules. A QTM resonance appearing only at high temperatures demonstrates tunneling between excited states with spin projections differing by a multiple of three. This is dictated by the C3 molecular symmetry, which forbids pure tunneling from the lowest metastable state. Transverse field resonances are understood by correctly orienting the Jahn-Teller axes of the individual manganese ions and including transverse dipolar fields. These factors are likely to be important for QTM in all single-molecule magnets.

Biomimetic plasmonic color generated by the single-layer coaxial honeycomb nanostructure arrays

Science.gov (United States)

Zhao, Jiancun; Gao, Bo; Li, Haoyong; Yu, Xiaochang; Yang, Xiaoming; Yu, Yiting

2017-07-01

We proposed a periodic coaxial honeycomb nanostructure array patterned in a silver film to realize the plasmonic structural color, which was inspired from natural honeybee hives. The spectral characteristics of the structure with variant geometrical parameters are investigated by employing a finite-difference time-domain method, and the corresponding colors are thus derived by calculating XYZ tristimulus values corresponding with the transmission spectra. The study demonstrates that the suggested structure with only a single layer has high transmission, narrow full-width at half-maximum, and wide color tunability by changing geometrical parameters. Therefore, the plasmonic colors realized possess a high color brightness, saturation, as well as a wide color gamut. In addition, the strong polarization independence makes it more attractive for practical applications. These results indicate that the recommended color-generating plasmonic structure has various potential applications in highly integrated optoelectronic devices, such as color filters and high-definition displays.

Excitonic Coupling in Linear and Trefoil Trimer Perylenediimide Molecules Probed by Single-Molecule Spectroscopy

KAUST Repository

Yoo, Hyejin

2012-10-25

Perylenediimide (PDI) molecules are promising building blocks for photophysical studies of electronic interactions within multichromophore arrays. Such PDI arrays are important materials for fabrication of molecular nanodevices such as organic light-emitting diodes, organic semiconductors, and biosensors because of their high photostability, chemical and physical inertness, electron affinity, and high tinctorial strength over the entire visible spectrum. In this work, PDIs have been organized into linear (L3) and trefoil (T3) trimer molecules and investigated by single-molecule fluorescence microscopy to probe the relationship between molecular structures and interchromophoric electronic interactions. We found a broad distribution of coupling strengths in both L3 and T3 and hence strong/weak coupling between PDI units by monitoring spectral peak shifts in single-molecule fluorescence spectra upon sequential photobleaching of each constituent chromophore. In addition, we used a wide-field defocused imaging technique to resolve heterogeneities in molecular structures of L3 and T3 embedded in a PMMA polymer matrix. A systematic comparison between the two sets of experimental results allowed us to infer the correlation between intermolecular interactions and molecular structures. Our results show control of the PDI intermolecular interactions using suitable multichromophoric structures. © 2012 American Chemical Society.

Excitonic Coupling in Linear and Trefoil Trimer Perylenediimide Molecules Probed by Single-Molecule Spectroscopy

KAUST Repository

Yoo, Hyejin; Furumaki, Shu; Yang, Jaesung; Lee, Ji-Eun; Chung, Heejae; Oba, Tatsuya; Kobayashi, Hiroyuki; Rybtchinski, Boris; Wilson, Thea M.; Wasielewski, Michael R.; Vacha, Martin; Kim, Dongho

2012-01-01

Perylenediimide (PDI) molecules are promising building blocks for photophysical studies of electronic interactions within multichromophore arrays. Such PDI arrays are important materials for fabrication of molecular nanodevices such as organic light-emitting diodes, organic semiconductors, and biosensors because of their high photostability, chemical and physical inertness, electron affinity, and high tinctorial strength over the entire visible spectrum. In this work, PDIs have been organized into linear (L3) and trefoil (T3) trimer molecules and investigated by single-molecule fluorescence microscopy to probe the relationship between molecular structures and interchromophoric electronic interactions. We found a broad distribution of coupling strengths in both L3 and T3 and hence strong/weak coupling between PDI units by monitoring spectral peak shifts in single-molecule fluorescence spectra upon sequential photobleaching of each constituent chromophore. In addition, we used a wide-field defocused imaging technique to resolve heterogeneities in molecular structures of L3 and T3 embedded in a PMMA polymer matrix. A systematic comparison between the two sets of experimental results allowed us to infer the correlation between intermolecular interactions and molecular structures. Our results show control of the PDI intermolecular interactions using suitable multichromophoric structures. © 2012 American Chemical Society.

Chemical Principles and Interference in the Electrical Conductance of Single Molecules

DEFF Research Database (Denmark)

Borges, Anders Christian

, the conductance of molecules can vary orders of magnitude and the concept of interference is believed to play a major role in this. This thesis investigates the links between single molecule conductance, chemistry and interference effects in short organic molecules. It is investigated to which extent...... the conductance can be understood in terms of separate contributions and when the effects of interference are important. Links between chemical principles and constructive- and destructive interference effects are demonstrated using a combination of simple models, atomistic calculations and Scanning......-Tunneling Microscope Break-Junction experiments (STM-BJ). It is demonstrated that these links can be used to design molecules exhibiting surprising interference effects and to interpret and predict the trends in the characteristic conductance of single molecules without resorting to numerical computational methods...

FRET Sensor for Erythrosine Dye Based on Organic Nanoparticles: Application to Analysis of Food Stuff.

Science.gov (United States)

Mahajan, Prasad G; Bhopate, Dhanaji P; Kolekar, Govind B; Patil, Shivajirao R

2016-07-01

An aqueous suspension of fluorescent nanoparticles (PHNNPs) of naphthol based fluorescent organic compound 1-[(Z)-(2-phenylhydrazinylidene) methyl] naphthalene -2-ol (PHN) were prepared using reprecipitation method shows bathochromically shifted aggregation induced enhanced emission (AIEE) in the spectral region where erythrosine (ETS) food dye absorbs strongly. The average size of 72.6 nm of aqueous suspension of PHNNPs obtained by Dynamic light scattering results shows a narrow particle size distribution. The negative zeta potential of nano probe (-22.6 mV) responsible to adsorb oppositely charged analyte on its surface and further permit to bind nano probe and analyte within the close distance proximity required for efficient fluorescence resonance energy transfer (FRET) to take place from donor (PHNNPs) to acceptor (ETS). Systematic FRET experiments performed by measuring fluorescence quenching of PHNNPs with successive addition of ETS solution exploited the use of the PHNNPs as a novel nano probe for the detection of ETS in aqueous solution with extremely lower limit of detection equal to 3.6 nM (3.1 ng/mL). The estimation of photo kinetic and thermodynamic parameters such as quenching rate constant, enthalpy change (∆H), Gibbs free energy change (∆G) and entropy change (∆S) was obtained by the quenching results obtained at different constant temperatures which were found to fit the well-known Stern-Volmer relation. The mechanism of binding and fluorescence quenching of PHNNPs by ETS food dye is proposed on the basis of results obtained in photophysical studies, thermodynamic parameter, energy transfer efficiency, critical energy transfer distance (R0) and distance of approach between donor-acceptor molecules (r). The proposed FRET method based on fluorescence quenching of PHNNPs was successfully applied to develop an analytical method for estimation of ETS from food stuffs without interference of other complex ingredients. Graphical Abstract A

Manifestation of Spin Selection Rules on the Quantum Tunneling of Magnetization in a Single Molecule Magnet

OpenAIRE

Henderson, J. J.; Koo, C.; Feng, P. L.; del Barco, E.; Hill, S.; Tupitsyn, I. S.; Stamp, P. C. E.; Hendrickson, D. N.

2009-01-01

We present low temperature magnetometry measurements on a new Mn3 single-molecule magnet (SMM) in which the quantum tunneling of magnetization (QTM) displays clear evidence for quantum mechanical selection rules. A QTM resonance appearing only at elevated temperatures demonstrates tunneling between excited states with spin projections differing by a multiple of three: this is dictated by the C3 symmetry of the molecule, which forbids pure tunneling from the lowest metastable state. Resonances...

Spectrally resolved single-molecule electrometry

Science.gov (United States)

Ruggeri, F.; Krishnan, M.

2018-03-01

Escape-time electrometry is a recently developed experimental technique that offers the ability to measure the effective electrical charge of a single biomolecule in solution with sub-elementary charge precision. The approach relies on measuring the average escape-time of a single charged macromolecule or molecular species transiently confined in an electrostatic fluidic trap. Comparing the experiments with the predictions of a mean-field model of molecular electrostatics, we have found that the measured effective charge even reports on molecular conformation, e.g., folded or disordered state, and non-uniform charge distribution in disordered proteins or polyelectrolytes. Here we demonstrate the ability to use the spectral dimension to distinguish minute differences in electrical charge between individual molecules or molecular species in a single simultaneous measurement, under identical experimental conditions. Using one spectral channel for referenced measurement, this kind of photophysical distinguishability essentially eliminates the need for accurate knowledge of key experimental parameters, otherwise obtained through intensive characterization of the experimental setup. As examples, we demonstrate the ability to detect small differences (˜5%) in the length of double-stranded DNA fragments as well as single amino acid exchange in an intrinsically disordered protein, prothymosin α.

Turbulence induced Fretting-wear characteristics of steam generator helical tubes

International Nuclear Information System (INIS)

Jhung, Myung Jo; Jo, Jong Chull; Kim, Hho Jung; Yune, Young Gill; Yu, Seon Oh

2005-01-01

This study addresses safety assessment of the potential for fretting-wear damages on steam generator helical tubes due to turbulence-induced vibration in operating nuclear power plants. To get the natural frequency, corresponding mode shape and participation factor, modal analyses are performed for helical type tubes with various conditions. Special emphases are put on the effects of coil diameter and the number of turns on the modal and fretting wear characteristics of tubes. Also, investigated are the effects of external pressure on the tube modal characteristics as well as the effects of turbulence induced vibration on the fretting-wear characteristics of tubes

2012 Gordon Research Conference, Single molecule approaches to biology, July 15-20 2012

Energy Technology Data Exchange (ETDEWEB)

Fernandez, Julio M. [Columbia Univ., New York, NY (United States)

2012-04-20

Single molecule techniques are rapidly occupying a central role in biological research at all levels. This transition was made possible by the availability and dissemination of robust techniques that use fluorescence and force probes to track the conformation of molecules one at a time, in vitro as well as in live cells. Single-molecule approaches have changed the way many biological problems are studied. These novel techniques provide previously unobtainable data on fundamental biochemical processes that are essential for all forms of life. The ability of single-molecule approaches to avoid ensemble averaging and to capture transient intermediates and heterogeneous behavior renders them particularly powerful in elucidating mechanisms of the molecular systems that underpin the functioning of living cells. Hence, our conference seeks to disseminate the implementation and use of single molecule techniques in the pursuit of new biological knowledge. Topics covered include: Molecular Motors on the Move; Origin And Fate Of Proteins; Physical Principles Of Life; Molecules and Super-resolution Microscopy; Nanoswitches In Action; Active Motion Or Random Diffusion?; Building Blocks Of Living Cells; From Molecular Mechanics To Physiology; Tug-of-war: Force Spectroscopy Of Single Proteins.

Blinking effect and the use of quantum dots in single molecule spectroscopy

International Nuclear Information System (INIS)

Rombach-Riegraf, Verena; Oswald, Peter; Bienert, Roland; Petersen, Jan; Domingo, M.P.; Pardo, Julian; Gräber, P.; Galvez, E.M.

2013-01-01

Highlights: ► It is possible to eliminate the blinking effect of a water-soluble QD. ► We provide a direct method to study protein function and dynamics at the single level. ► QD, potent tool for single molecule studies of biochemical and biological processes. -- Abstract: Luminescent semiconductor nanocrystals (quantum dots, QD) have unique photo-physical properties: high photostability, brightness and narrow size-tunable fluorescence spectra. Due to their unique properties, QD-based single molecule studies have become increasingly more popular during the last years. However QDs show a strong blinking effect (random and intermittent light emission), which may limit their use in single molecule fluorescence studies. QD blinking has been widely studied and some hypotheses have been done to explain this effect. Here we summarise what is known about the blinking effect in QDs, how this phenomenon may affect single molecule studies and, on the other hand, how the “on”/“off” states can be exploited in diverse experimental settings. In addition, we present results showing that site-directed binding of QD to cysteine residues of proteins reduces the blinking effect. This option opens a new possibility of using QDs to study protein–protein interactions and dynamics by single molecule fluorescence without modifying the chemical composition of the solution or the QD surface.

Blinking effect and the use of quantum dots in single molecule spectroscopy

Energy Technology Data Exchange (ETDEWEB)

Rombach-Riegraf, Verena; Oswald, Peter; Bienert, Roland; Petersen, Jan [Albert-Ludwigs-Universitaet Freiburg, Institut fuer Physikalische Chemie, Albertstrasse 23a, 79104 Freiburg (Germany); Domingo, M.P. [Instituto de Carboquimica (CSIC), Miguel Luesma 4, 50018 Zaragoza (Spain); Pardo, Julian [Grupo Apoptosis, Inmunidad y Cancer, Departamento Bioquimica y Biologia Molecular y Celular, Fac. Ciencias, Universidad de Zaragoza, Zaragoza (Spain); Fundacion Aragon I-D (ARAID), Gobierno de Aragon, Zaragoza (Spain); Immune Effector Cells Group, Aragon Health Research Institute (IIS Aragon), Biomedical Research Centre of Aragon (CIBA) Fundacion Aragon I-D - ARAID, Gobierno de Aragon, Zaragoza (Spain); Graeber, P. [Albert-Ludwigs-Universitaet Freiburg, Institut fuer Physikalische Chemie, Albertstrasse 23a, 79104 Freiburg (Germany); Galvez, E.M., E-mail: eva@icb.csic.es [Instituto de Carboquimica (CSIC), Miguel Luesma 4, 50018 Zaragoza (Spain); Immune Effector Cells Group, Aragon Health Research Institute (IIS Aragon), Biomedical Research Centre of Aragon (CIBA) Fundacion Aragon I-D - ARAID, Gobierno de Aragon, Zaragoza (Spain)

2013-01-04

Highlights: Black-Right-Pointing-Pointer It is possible to eliminate the blinking effect of a water-soluble QD. Black-Right-Pointing-Pointer We provide a direct method to study protein function and dynamics at the single level. Black-Right-Pointing-Pointer QD, potent tool for single molecule studies of biochemical and biological processes. -- Abstract: Luminescent semiconductor nanocrystals (quantum dots, QD) have unique photo-physical properties: high photostability, brightness and narrow size-tunable fluorescence spectra. Due to their unique properties, QD-based single molecule studies have become increasingly more popular during the last years. However QDs show a strong blinking effect (random and intermittent light emission), which may limit their use in single molecule fluorescence studies. QD blinking has been widely studied and some hypotheses have been done to explain this effect. Here we summarise what is known about the blinking effect in QDs, how this phenomenon may affect single molecule studies and, on the other hand, how the 'on'/'off' states can be exploited in diverse experimental settings. In addition, we present results showing that site-directed binding of QD to cysteine residues of proteins reduces the blinking effect. This option opens a new possibility of using QDs to study protein-protein interactions and dynamics by single molecule fluorescence without modifying the chemical composition of the solution or the QD surface.

Plasmonic tunnel junctions for single-molecule redox chemistry.

Science.gov (United States)

de Nijs, Bart; Benz, Felix; Barrow, Steven J; Sigle, Daniel O; Chikkaraddy, Rohit; Palma, Aniello; Carnegie, Cloudy; Kamp, Marlous; Sundararaman, Ravishankar; Narang, Prineha; Scherman, Oren A; Baumberg, Jeremy J

2017-10-20

Nanoparticles attached just above a flat metallic surface can trap optical fields in the nanoscale gap. This enables local spectroscopy of a few molecules within each coupled plasmonic hotspot, with near thousand-fold enhancement of the incident fields. As a result of non-radiative relaxation pathways, the plasmons in such sub-nanometre cavities generate hot charge carriers, which can catalyse chemical reactions or induce redox processes in molecules located within the plasmonic hotspots. Here, surface-enhanced Raman spectroscopy allows us to track these hot-electron-induced chemical reduction processes in a series of different aromatic molecules. We demonstrate that by increasing the tunnelling barrier height and the dephasing strength, a transition from coherent to hopping electron transport occurs, enabling observation of redox processes in real time at the single-molecule level.

SH2 Domain-Based FRET Biosensor for Measuring BCR-ABL Activity in Living CML Cells.

Science.gov (United States)

Fujioka, Mari; Asano, Yumi; Nakada, Shigeyuki; Ohba, Yusuke

2017-01-01

Fluorescent proteins (FPs) displaying distinct spectra have shed their light on a wide range of biological functions. Moreover, sophisticated biosensors engineered to contain single or multiple FPs, including Förster resonance energy transfer (FRET)-based biosensors, spatiotemporally reveal the molecular mechanisms underlying a variety of pathophysiological processes. However, their usefulness for applied life sciences has yet to be fully explored. Recently, our research group has begun to expand the potential of FPs from basic biological research to the clinic. Here, we describe a method to evaluate the responsiveness of leukemia cells from patients to tyrosine kinase inhibitors using a biosensor based on FP technology and the principle of FRET. Upon phosphorylation of the tyrosine residue of the biosensor, binding of the SH2 domain to phosphotyrosine induces conformational change of the biosensor and brings the donor and acceptor FPs into close proximity. Therefore, kinase activity and response to kinase inhibitors can be monitored by an increase and a decrease in FRET efficiency, respectively. As in basic research, this biosensor resolves hitherto arduous tasks and may provide innovative technological advances in clinical laboratory examinations. State-of-the-art detection devices that enable such innovation are also introduced.

A systematic investigation of differential effects of cell culture substrates on the extent of artifacts in single-molecule tracking.

Directory of Open Access Journals (Sweden)

Laura C Zanetti-Domingues

Full Text Available Single-molecule techniques are being increasingly applied to biomedical investigation, notwithstanding the numerous challenges they pose in terms of signal-to-noise ratio issues. Non-specific binding of probes to glass substrates, in particular, can produce experimental artifacts due to spurious molecules on glass, which can be particularly deleterious in live-cell tracking experiments. In order to resolve the issue of non-specific probe binding to substrates, we performed systematic testing of a range of available surface coatings, using three different proteins, and then extended our assessment to the ability of these coatings to foster cell growth and retain non-adhesive properties. Linear PEG, a passivating agent commonly used both in immobilized-molecule single-molecule techniques and in tissue engineering, is able to both successfully repel non-specific adhesion of fluorescent probes and to foster cell growth when functionalized with appropriate adhesive peptides. Linear PEG treatment results in a significant reduction of tracking artifacts in EGFR tracking with Affibody ligands on a cell line expressing EGFR-eGFP. The findings reported herein could be beneficial to a large number of experimental situations where single-molecule or single-particle precision is required.

Derivation of Elastic Stress Concentration Factor Equations for Debris Fretting Flaws in Pressure Tubes of Pressurized Heavy Water Reactors

International Nuclear Information System (INIS)

Kim, Jong Sung; Oh, Young Jin

2014-01-01

If volumetric flaws such as bearing pad fretting flaws and debris fretting flaws are detected in the pressure tubes of pressurized heavy water reactors during in-service inspection, the initiation of fatigue cracks and delayed hydrogen cracking from the detected volumetric flaws shall be assessed by using elastic stress concentration factors in accordance with CSA N285.8-05. The CSA N285.8-05 presents only an approximate formula based on linear elastic fracture mechanics for the debris fretting flaw. In this study, an engineering formula considering the geometric characteristics of the debris fretting flaw in detail was derived using two-dimensional finite element analysis and Kinectrics, Inc.'s engineering procedure with slight modifications. Comparing the application results obtained using the derived formula with the three-dimensional finite element analysis results, it is found that the results obtained using the derived formula agree well with the results of the finite element analysis

Viruses and Tetraspanins: Lessons from Single Molecule Approaches

Science.gov (United States)

Dahmane, Selma; Rubinstein, Eric; Milhiet, Pierre-Emmanuel

2014-01-01

Tetraspanins are four-span membrane proteins that are widely distributed in multi-cellular organisms and involved in several infectious diseases. They have the unique property to form a network of protein-protein interaction within the plasma membrane, due to the lateral associations with one another and with other membrane proteins. Tracking tetraspanins at the single molecule level using fluorescence microscopy has revealed the membrane behavior of the tetraspanins CD9 and CD81 in epithelial cell lines, providing a first dynamic view of this network. Single molecule tracking highlighted that these 2 proteins can freely diffuse within the plasma membrane but can also be trapped, permanently or transiently, in tetraspanin-enriched areas. More recently, a similar strategy has been used to investigate tetraspanin membrane behavior in the context of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) infection. In this review we summarize the main results emphasizing the relationship in terms of membrane partitioning between tetraspanins, some of their partners such as Claudin-1 and EWI-2, and viral proteins during infection. These results will be analyzed in the context of other membrane microdomains, stressing the difference between raft and tetraspanin-enriched microdomains, but also in comparison with virus diffusion at the cell surface. New advanced single molecule techniques that could help to further explore tetraspanin assemblies will be also discussed. PMID:24800676

Transport mirages in single-molecule devices

Science.gov (United States)

Gaudenzi, R.; Misiorny, M.; Burzurí, E.; Wegewijs, M. R.; van der Zant, H. S. J.

2017-03-01

Molecular systems can exhibit a complex, chemically tailorable inner structure which allows for targeting of specific mechanical, electronic, and optical properties. At the single-molecule level, two major complementary ways to explore these properties are molecular quantum-dot structures and scanning probes. This article outlines comprehensive principles of electron-transport spectroscopy relevant to both these approaches and presents a new, high-resolution experiment on a high-spin single-molecule junction exemplifying these principles. Such spectroscopy plays a key role in further advancing our understanding of molecular and atomic systems, in particular, the relaxation of their spin. In this joint experimental and theoretical analysis, particular focus is put on the crossover between the resonant regime [single-electron tunneling] and the off-resonant regime [inelastic electron (co)tunneling spectroscopy (IETS)]. We show that the interplay of these two processes leads to unexpected mirages of resonances not captured by either of the two pictures alone. Although this turns out to be important in a large fraction of the possible regimes of level positions and bias voltages, it has been given little attention in molecular transport studies. Combined with nonequilibrium IETS—four-electron pump-probe excitations—these mirages provide crucial information on the relaxation of spin excitations. Our encompassing physical picture is supported by a master-equation approach that goes beyond weak coupling. The present work encourages the development of a broader connection between the fields of molecular quantum-dot and scanning probe spectroscopy.

Plasmonics and single-molecule detection in evaporated silver-island films

Energy Technology Data Exchange (ETDEWEB)

Moula, G.; Aroca, R.F. [Materials and Surface Science Group, University of Windsor, Ontario (Canada); Rodriguez-Oliveros, R.; Sanchez-Gil, J.A. [Instituto de Estructura de la Materia, Consejo Superior de Investigaciones Cientificas, Serrano 121, 28006 Madrid (Spain); Albella, P. [Centro de Fisica de Materiales (CSIC-UPV/EHU) and Donostia International Physics Center (DIPC), 20018 Donostia, San Sebastian (Spain)

2012-11-15

The plasmonic origin of surface-enhanced Raman scattering (SERS) leads to the concept of hotspots and plasmon coupling that can be realized in the interstitial regions, or on specially engineered, silver and gold nanostructures. It is also possible to achieve spatial locations of high local field or hotspots on silver-island films (SIF) allowing single-molecule detection (SMD). When a single monomolecular layer coating the SIFs contains dye molecules dispersed in it, single-molecule impurities, (with an average of one hundred dye molecules in 1 {mu}m{sup 2}, which is the field of view of the micro-Raman system), SMD is observed as a rare statistical event. Here, the SMD results for silver-island films are presented, with the same nominal mass thickness, but differing in the localized surface plasmon resonance that is a function of the temperature of substrate during deposition. A blue-shifted plasmon can be seen as a decrease in plasmon coupling for deposition at higher temperature. A simple two-particle model for localized plasmon resonance coupling calculations, including the shape and substrate effects seems to explain the trend of observations. (Copyright copyright 2012 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

Investigation on ionic states of 1,2-Dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC) using organic laser dyes: A FRET study

Energy Technology Data Exchange (ETDEWEB)

Roy, Arpan Datta; Saha, Jaba; Dey, D.; Bhattacharjee, D.; Hussain, Syed Arshad, E-mail: sa_h153@hotmail.com

2017-05-15

Fluorescence Resonance Energy Transfer (FRET) between two organic dyes Fluorescein and Rhodamine 6G were successfully investigated in aqueous solution in presence and absence of 1,2-Dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC) at different pH. Spectroscopic studies suggest that both the dyes were present mainly as monomer in solution. FRET occurred from Fluorescein to Rhodamine 6G in solutions. Energy transfer efficiency increases in presence of DPPC and the maximum efficiency was 59.3% when the concentration of DPPC was 1.4×10{sup −4} M at ambient condition. pH plays a crucial role in this investigation as the energy transfer efficiency was found to change in presence of DPPC at different pH. It has been demonstrated that with proper calibration it is possible to use the present system under investigation to realize various ionic states of DPPC by observing the change in FRET efficiency between these two dyes. - Graphical abstract: Electrostatic interaction between anionic Flu and cationic R6G molecules in presence and absence of DPPC at different pH. Here pH of DPPC was changed, not the pH of individual dyes.

Fretting fatigue behaviour of Ni-free high-nitrogen stainless steel in a simulated body fluid

Directory of Open Access Journals (Sweden)

Norio Maruyama, Sachiko Hiromoto, Eiji Akiyama and Morihiko Nakamura

2013-01-01

Full Text Available Fretting fatigue behaviour of Ni-free high-nitrogen steel (HNS with a yield strength of about 800 MPa, which was prepared by nitrogen gas pressurized electroslag remelting, was studied in air and in phosphate-buffered saline (PBS(-. For comparison, fretting fatigue behaviour of cold-rolled SUS316L steel (SUS316L(CR with similar yield strength was examined. The plain fatigue limit of HNS was slightly lower than that of SUS316L(CR although the former had a higher tensile strength than the latter. The fretting fatigue limit of HNS was higher than that of SUS316L(CR both in air and in PBS(-. A decrease in fatigue limit of HNS by fretting was significantly smaller than that of SUS316L(CR in both environments, indicating that HNS has better fretting fatigue resistance than SUS316L(CR. The decrease in fatigue limit by fretting is discussed taking into account the effect of friction stress due to fretting and the additional influences of wear, tribocorrosion and plastic deformation in the fretted area.

Experimental demonstration of a single-molecule electric motor.

Science.gov (United States)

Tierney, Heather L; Murphy, Colin J; Jewell, April D; Baber, Ashleigh E; Iski, Erin V; Khodaverdian, Harout Y; McGuire, Allister F; Klebanov, Nikolai; Sykes, E Charles H

2011-09-04

For molecules to be used as components in molecular machines, methods that couple individual molecules to external energy sources and that selectively excite motion in a given direction are required. Significant progress has been made in the construction of molecular motors powered by light and by chemical reactions, but electrically driven motors have not yet been built, despite several theoretical proposals for such motors. Here we report that a butyl methyl sulphide molecule adsorbed on a copper surface can be operated as a single-molecule electric motor. Electrons from a scanning tunnelling microscope are used to drive the directional motion of the molecule in a two-terminal setup. Moreover, the temperature and electron flux can be adjusted to allow each rotational event to be monitored at the molecular scale in real time. The direction and rate of the rotation are related to the chiralities of both the molecule and the tip of the microscope (which serves as the electrode), illustrating the importance of the symmetry of the metal contacts in atomic-scale electrical devices.

Methodological considerations for global analysis of cellular FLIM/FRET measurements

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