Single-molecule spectroscopic methods.

PubMed

Haustein, Elke; Schwille, Petra

2004-10-01

Being praised for the mere fact of enabling the detection of individual fluorophores a dozen years ago, single-molecule techniques nowadays represent standard methods for the elucidation of the structural rearrangements of biologically relevant macromolecules. Single-molecule-sensitive techniques, such as fluorescence correlation spectroscopy, allow real-time access to a multitude of molecular parameters (e.g. diffusion coefficients, concentration and molecular interactions). As a result of various recent advances, this technique shows promise even for intracellular applications. Fluorescence imaging can reveal the spatial localization of fluorophores on nanometer length scales, whereas fluorescence resonance energy transfer supports a wide range of different applications, including real-time monitoring of conformational rearrangements (as in protein folding). Still in their infancy, single-molecule spectroscopic methods thus provide unprecedented insights into basic molecular mechanisms. Copyright 2004 Elsevier Ltd.

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

PubMed

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.

Single Molecule Enzymology via Nanoelectronic Circuits

NASA Astrophysics Data System (ADS)

Collins, Philip

Traditional single-molecule techniques rely on fluorescence or force transduction to monitor conformational changes and biochemical activity. Recent demonstrations of single-molecule monitoring with electronic transistors are poised to add to the single-molecule research toolkit. The transistor-based technique is sensitive to the motion of single charged side chain residues and can transduce those motions with microsecond resolution, opening the doors to single-molecule enzymology with unprecedented resolution. Furthermore, the solid-state platform provides opportunities for parallelization in arrays and long-duration monitoring of one molecule's activity or processivity, all without the limitations caused by photo-oxidation or mutagenic fluorophore incorporation. This presentation will review some of these advantages and their particular application to DNA polymerase I processing single-stranded DNA templates. This research was supported financially by the NIH NCI (R01 CA133592-01), the NIH NIGMS (1R01GM106957-01) and the NSF (DMR-1104629 and ECCS-1231910).

Single-molecule electrocatalysis by single-walled carbon nanotubes.

PubMed

Xu, Weilin; Shen, Hao; Kim, Yoon Ji; Zhou, Xiaochun; Liu, Guokun; Park, Jiwoong; Chen, Peng

2009-12-01

We report a single-molecule fluorescence study of electrocatalysis by single-walled carbon nanotubes (SWNTs) at single-reaction resolution. Applying super-resolution optical imaging, we find that the electrocatalysis occurs at discrete, nanometer-dimension sites on SWNTs. Single-molecule kinetic analysis leads to an electrocatalytic mechanism, allowing quantification of the reactivity and heterogeneity of individual reactive sites. Combined with conductivity measurements, this approach will be powerful to interrogate how the electronic structure of SWNTs affects the electrocatalytic interfacial charge transfer, a process fundamental to photoelectrochemical cells.

Extracting Models in Single Molecule Experiments

NASA Astrophysics Data System (ADS)

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.

Quantitative Aspects of Single Molecule Microscopy

PubMed Central

Ober, Raimund J.; Tahmasbi, Amir; Ram, Sripad; Lin, Zhiping; Ward, E. Sally

2015-01-01

Single molecule microscopy is a relatively new optical microscopy technique that allows the detection of individual molecules such as proteins in a cellular context. This technique has generated significant interest among biologists, biophysicists and biochemists, as it holds the promise to provide novel insights into subcellular processes and structures that otherwise cannot be gained through traditional experimental approaches. Single molecule experiments place stringent demands on experimental and algorithmic tools due to the low signal levels and the presence of significant extraneous noise sources. Consequently, this has necessitated the use of advanced statistical signal and image processing techniques for the design and analysis of single molecule experiments. In this tutorial paper, we provide an overview of single molecule microscopy from early works to current applications and challenges. Specific emphasis will be on the quantitative aspects of this imaging modality, in particular single molecule localization and resolvability, which will be discussed from an information theoretic perspective. We review the stochastic framework for image formation, different types of estimation techniques and expressions for the Fisher information matrix. We also discuss several open problems in the field that demand highly non-trivial signal processing algorithms. PMID:26167102

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

PubMed

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.

Single-molecule conductance studies of photo-active and photochromic molecules

NASA Astrophysics Data System (ADS)

Tam, E. S.; Parks, J. J.; Santiago-Berrios, M. B.; Zhong, Y.-W.; Abruna, H. D.; Ralph, D. C.

2010-03-01

We perform statistical measurements of single molecule conductance in repeatedly-formed metal-molecule-metal junctions at room temperature. Our results on diaminoalkanes are consistent with those reported by the Venkataraman group. We focus on photo-active and photochromic molecules, including a series of transition-metal complexes with different metal centers and endgroups. We compare the trend in conductance across the family of complexes with that expected from electrochemical measurements. We will also report initial results on the voltage dependence of single-molecule conductances and the effects of optical excitations.

A Single-Level Tunnel Model to Account for Electrical Transport through Single Molecule- and Self-Assembled Monolayer-based Junctions

PubMed Central

Garrigues, Alvar R.; Yuan, Li; Wang, Lejia; Mucciolo, Eduardo R.; Thompon, Damien; del Barco, Enrique; Nijhuis, Christian A.

2016-01-01

We present a theoretical analysis aimed at understanding electrical conduction in molecular tunnel junctions. We focus on discussing the validity of coherent versus incoherent theoretical formulations for single-level tunneling to explain experimental results obtained under a wide range of experimental conditions, including measurements in individual molecules connecting the leads of electromigrated single-electron transistors and junctions of self-assembled monolayers (SAM) of molecules sandwiched between two macroscopic contacts. We show that the restriction of transport through a single level in solid state junctions (no solvent) makes coherent and incoherent tunneling formalisms indistinguishable when only one level participates in transport. Similar to Marcus relaxation processes in wet electrochemistry, the thermal broadening of the Fermi distribution describing the electronic occupation energies in the electrodes accounts for the exponential dependence of the tunneling current on temperature. We demonstrate that a single-level tunnel model satisfactorily explains experimental results obtained in three different molecular junctions (both single-molecule and SAM-based) formed by ferrocene-based molecules. Among other things, we use the model to map the electrostatic potential profile in EGaIn-based SAM junctions in which the ferrocene unit is placed at different positions within the molecule, and we find that electrical screening gives rise to a strongly non-linear profile across the junction. PMID:27216489

Single-Molecule Studies of Actin Assembly and Disassembly Factors

PubMed Central

Smith, Benjamin A.; Gelles, Jeff; Goode, Bruce L.

2014-01-01

The actin cytoskeleton is very dynamic and highly regulated by multiple associated proteins in vivo. Understanding how this system of proteins functions in the processes of actin network assembly and disassembly requires methods to dissect the mechanisms of activity of individual factors and of multiple factors acting in concert. The advent of single-filament and single-molecule fluorescence imaging methods has provided a powerful new approach to discovering actin-regulatory activities and obtaining direct, quantitative insights into the pathways of molecular interactions that regulate actin network architecture and dynamics. Here we describe techniques for acquisition and analysis of single-molecule data, applied to the novel challenges of studying the filament assembly and disassembly activities of actin-associated proteins in vitro. We discuss the advantages of single-molecule analysis in directly visualizing the order of molecular events, measuring the kinetic rates of filament binding and dissociation, and studying the coordination among multiple factors. The methods described here complement traditional biochemical approaches in elucidating actin-regulatory mechanisms in reconstituted filamentous networks. PMID:24630103

Single-Molecule Spectroscopy and Imaging Over the Decades

PubMed Central

Moerner, W. E.; Shechtman, Yoav; Wang, Quan

2016-01-01

As of 2015, it has been 26 years since the first optical detection and spectroscopy of single molecules in condensed matter. This area of science has expanded far beyond the early low temperature studies in crystals to include single molecules in cells, polymers, and in solution. The early steps relied upon high-resolution spectroscopy of inhomogeneously broadened optical absorption profiles of molecular impurities in solids at low temperatures. Spectral fine structure arising directly from the position-dependent fluctuations of the number of molecules in resonance led to the attainment of the single-molecule limit in 1989 using frequency-modulation laser spectroscopy. In the early 1990's, a variety of fascinating physical effects were observed for individual molecules, including imaging of the light from single molecules as well as observations of spectral diffusion, optical switching and the ability to select different single molecules in the same focal volume simply by tuning the pumping laser frequency. In the room temperature regime, researchers showed that bursts of light from single molecules could be detected in solution, leading to imaging and microscopy by a variety of methods. Studies of single copies of the green fluorescent protein also uncovered surprises, especially the blinking and photoinduced recovery of emitters, which stimulated further development of photoswitchable fluorescent protein labels. All of these early steps provided important fundamentals underpinning the development of super-resolution microscopy based on single-molecule localization and active control of emitting concentration. Current thrust areas include extensions to three-dimensional imaging with high precision, orientational analysis of single molecules, and direct measurements of photodynamics and transport properties for single molecules trapped in solution by suppression of Brownian motion. Without question, a huge variety of studies of single molecules performed by many

Digital microfluidics-enabled single-molecule detection by printing and sealing single magnetic beads in femtoliter droplets.

PubMed

Witters, Daan; Knez, Karel; Ceyssens, Frederik; Puers, Robert; Lammertyn, Jeroen

2013-06-07

Digital microfluidics is introduced as a novel platform with unique advantages for performing single-molecule detection. We demonstrate how superparamagnetic beads, used for capturing single protein molecules, can be printed with unprecedentedly high loading efficiency and single bead resolution on an electrowetting-on-dielectric-based digital microfluidic chip by micropatterning the Teflon-AF surface of the device. By transporting droplets containing suspended superparamagnetic beads over a hydrophilic-in-hydrophobic micropatterned Teflon-AF surface, single beads are trapped inside the hydrophilic microwells due to their selective wettability and tailored dimensions. Digital microfluidics presents the following advantages for printing and sealing magnetic beads for single-molecule detection: (i) droplets containing suspended beads can be transported back and forth over the array of hydrophilic microwells to obtain high loading efficiencies of microwells with single beads, (ii) the use of hydrophilic-in-hydrophobic patterns permits the use of a magnet to speed up the bead transfer process to the wells, while the receding droplet meniscus removes excess beads off the chip surface and thereby shortens the bead patterning time, and (iii) reagents can be transported over the printed beads multiple times, while capillary forces and a magnet hold the printed beads in place. High loading efficiencies (98% with a CV of 0.9%) of single beads in microwells were obtained by transporting droplets of suspended beads over the array 10 times in less than 1 min, which is much higher than previously reported methods (40-60%), while the total surface area needed for performing single-molecule detection can be decreased. The performance of the device was demonstrated by fluorescent detection of the presence of the biotinylated enzyme β-galactosidase on streptavidin-coated beads with a linear dynamic range of 4 orders of magnitude ranging from 10 aM to 90 fM.

Single-Molecule Interfacial Electron Transfer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ho, Wilson

Interfacial electron transfer (ET) plays an important role in many chemical and biological processes. Specifically, interfacial ET in TiO 2-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 andmore » TiO 2 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, solar

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

PubMed

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.

Single-Cell and Single-Molecule Analysis of Gene Expression Regulation.

PubMed

Vera, Maria; Biswas, Jeetayu; Senecal, Adrien; Singer, Robert H; Park, Hye Yoon

2016-11-23

Recent advancements in single-cell and single-molecule imaging technologies have resolved biological processes in time and space that are fundamental to understanding the regulation of gene expression. Observations of single-molecule events in their cellular context have revealed highly dynamic aspects of transcriptional and post-transcriptional control in eukaryotic cells. This approach can relate transcription with mRNA abundance and lifetimes. Another key aspect of single-cell analysis is the cell-to-cell variability among populations of cells. Definition of heterogeneity has revealed stochastic processes, determined characteristics of under-represented cell types or transitional states, and integrated cellular behaviors in the context of multicellular organisms. In this review, we discuss novel aspects of gene expression of eukaryotic cells and multicellular organisms revealed by the latest advances in single-cell and single-molecule imaging technology.

Electrostatic placement of single ferritin molecules

NASA Astrophysics Data System (ADS)

Kumagai, Shinya; Yoshii, Shigeo; Yamada, Kiyohito; Matsukawa, Nozomu; Fujiwara, Isamu; Iwahori, Kenji; Yamashita, Ichiro

2006-04-01

We electrostatically placed a single ferritin molecule on a nanometric 3-aminopropyltriethoxysilane (APTES) pattern that was on an oxidized Si substrate. The numerical analysis of the total interaction free energy for ferritin predicted that a quadrilateral array of 15nm diameter APTES nanodisks placed at intervals of 100nm would accommodate a single molecule of ferritin in each disk under a Debye length of 14nm. The experiments we conducted conformed to theoretical predictions and we successfully placed a single ferritin molecule on each ATPES disk without ferritin adsorbing on the SiO2 substrate surface.

Molecular vibrations in metal-single-molecule-metal junctions

NASA Astrophysics Data System (ADS)

Yokota, Kazumichi; Taniguchi, Masateru; Kawai, Tomoji

2010-03-01

Molecular vibrations in a metal-single-molecule-metal junction were studied based on density functional theory using a single benzenedithiolate molecule connected between gold clusters. We found that the difference in vibrational energy between an isolated benzenedithiol and the single-molecule junction is less than 3% in the energy range above 540 cm -1, where sulfur atoms contribute little to molecular vibrations. The finding implies that we can predict the peak energy in the inelastic electron tunneling spectrum of the single-molecule junction in the high energy range by vibrational analyses of isolated molecules.

Optical-nanofiber-based interface for single molecules

NASA Astrophysics Data System (ADS)

Skoff, Sarah M.; Papencordt, David; Schauffert, Hardy; Bayer, Bernhard C.; Rauschenbeutel, Arno

2018-04-01

Optical interfaces for quantum emitters are a prerequisite for implementing quantum networks. Here, we couple single molecules to the guided modes of an optical nanofiber. The molecules are embedded within a crystal that provides photostability and, due to the inhomogeneous broadening, a means to spectrally address single molecules. Single molecules are excited and detected solely via the nanofiber interface without the requirement of additional optical access. In this way, we realize a fully fiber-integrated system that is scalable and may become a versatile constituent for quantum hybrid systems.

Single-Molecule Probing of Adsorption and Diffusion on Silica Surfaces

NASA Astrophysics Data System (ADS)

Wirth, Mary J.; Legg, Michael A.

2007-05-01

Single-molecule spectroscopy has emerged as a valuable tool in probing kinetics and dynamic equilibria in adsorption because advances in instrumentation and technology have enabled researchers to obtain high signal-to-noise ratios for common dyes at room temperature. Single-molecule spectroscopy was applied to the study of an important problem in chromatography: peak broadening and asymmetry in the chromatograms of pharmaceuticals, peptides, and proteins. Using DiI, a cationic dye that exhibits the same problematic chromatographic behavior, investigators showed that the adsorption sites that cause chromatographic problems are located at defects on the silica crystal surface.

Single Molecule Raman Spectroscopy Under High Pressure

NASA Astrophysics Data System (ADS)

Fu, Yuanxi; Dlott, Dana

2014-06-01

Pressure effects on surface-enhanced Raman scattering spectra of Rhdoamine 6G adsorbed on silver nanoparticle surfaces was studied using a confocal Raman microscope. Colloidal silver nanoparticles were treated with Rhodamine 6G (R6G) and its isotopically substituted partner, R6G-d4. Mixed isotopomers let us identify single-molecule spectra, since multiple-molecule spectra would show vibrational transitions from both species. The nanoparticles were embedded into a poly vinyl alcohol film, and loaded into a diamond anvil cell for the high-pressure Raman scattering measurement. Argon was the pressure medium. Ambient pressure Raman scattering spectra showed few single-molecule spectra. At moderately high pressure ( 1GPa), a surprising effect was observed. The number of sites with observable spectra decreased dramatically, and most of the spectra that could be observed were due to single molecules. The effects of high pressure suppressed the multiple-molecule Raman sites, leaving only the single-molecule sites to be observed.

The symmetry of single-molecule conduction.

PubMed

Solomon, Gemma C; Gagliardi, Alessio; Pecchia, Alessandro; Frauenheim, Thomas; Di Carlo, Aldo; Reimers, Jeffrey R; Hush, Noel S

2006-11-14

We introduce the conductance point group which defines the symmetry of single-molecule conduction within the nonequilibrium Green's function formalism. It is shown, either rigorously or to within a very good approximation, to correspond to a molecular-conductance point group defined purely in terms of the properties of the conducting molecule. This enables single-molecule conductivity to be described in terms of key qualitative chemical descriptors that are independent of the nature of the molecule-conductor interfaces. We apply this to demonstrate how symmetry controls the conduction through 1,4-benzenedithiol chemisorbed to gold electrodes as an example system, listing also the molecular-conductance point groups for a range of molecules commonly used in molecular electronics research.

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

PubMed

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

Single-molecule detection: applications to ultrasensitive biochemical analysis

NASA Astrophysics Data System (ADS)

Castro, Alonso; Shera, E. Brooks

1995-06-01

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

Direct single-molecule dynamic detection of chemical reactions.

PubMed

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.

Direct single-molecule dynamic detection of chemical reactions

PubMed Central

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

2018-01-01

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. PMID:29487914

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?

NASA Astrophysics Data System (ADS)

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.

PubMed

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.

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

NASA Astrophysics Data System (ADS)

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

NASA Astrophysics Data System (ADS)

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.

Spectrally resolved single-molecule electrometry

NASA Astrophysics Data System (ADS)

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.

Inferring diffusion in single live cells at the single-molecule level

PubMed Central

Robson, Alex; Burrage, Kevin; Leake, Mark C.

2013-01-01

The movement of molecules inside living cells is a fundamental feature of biological processes. The ability to both observe and analyse the details of molecular diffusion in vivo at the single-molecule and single-cell level can add significant insight into understanding molecular architectures of diffusing molecules and the nanoscale environment in which the molecules diffuse. The tool of choice for monitoring dynamic molecular localization in live cells is fluorescence microscopy, especially so combining total internal reflection fluorescence with the use of fluorescent protein (FP) reporters in offering exceptional imaging contrast for dynamic processes in the cell membrane under relatively physiological conditions compared with competing single-molecule techniques. There exist several different complex modes of diffusion, and discriminating these from each other is challenging at the molecular level owing to underlying stochastic behaviour. Analysis is traditionally performed using mean square displacements of tracked particles; however, this generally requires more data points than is typical for single FP tracks owing to photophysical instability. Presented here is a novel approach allowing robust Bayesian ranking of diffusion processes to discriminate multiple complex modes probabilistically. It is a computational approach that biologists can use to understand single-molecule features in live cells. PMID:23267182

Indistinguishable near-infrared single photons from an individual organic molecule

DOE Office of Scientific and Technical Information (OSTI.GOV)

Trebbia, Jean-Baptiste; Tamarat, Philippe; Lounis, Brahim

2010-12-15

By using the zero-phonon line emission of an individual organic molecule, we realized a source of indistinguishable single photons in the near infrared. A Hong-Ou-Mandel interference experiment is performed and a two-photon coalescence probability higher than 50% at 2 K is obtained. The contribution of the temperature-dependent dephasing processes to the two-photon interference contrast is studied. We show that the molecule delivers nearly ideal indistinguishable single photons at the lowest temperatures when the dephasing is nearly lifetime limited. This source is used to generate postselected polarization-entangled photon pairs as a test bench for applications in quantum information.

Single molecule detection, thermal fluctuation and life

PubMed Central

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

Surface Passivation for Single-molecule Protein Studies

PubMed Central

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

Multi-Scale Modeling to Improve Single-Molecule, Single-Cell Experiments

NASA Astrophysics Data System (ADS)

Munsky, Brian; Shepherd, Douglas

2014-03-01

Single-cell, single-molecule experiments are producing an unprecedented amount of data to capture the dynamics of biological systems. When integrated with computational models, observations of spatial, temporal and stochastic fluctuations can yield powerful quantitative insight. We concentrate on experiments that localize and count individual molecules of mRNA. These high precision experiments have large imaging and computational processing costs, and we explore how improved computational analyses can dramatically reduce overall data requirements. In particular, we show how analyses of spatial, temporal and stochastic fluctuations can significantly enhance parameter estimation results for small, noisy data sets. We also show how full probability distribution analyses can constrain parameters with far less data than bulk analyses or statistical moment closures. Finally, we discuss how a systematic modeling progression from simple to more complex analyses can reduce total computational costs by orders of magnitude. We illustrate our approach using single-molecule, spatial mRNA measurements of Interleukin 1-alpha mRNA induction in human THP1 cells following stimulation. Our approach could improve the effectiveness of single-molecule gene regulation analyses for many other process.

Quantitative analysis of single-molecule superresolution images

PubMed Central

Coltharp, Carla; Yang, Xinxing; Xiao, Jie

2014-01-01

This review highlights the quantitative capabilities of single-molecule localization-based superresolution imaging methods. In addition to revealing fine structural details, the molecule coordinate lists generated by these methods provide the critical ability to quantify the number, clustering, and colocalization of molecules with 10 – 50 nm resolution. Here we describe typical workflows and precautions for quantitative analysis of single-molecule superresolution images. These guidelines include potential pitfalls and essential control experiments, allowing critical assessment and interpretation of superresolution images. PMID:25179006

Single-Molecule Plasmon Sensing: Current Status and Future Prospects

PubMed Central

2017-01-01

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. PMID:28762723

Single molecule views of Nature's nano-machines

NASA Astrophysics Data System (ADS)

Ha, Taekjip

2006-03-01

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

Single molecule techniques in DNA repair: A primer

PubMed Central

Hughes, Craig D.; Simons, Michelle; Mackenzie, Cassidy E.; Van Houten, Bennett; Kad, Neil M.

2016-01-01

A powerful new approach has become much more widespread and offers insights into aspects of DNA repair unattainable with billions of molecules. Single molecule techniques can be used to image, manipulate or characterize the action of a single repair protein on a single strand of DNA. This allows search mechanisms to be probed, and the effects of force to be understood. These physical aspects can dominate a biochemical reaction, where at the ensemble level their nuances are obscured. In this paper we discuss some of the many technical advances that permit study at the single molecule level. We focus on DNA repair to which these techniques are actively being applied. DNA repair is also a process that encompasses so much of what single molecule studies benefit – searching for targets, complex formation, sequential biochemical reactions and substrate hand-off to name just a few. We discuss how single molecule biophysics is poised to transform our understanding of biological systems, in particular DNA repair. PMID:24819596

Reversible Aptamer-Au Plasmon Rulers for Secreted Single Molecules

DOE PAGES

Lee, Somin Eunice; Chen, Qian; Bhat, Ramray; ...

2015-06-03

Plasmon rulers, consisting of pairs of gold nanoparticles, allow single-molecule analysis without photobleaching or blinking; however, current plasmon rulers are irreversible, restricting detection to only single events. Here, we present a reversible plasmon ruler, comprised of coupled gold nanoparticles linked by a single aptamer, capable of binding individual secreted molecules with high specificity. We show that the binding of target secreted molecules to the reversible plasmon ruler is characterized by single-molecule sensitivity, high specificity, and reversibility. Lastly, such reversible plasmon rulers should enable dynamic and adaptive live-cell measurement of secreted single molecules in their local microenvironment.

Investigating single molecule adhesion by atomic force spectroscopy.

PubMed

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.

Investigating Single Molecule Adhesion by Atomic Force Spectroscopy

PubMed Central

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

2015-01-01

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. PMID:25867282

Computer systems for annotation of single molecule fragments

DOEpatents

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.

Single Molecule and Single Cell Epigenomics

PubMed Central

Hyun, Byung-Ryool; McElwee, John L.; Soloway, Paul D.

2014-01-01

Dynamically regulated changes in chromatin states are vital for normal development and can produce disease when they go awry. Accordingly, much effort has been devoted to characterizing these states under normal and pathological conditions. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is the most widely used method to characterize where in the genome transcription factors, modified histones, modified nucleotides and chromatin binding proteins are found; bisulfite sequencing (BS-seq) and its variants are commonly used to characterize the locations of DNA modifications. Though very powerful, these methods are not without limitations. Notably, they are best at characterizing one chromatin feature at a time, yet chromatin features arise and function in combination. Investigators commonly superimpose separate ChIP-seq or BS-seq datasets, and then infer where chromatin features are found together. While these inferences might be correct, they can be misleading when the chromatin source has distinct cell types, or when a given cell type exhibits any cell to cell variation in chromatin state. These ambiguities can be eliminated by robust methods that directly characterize the existence and genomic locations of combinations of chromatin features in very small inputs of cells or ideally, single cells. Here we review single molecule epigenomic methods under development to overcome these limitations, the technical challenges associated with single molecule methods and their potential application to single cells. PMID:25204781

Probing molecular choreography through single-molecule biochemistry.

PubMed

van Oijen, Antoine M; Dixon, Nicholas E

2015-12-01

Single-molecule approaches are having a dramatic impact on views of how proteins work. The ability to observe molecular properties at the single-molecule level allows characterization of subpopulations and acquisition of detailed kinetic information that would otherwise be hidden in the averaging over an ensemble of molecules. In this Perspective, we discuss how such approaches have successfully been applied to in vitro-reconstituted systems of increasing complexity.

Transition paths in single-molecule force spectroscopy

NASA Astrophysics Data System (ADS)

Cossio, Pilar; Hummer, Gerhard; Szabo, Attila

2018-03-01

In a typical single-molecule force spectroscopy experiment, the ends of the molecule of interest are connected by long polymer linkers to a pair of mesoscopic beads trapped in the focus of two laser beams. At constant force load, the total extension, i.e., the end-to-end distance of the molecule plus linkers, is measured as a function of time. In the simplest systems, the measured extension fluctuates about two values characteristic of folded and unfolded states, with occasional transitions between them. We have recently shown that molecular (un)folding rates can be recovered from such trajectories, with a small linker correction, as long as the characteristic time of the bead fluctuations is shorter than the residence time in the unfolded (folded) state. Here, we show that accurate measurements of the molecular transition path times require an even faster apparatus response. Transition paths, the trajectory segments in which the molecule (un)folds, are properly resolved only if the beads fluctuate more rapidly than the end-to-end distance of the molecule. Therefore, over a wide regime, the measured rates may be meaningful but not the transition path times. Analytic expressions for the measured mean transition path times are obtained for systems diffusing anisotropically on a two-dimensional free energy surface. The transition path times depend on the properties both of the molecule and of the pulling device.

Organization of Single Molecule Magnets on Surfaces

NASA Astrophysics Data System (ADS)

Sessoli, Roberta

2006-03-01

The field of magnetic molecular clusters showing slow relaxation of the magnetization has attracted a great interest for the spectacular quantum effects in the dynamics of the magnetization that range from resonant quantum tunneling to topological interferences. Recently these systems, known as Single Molecule Magnets (SMMs), have also been proposed as model systems for the investigation of flame propagation in flammable substances. A renewed interest in SMMs also comes from the possibility to exploit their rich and complex magnetic behavior in nano-spintronics. However, at the crystalline state these molecular materials are substantially insulating. They can however exhibit significant transport properties if the conduction occurs through one molecule connected to two metal electrodes, or through a tunneling mechanism when the SMM is grafted on a conducting surface, as occurs in scanning tunnel microscopy experiments. Molecular compounds can be organized on surfaces thanks to the self assembly technique that exploits the strong affinity of some groups for the surface, e.g. thiols for gold surfaces. However the deposition of large molecules mainly comprising relatively weak coordinative bonds is far from trivial. Several different approaches have started to be investigated. We will briefly review here the strategies developed in a collaboration between the Universities of Florence and Modena. Well isolated molecules on Au(111) surfaces have been obtained with sub-monolayer coverage and different spacers. Organization on a large scale of micrometric structures has been obtained thanks to micro-contact printing. The magnetic properties of the grafted molecules have been investigated through magneto-optical techniques and the results show a significant change in the magnetization dynamics whose origin is still object of investigations.

Spectroscopic characterization of Venus at the single molecule level.

PubMed

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

DNA curtains for high-throughput single-molecule optical imaging.

PubMed

Greene, Eric C; Wind, Shalom; Fazio, Teresa; Gorman, Jason; Visnapuu, Mari-Liis

2010-01-01

Single-molecule approaches provide a valuable tool in the arsenal of the modern biologist, and new discoveries continue to be made possible through the use of these state-of-the-art technologies. However, it can be inherently difficult to obtain statistically relevant data from experimental approaches specifically designed to probe individual reactions. This problem is compounded with more complex biochemical reactions, heterogeneous systems, and/or reactions requiring the use of long DNA substrates. Here we give an overview of a technology developed in our laboratory, which relies upon simple micro- or nanofabricated structures in combination with "bio-friendly" lipid bilayers, to align thousands of long DNA molecules into defined patterns on the surface of a microfluidic sample chamber. We call these "DNA curtains," and we have developed several different versions varying in complexity and DNA substrate configuration, which are designed to meet different experimental needs. This novel approach to single-molecule imaging provides a powerful experimental platform that offers the potential for concurrent observation of hundreds or even thousands of protein-DNA interactions in real time. Copyright 2010 Elsevier Inc. All rights reserved.

Cucurbituril mediated single molecule detection and identification via recognition tunneling.

PubMed

Xiao, Bohuai; Liang, Feng; Liu, Simin; Im, JongOne; Li, Yunchuan; Liu, Jing; Zhang, Bintian; Zhou, Jianghao; He, Jin; Chang, Shuai

2018-06-08

Recognition tunneling (RT) is an emerging technique for investigating single molecules in a tunnel junction. We have previously demonstrated its capability of single molecule detection and identification, as well as probing the dynamics of intermolecular bonding at the single molecule level. Here by introducing cucurbituril as a new class of recognition molecule, we demonstrate a powerful platform for electronically investigating the host-guest chemistry at single molecule level. In this report, we first investigated the single molecule electrical properties of cucurbituril in a tunnel junction. Then we studied two model guest molecules, aminoferrocene and amantadine, which were encapsulated by cucurbituril. Small differences in conductance and lifetime can be recognized between the host-guest complexes with the inclusion of different guest molecules. By using a machine learning algorithm to classify the RT signals in a hyper dimensional space, the accuracy of guest molecule recognition can be significantly improved, suggesting the possibility of using cucurbituril molecule for single molecule identification. This work enables a new class of recognition molecule for RT technique and opens the door for detecting a vast variety of small molecules by electrical measurements.

Controlled chain polymerisation and chemical soldering for single-molecule electronics.

PubMed

Okawa, Yuji; Akai-Kasaya, Megumi; Kuwahara, Yuji; Mandal, Swapan K; Aono, Masakazu

2012-05-21

Single functional molecules offer great potential for the development of novel nanoelectronic devices with capabilities beyond today's silicon-based devices. To realise single-molecule electronics, the development of a viable method for connecting functional molecules to each other using single conductive polymer chains is required. The method of initiating chain polymerisation using the tip of a scanning tunnelling microscope (STM) is very useful for fabricating single conductive polymer chains at designated positions and thereby wiring single molecules. In this feature article, developments in the controlled chain polymerisation of diacetylene compounds and the properties of polydiacetylene chains are summarised. Recent studies of "chemical soldering", a technique enabling the covalent connection of single polydiacetylene chains to single functional molecules, are also introduced. This represents a key step in advancing the development of single-molecule electronics.

Single molecule and single cell epigenomics.

PubMed

Hyun, Byung-Ryool; McElwee, John L; Soloway, Paul D

2015-01-15

Dynamically regulated changes in chromatin states are vital for normal development and can produce disease when they go awry. Accordingly, much effort has been devoted to characterizing these states under normal and pathological conditions. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is the most widely used method to characterize where in the genome transcription factors, modified histones, modified nucleotides and chromatin binding proteins are found; bisulfite sequencing (BS-seq) and its variants are commonly used to characterize the locations of DNA modifications. Though very powerful, these methods are not without limitations. Notably, they are best at characterizing one chromatin feature at a time, yet chromatin features arise and function in combination. Investigators commonly superimpose separate ChIP-seq or BS-seq datasets, and then infer where chromatin features are found together. While these inferences might be correct, they can be misleading when the chromatin source has distinct cell types, or when a given cell type exhibits any cell to cell variation in chromatin state. These ambiguities can be eliminated by robust methods that directly characterize the existence and genomic locations of combinations of chromatin features in very small inputs of cells or ideally, single cells. Here we review single molecule epigenomic methods under development to overcome these limitations, the technical challenges associated with single molecule methods and their potential application to single cells. Copyright © 2014 Elsevier Inc. All rights reserved.

Single Molecule Nano-Metronome

PubMed Central

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

NASA Astrophysics Data System (ADS)

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

2015-07-01

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.

DNA-psoralen interaction: a single molecule experiment.

PubMed

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.

Common fluorescent proteins for single-molecule localization microscopy

NASA Astrophysics Data System (ADS)

Klementieva, Natalia V.; Bozhanova, Nina G.; Mishina, Natalie M.; Zagaynova, Elena V.; Lukyanov, Konstantin A.; Mishin, Alexander S.

2015-07-01

Super-resolution techniques for breaking the diffraction barrier are spread out over multiple studies nowadays. Single-molecule localization microscopy such as PALM, STORM, GSDIM, etc allow to get super-resolved images of cell ultrastructure by precise localization of individual fluorescent molecules via their temporal isolation. However, these methods are supposed the use of fluorescent dyes and proteins with special characteristics (photoactivation/photoconversion). At the same time, there is a need for retaining high photostability of fluorophores during long-term acquisition. Here, we first showed the potential of common red fluorescent protein for single-molecule localization microscopy based on spontaneous intrinsic blinking. Also, we assessed the effect of different imaging media on photobleaching of these fluorescent proteins. Monomeric orange and red fluorescent proteins were examined for stochastic switching from a dark state to a bright fluorescent state. We studied fusions with cytoskeletal proteins in NIH/3T3 and HeLa cells. Imaging was performed on the Nikon N-STORM system equipped with EMCCD camera. To define the optimal imaging conditions we tested several types of cell culture media and buffers. As a result, high-resolution images of cytoskeleton structure were obtained. Essentially, low-intensity light was sufficient to initiate the switching of tested red fluorescent protein reducing phototoxicity and provide long-term live-cell imaging.

Advances in single-molecule magnet surface patterning through microcontact printing.

PubMed

Mannini, Matteo; Bonacchi, Daniele; Zobbi, Laura; Piras, Federica M; Speets, Emiel A; Caneschi, Andrea; Cornia, Andrea; Magnani, Agnese; Ravoo, Bart Jan; Reinhoudt, David N; Sessoli, Roberta; Gatteschi, Dante

2005-07-01

We present an implementation of strategies to deposit single-molecule magnets (SMMs) using microcontact printing microCP). We describe different approaches of microCP to print stripes of a sulfur-functionalized dodecamanganese (III, IV) cluster on gold surfaces. Comparison by atomic force microscopy profile analysis of the patterned structures confirms the formation of a chemically stable single layer of SMMs. Images based on chemical contrast, obtained by time-of-flight secondary ion mass spectrometry, confirm the patterned structure.

Zero-mode waveguide nanophotonic structures for single molecule characterization

NASA Astrophysics Data System (ADS)

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.

Hydrogel Droplet Microfluidics for High-Throughput Single Molecule/Cell Analysis.

PubMed

Zhu, Zhi; Yang, Chaoyong James

2017-01-17

Heterogeneity among individual molecules and cells has posed significant challenges to traditional bulk assays, due to the assumption of average behavior, which would lose important biological information in heterogeneity and result in a misleading interpretation. Single molecule/cell analysis has become an important and emerging field in biological and biomedical research for insights into heterogeneity between large populations at high resolution. Compared with the ensemble bulk method, single molecule/cell analysis explores the information on time trajectories, conformational states, and interactions of individual molecules/cells, all key factors in the study of chemical and biological reaction pathways. Various powerful techniques have been developed for single molecule/cell analysis, including flow cytometry, atomic force microscopy, optical and magnetic tweezers, single-molecule fluorescence spectroscopy, and so forth. However, some of them have the low-throughput issue that has to analyze single molecules/cells one by one. Flow cytometry is a widely used high-throughput technique for single cell analysis but lacks the ability for intercellular interaction study and local environment control. Droplet microfluidics becomes attractive for single molecule/cell manipulation because single molecules/cells can be individually encased in monodisperse microdroplets, allowing high-throughput analysis and manipulation with precise control of the local environment. Moreover, hydrogels, cross-linked polymer networks that swell in the presence of water, have been introduced into droplet microfluidic systems as hydrogel droplet microfluidics. By replacing an aqueous phase with a monomer or polymer solution, hydrogel droplets can be generated on microfluidic chips for encapsulation of single molecules/cells according to the Poisson distribution. The sol-gel transition property endows the hydrogel droplets with new functionalities and diversified applications in single

Polymer physics experiments with single DNA molecules

NASA Astrophysics Data System (ADS)

Smith, Douglas E.

1999-11-01

Bacteriophage DNA molecules were taken as a model flexible polymer chain for the experimental study of polymer dynamics at the single molecule level. Video fluorescence microscopy was used to directly observe the conformational dynamics of fluorescently labeled molecules, optical tweezers were used to manipulate individual molecules, and micro-fabricated flow cells were used to apply controlled hydrodynamic strain to molecules. These techniques constitute a powerful new experimental approach in the study of basic polymer physics questions. I have used these techniques to study the diffusion and relaxation of isolated and entangled polymer molecules and the hydrodynamic deformation of polymers in elongational and shear flows. These studies revealed a rich, and previously unobserved, ``molecular individualism'' in the dynamical behavior of single molecules. Individual measurements on ensembles of identical molecules allowed the average conformation to be determined as well as the underlying probability distributions for molecular conformation. Scaling laws, that predict the dependence of properties on chain length and concentration, were also tested. The basic assumptions of the reptation model were directly confirmed by visualizing the dynamics of entangled chains.

Analyzing Single-Molecule Time Series via Nonparametric Bayesian Inference

PubMed Central

Hines, Keegan E.; Bankston, John R.; Aldrich, Richard W.

2015-01-01

The ability to measure the properties of proteins at the single-molecule level offers an unparalleled glimpse into biological systems at the molecular scale. The interpretation of single-molecule time series has often been rooted in statistical mechanics and the theory of Markov processes. While existing analysis methods have been useful, they are not without significant limitations including problems of model selection and parameter nonidentifiability. To address these challenges, we introduce the use of nonparametric Bayesian inference for the analysis of single-molecule time series. These methods provide a flexible way to extract structure from data instead of assuming models beforehand. We demonstrate these methods with applications to several diverse settings in single-molecule biophysics. This approach provides a well-constrained and rigorously grounded method for determining the number of biophysical states underlying single-molecule data. PMID:25650922

Robust nonparametric quantification of clustering density of molecules in single-molecule localization microscopy

PubMed Central

Jiang, Shenghang; Park, Seongjin; Challapalli, Sai Divya; Fei, Jingyi; Wang, Yong

2017-01-01

We report a robust nonparametric descriptor, J′(r), for quantifying the density of clustering molecules in single-molecule localization microscopy. J′(r), based on nearest neighbor distribution functions, does not require any parameter as an input for analyzing point patterns. We show that J′(r) displays a valley shape in the presence of clusters of molecules, and the characteristics of the valley reliably report the clustering features in the data. Most importantly, the position of the J′(r) valley (rJm′) depends exclusively on the density of clustering molecules (ρc). Therefore, it is ideal for direct estimation of the clustering density of molecules in single-molecule localization microscopy. As an example, this descriptor was applied to estimate the clustering density of ptsG mRNA in E. coli bacteria. PMID:28636661

Room-temperature ultrafast nonlinear spectroscopy of a single molecule

NASA Astrophysics Data System (ADS)

Liebel, Matz; Toninelli, Costanza; van Hulst, Niek F.

2018-01-01

Single-molecule spectroscopy aims to unveil often hidden but potentially very important contributions of single entities to a system's ensemble response. Albeit contributing tremendously to our ever growing understanding of molecular processes, the fundamental question of temporal evolution, or change, has thus far been inaccessible, thus painting a static picture of a dynamic world. Here, we finally resolve this dilemma by performing ultrafast time-resolved transient spectroscopy on a single molecule. By tracing the femtosecond evolution of excited electronic state spectra of single molecules over hundreds of nanometres of bandwidth at room temperature, we reveal their nonlinear ultrafast response in an effective three-pulse scheme with fluorescence detection. A first excitation pulse is followed by a phase-locked de-excitation pulse pair, providing spectral encoding with 25 fs temporal resolution. This experimental realization of true single-molecule transient spectroscopy demonstrates that two-dimensional electronic spectroscopy of single molecules is experimentally within reach.

Single Molecule Conductance of Oligothiophene Derivatives

NASA Astrophysics Data System (ADS)

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

Single-Molecule Electronic Measurements with Metal Electrodes

ERIC Educational Resources Information Center

Lindsay, Stuart

2005-01-01

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

Dependence of magnetic field and electronic transport of Mn4 Single-molecule magnet in a Single-Electron Transistor

NASA Astrophysics Data System (ADS)

Rodriguez, Alvar; Singh, Simranjeet; Haque, Firoze; Del Barco, Enrique; Nguyen, Tu; Christou, George

2012-02-01

Dependence of magnetic field and electronic transport of Mn4 Single-molecule magnet in a Single-Electron Transistor A. Rodriguez, S. Singh, F. Haque and E. del Barco Department of Physics, University of Central Florida, 4000 Central Florida Blvd., Orlando, Florida 32816 USA T. Nguyen and G. Christou Department of Chemistry, University of Florida, Gainesville, Florida 32611 USA Abstract We have performed single-electron transport measurements on a series of Mn-based low-nuclearity single-molecule magnets (SMM) observing Coulomb blockade. SMMs with well isolated and low ground spin states, i.e. S = 9/2 (Mn4) and S = 6 (Mn3) were chosen for these studies, such that the ground spin multiplet does not mix with levels of other excited spin states for the magnetic fields (H = 0-8 T) employed in the experiments. Different functionalization groups were employed to change the mechanical, geometrical and transport characteristics of the molecules when deposited from liquid solution on the transistors. Electromigration-broken three-terminal single-electron transistors were used. Results obtained at temperatures down to 240 mK and in the presence of high magnetic fields will be shown.

Nanomanipulation of Single RNA Molecules by Optical Tweezers

PubMed Central

Stephenson, William; Wan, Gorby; Tenenbaum, Scott A.; Li, Pan T. X.

2014-01-01

A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be increasingly important. As RNA function often depends on its ability to adopt alternative structures, it is difficult to predict RNA three-dimensional structures directly from sequence. Single-molecule approaches show potentials to solve the problem of RNA structural polymorphism by monitoring molecular structures one molecule at a time. This work presents a method to precisely manipulate the folding and structure of single RNA molecules using optical tweezers. First, methods to synthesize molecules suitable for single-molecule mechanical work are described. Next, various calibration procedures to ensure the proper operations of the optical tweezers are discussed. Next, various experiments are explained. To demonstrate the utility of the technique, results of mechanically unfolding RNA hairpins and a single RNA kissing complex are used as evidence. In these examples, the nanomanipulation technique was used to study folding of each structural domain, including secondary and tertiary, independently. Lastly, the limitations and future applications of the method are discussed. PMID:25177917

Improved Dye Stability in Single-Molecule Fluorescence Experiments

NASA Astrophysics Data System (ADS)

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.

Cell biochemistry studied by single-molecule imaging.

PubMed

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.

Single molecule image formation, reconstruction and processing: introduction.

PubMed

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.

A method to obtain static potential for electron-molecule scattering

NASA Astrophysics Data System (ADS)

Srivastava, Rajesh; Das, Tapasi; Stauffer, Allan

2014-05-01

Electron scattering from molecules is complicated by the fact that molecules are a multi-centered target with the nuclei of the constituent atoms being a center of charge. One of the most important parts of a scattering calculation is to obtain the static potential which represents the interaction of the incident electron with the unperturbed charge distribution of the molecule. A common way to represent the charge distribution of molecules is with Gaussian orbitals centered on the various nuclei. We have derived a way to calculate spherically-averaged molecular static potentials using this form of molecular wave function which is mostly analytic. This method has been applied to elastic electron scattering from water molecules and we obtained differential cross sections which are compared with previous experimental and theoretical results. The method can be extended to more complex molecules. One of us (RS) is thankful to IAEA, Vienna, Austria and DAE-BRNS, Mumbai, India for financial support.

Fluorescence Microscopy of Single Molecules

ERIC Educational Resources Information Center

Zimmermann, Jan; van Dorp, Arthur; Renn, Alois

2004-01-01

The investigation of photochemistry and photophysics of individual quantum systems is described with the help of a wide-field fluorescence microscopy approach. The fluorescence single molecules are observed in real time.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sotthewes, Kai; Heimbuch, René, E-mail: r.heimbuch@utwente.nl; Kumar, Avijit

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 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 dropmore » 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.« less

Watching single molecules dance

NASA Astrophysics Data System (ADS)

Mehta, Amit Dinesh

Molecular motors convert chemical energy, from ATP hydrolysis or ion flow, into mechanical motion. A variety of increasingly precise mechanical probes have been developed to monitor and perturb these motors at the single molecule level. Several outstanding questions can be best approached at the single molecule level. These include: how far does a motor progress per energy quanta consumed? how does its reaction cycle respond to load? how many productive catalytic cycles can it undergo per diffusional encounter with its track? and what is the mechanical stiffness of a single molecule connection? A dual beam optical trap, in conjunction with in vitro ensemble motility assays, has been used to characterize two members of the myosin superfamily: muscle myosin II and chick brain myosin V. Both move the helical polymer actin, but myosin II acts in large ensembles to drive muscle contraction or cytokinesis, while myosin V acts in small numbers to transport vesicles. An optical trapping apparatus was rendered sufficiently precise to identify a myosin working stroke with 1nm or so, barring systematic errors such as those perhaps due to random protein orientations. This and other light microscopic motility assays were used to characterize myosin V: unlike myosin II this vesicle transport protein moves through many increments of travel while remaining strongly bound to a single actin filament. The step size, stall force, and travel distance of myosin V reveal a remarkably efficient motor capable of moving along a helical track for over a micrometer without significantly spiraling around it. Such properties are fully consistent with the putative role of an organelle transport motor, present in small numbers to maintain movement over long ranges relative to cellular size scales. The contrast between myosin II and myosin V resembles that between a human running on the moon and one walking on earth, where the former allows for faster motion when in larger ensembles but for less

Single-molecule nanopore enzymology

PubMed Central

Wloka, Carsten; Maglia, Giovanni

2017-01-01

Biological nanopores are a class of membrane proteins that open nanoscale water-conduits in biological membranes. When they are reconstituted in artificial membranes and a bias voltage is applied across the membrane, the ionic current passing through individual nanopores can be used to monitor chemical reactions, to recognize individual molecules and, of most interest, to sequence DNA. More recently, proteins and enzymes have started being analysed with nanopores. Monitoring enzymatic reactions with nanopores, i.e. nanopore enzymology, has the unique advantage that it allows long-timescale observations of native proteins at the single-molecule level. Here we describe the approaches and challenges in nanopore enzymology. PMID:28630164

SINGLE STRAND-CONTAINING REPLICATING MOLECULES OF CIRCULAR MITOCHONDRIAL DNA

PubMed Central

Wolstenholme, David R.; Koike, Katsuro; Cochran-Fouts, Patricia

1973-01-01

Mitochondrial DNAs (mtDNAs) from Chang rat solid hepatomas and Novikoff rat ascites hepatomas were examined in the electron microscope after preparation by the aqueous and by the formamide protein monolayer techniques. MtDNAs from both tumors were found to include double-forked circular molecules with a form and size suggesting they were replicative intermediates. These molecules were of two classes. In molecules of one class, all three segments were apparently totally double stranded. Molecules of the second class were distinguished by the fact that one of the segments spanning the region between the forks in which replication had occurred (the daughter segments) was either totally single stranded, or contained a single-stranded region associated with one of the forks. Daughter segments of both totally double-stranded and single strand-containing replicating molecules varied in length from about 3 to about 80% of the circular contour length of the molecule. Similar classes of replicating molecules were found in mtDNA from regenerating rat liver and chick embryos, indicating them to be normal intermediates in the replication of mtDNA All of the mtDNAs examined included partially single-stranded simple (nonforked) circular molecules. A possible scheme for the replication of mtDNA is presented, based on the different molecular forms observed PMID:4345165

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

NASA Astrophysics Data System (ADS)

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

Single Molecules as Optical Probes for Structure and Dynamics

NASA Astrophysics Data System (ADS)

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.

Quantum design rules for single molecule logic gates.

PubMed

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.

Single-Molecule Reaction Chemistry in Patterned Nanowells

PubMed Central

2016-01-01

A new approach to synthetic chemistry is performed in ultraminiaturized, nanofabricated reaction chambers. Using lithographically defined nanowells, we achieve single-point covalent chemistry on hundreds of individual carbon nanotube transistors, providing robust statistics and unprecedented spatial resolution in adduct position. Each device acts as a sensor to detect, in real-time and through quantized changes in conductance, single-point functionalization of the nanotube as well as consecutive chemical reactions, molecular interactions, and molecular conformational changes occurring on the resulting single-molecule probe. In particular, we use a set of sequential bioconjugation reactions to tether a single-strand of DNA to the device and record its repeated, reversible folding into a G-quadruplex structure. The stable covalent tether allows us to measure the same molecule in different solutions, revealing the characteristic increased stability of the G-quadruplex structure in the presence of potassium ions (K+) versus sodium ions (Na+). Nanowell-confined reaction chemistry on carbon nanotube devices offers a versatile method to isolate and monitor individual molecules during successive chemical reactions over an extended period of time. PMID:27270004

Markov Chain Monte Carlo in the Analysis of Single-Molecule Experimental Data

NASA Astrophysics Data System (ADS)

Kou, S. C.; Xie, X. Sunney; Liu, Jun S.

2003-11-01

This article provides a Bayesian analysis of the single-molecule fluorescence lifetime experiment designed to probe the conformational dynamics of a single DNA hairpin molecule. The DNA hairpin's conformational change is initially modeled as a two-state Markov chain, which is not observable and has to be indirectly inferred. The Brownian diffusion of the single molecule, in addition to the hidden Markov structure, further complicates the matter. We show that the analytical form of the likelihood function can be obtained in the simplest case and a Metropolis-Hastings algorithm can be designed to sample from the posterior distribution of the parameters of interest and to compute desired estiamtes. To cope with the molecular diffusion process and the potentially oscillating energy barrier between the two states of the DNA hairpin, we introduce a data augmentation technique to handle both the Brownian diffusion and the hidden Ornstein-Uhlenbeck process associated with the fluctuating energy barrier, and design a more sophisticated Metropolis-type algorithm. Our method not only increases the estimating resolution by several folds but also proves to be successful for model discrimination.

Single-molecule Force Spectroscopy Approach to Enzyme Catalysis*

PubMed Central

Alegre-Cebollada, Jorge; Perez-Jimenez, Raul; Kosuri, Pallav; Fernandez, Julio M.

2010-01-01

Enzyme catalysis has been traditionally studied using a diverse set of techniques such as bulk biochemistry, x-ray crystallography, and NMR. Recently, single-molecule force spectroscopy by atomic force microscopy has been used as a new tool to study the catalytic properties of an enzyme. In this approach, a mechanical force ranging up to hundreds of piconewtons is applied to the substrate of an enzymatic reaction, altering the conformational energy of the substrate-enzyme interactions during catalysis. From these measurements, the force dependence of an enzymatic reaction can be determined. The force dependence provides valuable new information about the dynamics of enzyme catalysis with sub-angstrom resolution, a feat unmatched by any other current technique. To date, single-molecule force spectroscopy has been applied to gain insight into the reduction of disulfide bonds by different enzymes of the thioredoxin family. This minireview aims to present a perspective on this new approach to study enzyme catalysis and to summarize the results that have already been obtained from it. Finally, the specific requirements that must be fulfilled to apply this new methodology to any other enzyme will be discussed. PMID:20382731

Single-molecule force spectroscopy approach to enzyme catalysis.

PubMed

Alegre-Cebollada, Jorge; Perez-Jimenez, Raul; Kosuri, Pallav; Fernandez, Julio M

2010-06-18

Enzyme catalysis has been traditionally studied using a diverse set of techniques such as bulk biochemistry, x-ray crystallography, and NMR. Recently, single-molecule force spectroscopy by atomic force microscopy has been used as a new tool to study the catalytic properties of an enzyme. In this approach, a mechanical force ranging up to hundreds of piconewtons is applied to the substrate of an enzymatic reaction, altering the conformational energy of the substrate-enzyme interactions during catalysis. From these measurements, the force dependence of an enzymatic reaction can be determined. The force dependence provides valuable new information about the dynamics of enzyme catalysis with sub-angstrom resolution, a feat unmatched by any other current technique. To date, single-molecule force spectroscopy has been applied to gain insight into the reduction of disulfide bonds by different enzymes of the thioredoxin family. This minireview aims to present a perspective on this new approach to study enzyme catalysis and to summarize the results that have already been obtained from it. Finally, the specific requirements that must be fulfilled to apply this new methodology to any other enzyme will be discussed.

Improved maize reference genome with single-molecule technologies.

PubMed

Jiao, Yinping; Peluso, Paul; Shi, Jinghua; Liang, Tiffany; Stitzer, Michelle C; Wang, Bo; Campbell, Michael S; Stein, Joshua C; Wei, Xuehong; Chin, Chen-Shan; Guill, Katherine; Regulski, Michael; Kumari, Sunita; Olson, Andrew; Gent, Jonathan; Schneider, Kevin L; Wolfgruber, Thomas K; May, Michael R; Springer, Nathan M; Antoniou, Eric; McCombie, W Richard; Presting, Gernot G; McMullen, Michael; Ross-Ibarra, Jeffrey; Dawe, R Kelly; Hastie, Alex; Rank, David R; Ware, Doreen

2017-06-22

Complete and accurate reference genomes and annotations provide fundamental tools for characterization of genetic and functional variation. These resources facilitate the determination of biological processes and support translation of research findings into improved and sustainable agricultural technologies. Many reference genomes for crop plants have been generated over the past decade, but these genomes are often fragmented and missing complex repeat regions. Here we report the assembly and annotation of a reference genome of maize, a genetic and agricultural model species, using single-molecule real-time sequencing and high-resolution optical mapping. Relative to the previous reference genome, our assembly features a 52-fold increase in contig length and notable improvements in the assembly of intergenic spaces and centromeres. Characterization of the repetitive portion of the genome revealed more than 130,000 intact transposable elements, allowing us to identify transposable element lineage expansions that are unique to maize. Gene annotations were updated using 111,000 full-length transcripts obtained by single-molecule real-time sequencing. In addition, comparative optical mapping of two other inbred maize lines revealed a prevalence of deletions in regions of low gene density and maize lineage-specific genes.

Single-Molecule Imaging of Cellular Signaling

NASA Astrophysics Data System (ADS)

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

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

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

PubMed

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.

Coherent interaction of single molecules and plasmonic nanowires

NASA Astrophysics Data System (ADS)

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.

Mapping Nanoscale Hotspots with Single-Molecule Emitters Assembled into Plasmonic Nanocavities Using DNA Origami

PubMed Central

2017-01-01

Fabricating nanocavities in which optically active single quantum emitters are precisely positioned is crucial for building nanophotonic devices. Here we show that self-assembly based on robust DNA-origami constructs can precisely position single molecules laterally within sub-5 nm gaps between plasmonic substrates that support intense optical confinement. By placing single-molecules at the center of a nanocavity, we show modification of the plasmon cavity resonance before and after bleaching the chromophore and obtain enhancements of ≥4 × 103 with high quantum yield (≥50%). By varying the lateral position of the molecule in the gap, we directly map the spatial profile of the local density of optical states with a resolution of ±1.5 nm. Our approach introduces a straightforward noninvasive way to measure and quantify confined optical modes on the nanoscale. PMID:29166033

Mapping Nanoscale Hotspots with Single-Molecule Emitters Assembled into Plasmonic Nanocavities Using DNA Origami.

PubMed

Chikkaraddy, Rohit; Turek, V A; Kongsuwan, Nuttawut; Benz, Felix; Carnegie, Cloudy; van de Goor, Tim; de Nijs, Bart; Demetriadou, Angela; Hess, Ortwin; Keyser, Ulrich F; Baumberg, Jeremy J

2018-01-10

Fabricating nanocavities in which optically active single quantum emitters are precisely positioned is crucial for building nanophotonic devices. Here we show that self-assembly based on robust DNA-origami constructs can precisely position single molecules laterally within sub-5 nm gaps between plasmonic substrates that support intense optical confinement. By placing single-molecules at the center of a nanocavity, we show modification of the plasmon cavity resonance before and after bleaching the chromophore and obtain enhancements of ≥4 × 10 3 with high quantum yield (≥50%). By varying the lateral position of the molecule in the gap, we directly map the spatial profile of the local density of optical states with a resolution of ±1.5 nm. Our approach introduces a straightforward noninvasive way to measure and quantify confined optical modes on the nanoscale.

Mapping Nanoscale Hotspots with Single-Molecule Emitters Assembled into Plasmonic Nanocavities Using DNA Origami

NASA Astrophysics Data System (ADS)

Chikkaraddy, Rohit; Turek, V. A.; Kongsuwan, Nuttawut; Benz, Felix; Carnegie, Cloudy; van de Goor, Tim; de Nijs, Bart; Demetriadou, Angela; Hess, Ortwin; Keyser, Ulrich F.; Baumberg, Jeremy J.

2018-01-01

Fabricating nanocavities in which optically-active single quantum emitters are precisely positioned, is crucial for building nanophotonic devices. Here we show that self-assembly based on robust DNA-origami constructs can precisely position single molecules laterally within sub-5nm gaps between plasmonic substrates that support intense optical confinement. By placing single-molecules at the center of a nanocavity, we show modification of the plasmon cavity resonance before and after bleaching the chromophore, and obtain enhancements of $\\geq4\\times10^3$ with high quantum yield ($\\geq50$%). By varying the lateral position of the molecule in the gap, we directly map the spatial profile of the local density of optical states with a resolution of $\\pm1.5$ nm. Our approach introduces a straightforward non-invasive way to measure and quantify confined optical modes on the nanoscale.

Improving single-molecule FRET measurements by confining molecules in nanopipettes

NASA Astrophysics Data System (ADS)

Vogelsang, J.; Doose, S.; Sauer, M.; Tinnefeld, P.

2007-07-01

In recent years Fluorescence Resonance Energy Transfer (FRET) has been widely used to determine distances, observe distance dynamics, and monitor molecular binding at the single-molecule level. A basic constraint of single-molecule FRET studies is the limited distance resolution owing to low photon statistics. We demonstrate that by confining molecules in nanopipettes (50-100 nm diameter) smFRET can be measured with improved photon statistics reducing the width of FRET proximity ratio distributions (PRD). This increase in distance resolution makes it possible to reveal subpopulations and dynamics in biomolecular complexes. Our data indicate that the width of PRD is not only determined by photon statistics (shot noise) and distance distributions between the chromophores but that photoinduced dark states of the acceptor also contribute to the PRD width. Furthermore, acceptor dark states such as triplet states influence the accuracy of determined mean FRET values. In this context, we present a strategy for the correction of the shift of the mean PR that is related to triplet induced blinking of the acceptor using reference FCS measurements.

Choosing the right fluorophore for single-molecule fluorescence studies in a lipid environment.

PubMed

Zhang, Zhenfu; Yomo, Dan; Gradinaru, Claudiu

2017-07-01

Nonspecific interactions between lipids and fluorophores can alter the outcomes of single-molecule spectroscopy of membrane proteins in live cells, liposomes or lipid nanodiscs and of cytosolic proteins encapsulated in liposomes or tethered to supported lipid bilayers. To gain insight into these effects, we examined interactions between 9 dyes that are commonly used as labels for single-molecule fluorescence (SMF) and 6 standard lipids including cationic, zwitterionic and anionic types. The diffusion coefficients of dyes in the absence and presence of set amounts of lipid vesicles were measured by fluorescence correlation spectroscopy (FCS). The partition coefficients and the free energies of partitioning for different fluorophore-lipid pairs were obtained by global fitting of the titration FCS curves. Lipids with different charges, head groups and degrees of chain saturation were investigated, and interactions with dyes are discussed in terms of hydrophobic, electrostatic and steric contributions. Fluorescence imaging of individual fluorophores adsorbed on supported lipid bilayers provides visualization and additional quantification of the strength of dye-lipid interaction in the context of single-molecule measurements. By dissecting fluorophore-lipid interactions, our study provides new insights into setting up single-molecule fluorescence spectroscopy experiments with minimal interference from interactions between fluorescent labels and lipids in the environment. Copyright © 2017 Elsevier B.V. All rights reserved.

A reversible single-molecule switch based on activated antiaromaticity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yin, Xiaodong; Zang, Yaping; Zhu, Liangliang

Single-molecule electronic devices provide researchers with an unprecedented ability to relate novel physical phenomena to molecular chemical structures. Typically, conjugated aromatic molecular backbones are relied upon to create electronic devices, where the aromaticity of the building blocks is used to enhance conductivity. We capitalize on the classical physical organic chemistry concept of Hückel antiaromaticity by demonstrating a single-molecule switch that exhibits low conductance in the neutral state and, upon electrochemical oxidation, reversibly switches to an antiaromatic high-conducting structure. We form single-molecule devices using the scanning tunneling microscope–based break-junction technique and observe an on/off ratio of ~70 for a thiophenylidene derivativemore » that switches to an antiaromatic state with 6-4-6-p electrons. Through supporting nuclear magnetic resonance measurements, we show that the doubly oxidized core has antiaromatic character and we use density functional theory calculations to rationalize the origin of the high-conductance state for the oxidized single-molecule junction. Together, our work demonstrates how the concept of antiaromaticity can be exploited to create single-molecule devices that are highly conducting.« less

A reversible single-molecule switch based on activated antiaromaticity

DOE PAGES

Yin, Xiaodong; Zang, Yaping; Zhu, Liangliang; ...

2017-10-27

Single-molecule electronic devices provide researchers with an unprecedented ability to relate novel physical phenomena to molecular chemical structures. Typically, conjugated aromatic molecular backbones are relied upon to create electronic devices, where the aromaticity of the building blocks is used to enhance conductivity. We capitalize on the classical physical organic chemistry concept of Hückel antiaromaticity by demonstrating a single-molecule switch that exhibits low conductance in the neutral state and, upon electrochemical oxidation, reversibly switches to an antiaromatic high-conducting structure. We form single-molecule devices using the scanning tunneling microscope–based break-junction technique and observe an on/off ratio of ~70 for a thiophenylidene derivativemore » that switches to an antiaromatic state with 6-4-6-p electrons. Through supporting nuclear magnetic resonance measurements, we show that the doubly oxidized core has antiaromatic character and we use density functional theory calculations to rationalize the origin of the high-conductance state for the oxidized single-molecule junction. Together, our work demonstrates how the concept of antiaromaticity can be exploited to create single-molecule devices that are highly conducting.« less

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

PubMed

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

Torque Measurement at the Single Molecule Level

PubMed Central

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

Torque measurement at the single-molecule level.

PubMed

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

2013-01-01

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 that 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 that would be well suited for analysis with torsional measurement techniques.

Optical Modification of a Single Impurity Molecule in a Solid

DTIC Science & Technology

1991-10-17

have led to direct observations of the lifetime-limited homogeneous Iinewidth of a single pentacene molecule as well as the surprising observation of...advances in the optical detection and spectroscopy of single impurity centers in solids. For the system composed of pentacene impurity molecules in the...limited homogcncous linewidth of a single pentacene molecule as well as the surprising observation of spontaneous spectral diffusion in a crystal

Optimal Background Estimators in Single-Molecule FRET Microscopy.

PubMed

Preus, Søren; Hildebrandt, Lasse L; Birkedal, Victoria

2016-09-20

Single-molecule total internal reflection fluorescence (TIRF) microscopy constitutes an umbrella of powerful tools that facilitate direct observation of the biophysical properties, population heterogeneities, and interactions of single biomolecules without the need for ensemble synchronization. Due to the low signal/noise ratio in single-molecule TIRF microscopy experiments, it is important to determine the local background intensity, especially when the fluorescence intensity of the molecule is used quantitatively. Here we compare and evaluate the performance of different aperture-based background estimators used particularly in single-molecule Förster resonance energy transfer. We introduce the general concept of multiaperture signatures and use this technique to demonstrate how the choice of background can affect the measured fluorescence signal considerably. A new, to our knowledge, and simple background estimator is proposed, called the local statistical percentile (LSP). We show that the LSP background estimator performs as well as current background estimators at low molecular densities and significantly better in regions of high molecular densities. The LSP background estimator is thus suited for single-particle TIRF microscopy of dense biological samples in which the intensity itself is an observable of the technique. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Molecular electronics with single molecules in solid-state devices.

PubMed

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.

Model systems for single molecule polymer dynamics

PubMed Central

Latinwo, Folarin

2012-01-01

Double stranded DNA (dsDNA) has long served as a model system for single molecule polymer dynamics. However, dsDNA is a semiflexible polymer, and the structural rigidity of the DNA double helix gives rise to local molecular properties and chain dynamics that differ from flexible chains, including synthetic organic polymers. Recently, we developed single stranded DNA (ssDNA) as a new model system for single molecule studies of flexible polymer chains. In this work, we discuss model polymer systems in the context of “ideal” and “real” chain behavior considering thermal blobs, tension blobs, hydrodynamic drag and force–extension relations. In addition, we present monomer aspect ratio as a key parameter describing chain conformation and dynamics, and we derive dynamical scaling relations in terms of this molecular-level parameter. We show that asymmetric Kuhn segments can suppress monomer–monomer interactions, thereby altering global chain dynamics. Finally, we discuss ssDNA in the context of a new model system for single molecule polymer dynamics. Overall, we anticipate that future single polymer studies of flexible chains will reveal new insight into the dynamic behavior of “real” polymers, which will highlight the importance of molecular individualism and the prevalence of non-linear phenomena. PMID:22956980

Single DNA molecule detection using nanopipettes and nanoparticles.

PubMed

Karhanek, Miloslav; Kemp, Jennifer T; Pourmand, Nader; Davis, Ronald W; Webb, Chris D

2005-02-01

Single DNA molecules labeled with nanoparticles can be detected by blockades of ionic current as they are translocated through a nanopipette tip formed by a pulled glass capillary. The nanopipette detection technique can provide not only tools for detection and identification of single DNA and protein molecules but also deeper insight and understanding of stochastic interactions of various biomolecules with their environment.

Communication: Coordinate-dependent diffusivity from single molecule trajectories

NASA Astrophysics Data System (ADS)

Berezhkovskii, Alexander M.; Makarov, Dmitrii E.

2017-11-01

Single-molecule observations of biomolecular folding are commonly interpreted using the model of one-dimensional diffusion along a reaction coordinate, with a coordinate-independent diffusion coefficient. Recent analysis, however, suggests that more general models are required to account for single-molecule measurements performed with high temporal resolution. Here, we consider one such generalization: a model where the diffusion coefficient can be an arbitrary function of the reaction coordinate. Assuming Brownian dynamics along this coordinate, we derive an exact expression for the coordinate-dependent diffusivity in terms of the splitting probability within an arbitrarily chosen interval and the mean transition path time between the interval boundaries. This formula can be used to estimate the effective diffusion coefficient along a reaction coordinate directly from single-molecule trajectories.

Detectors for single-molecule fluorescence imaging and spectroscopy

PubMed Central

MICHALET, X.; SIEGMUND, O.H.W.; VALLERGA, J.V.; JELINSKY, P.; MILLAUD, J.E.; WEISS, S.

2010-01-01

Single-molecule observation, characterization and manipulation techniques have recently come to the forefront of several research domains spanning chemistry, biology and physics. Due to the exquisite sensitivity, specificity, and unmasking of ensemble averaging, single-molecule fluorescence imaging and spectroscopy have become, in a short period of time, important tools in cell biology, biochemistry and biophysics. These methods led to new ways of thinking about biological processes such as viral infection, receptor diffusion and oligomerization, cellular signaling, protein-protein or protein-nucleic acid interactions, and molecular machines. Such achievements require a combination of several factors to be met, among which detector sensitivity and bandwidth are crucial. We examine here the needed performance of photodetectors used in these types of experiments, the current state of the art for different categories of detectors, and actual and future developments of single-photon counting detectors for single-molecule imaging and spectroscopy. PMID:20157633

Single Molecule Sensing by Nanopores and Nanopore Devices

PubMed Central

Gu, Li-Qun; Shim, Ji Wook

2010-01-01

Molecular-scale pore structures, called nanopores, can be assembled by protein ion channels through genetic engineering or be artificially fabricated on solid substrates using fashion nanotechnology. When target molecules interact with the functionalized lumen of a nanopore, they characteristically block the ion pathway. The resulting conductance changes allow for identification of single molecules and quantification of target species in the mixture. In this review, we first overview nanopore-based sensory techniques that have been created for the detection of myriad biomedical targets, from metal ions, drug compounds, and cellular second messengers to proteins and DNA. Then we introduce our recent discoveries in nanopore single molecule detection: (1) using the protein nanopore to study folding/unfolding of the G-quadruplex aptamer; (2) creating a portable and durable biochip that is integrated with a single-protein pore sensor (this chip is compared with recently developed protein pore sensors based on stabilized bilayers on glass nanopore membranes and droplet interface bilayer); and (3) creating a glass nanopore-terminated probe for single-molecule DNA detection, chiral enantiomer discrimination, and identification of the bioterrorist agent ricin with an aptamer-encoded nanopore. PMID:20174694

Molecular electronics--resonant transport through single molecules.

PubMed

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.

Structural Information from Single-molecule FRET Experiments Using the Fast Nano-positioning System

PubMed Central

Röcker, Carlheinz; Nagy, Julia; Michaelis, Jens

2017-01-01

Single-molecule Förster Resonance Energy Transfer (smFRET) can be used to obtain structural information on biomolecular complexes in real-time. Thereby, multiple smFRET measurements are used to localize an unknown dye position inside a protein complex by means of trilateration. In order to obtain quantitative information, the Nano-Positioning System (NPS) uses probabilistic data analysis to combine structural information from X-ray crystallography with single-molecule fluorescence data to calculate not only the most probable position but the complete three-dimensional probability distribution, termed posterior, which indicates the experimental uncertainty. The concept was generalized for the analysis of smFRET networks containing numerous dye molecules. The latest version of NPS, Fast-NPS, features a new algorithm using Bayesian parameter estimation based on Markov Chain Monte Carlo sampling and parallel tempering that allows for the analysis of large smFRET networks in a comparably short time. Moreover, Fast-NPS allows the calculation of the posterior by choosing one of five different models for each dye, that account for the different spatial and orientational behavior exhibited by the dye molecules due to their local environment. Here we present a detailed protocol for obtaining smFRET data and applying the Fast-NPS. We provide detailed instructions for the acquisition of the three input parameters of Fast-NPS: the smFRET values, as well as the quantum yield and anisotropy of the dye molecules. Recently, the NPS has been used to elucidate the architecture of an archaeal open promotor complex. This data is used to demonstrate the influence of the five different dye models on the posterior distribution. PMID:28287526

Structural Information from Single-molecule FRET Experiments Using the Fast Nano-positioning System.

PubMed

Dörfler, Thilo; Eilert, Tobias; Röcker, Carlheinz; Nagy, Julia; Michaelis, Jens

2017-02-09

Single-molecule Förster Resonance Energy Transfer (smFRET) can be used to obtain structural information on biomolecular complexes in real-time. Thereby, multiple smFRET measurements are used to localize an unknown dye position inside a protein complex by means of trilateration. In order to obtain quantitative information, the Nano-Positioning System (NPS) uses probabilistic data analysis to combine structural information from X-ray crystallography with single-molecule fluorescence data to calculate not only the most probable position but the complete three-dimensional probability distribution, termed posterior, which indicates the experimental uncertainty. The concept was generalized for the analysis of smFRET networks containing numerous dye molecules. The latest version of NPS, Fast-NPS, features a new algorithm using Bayesian parameter estimation based on Markov Chain Monte Carlo sampling and parallel tempering that allows for the analysis of large smFRET networks in a comparably short time. Moreover, Fast-NPS allows the calculation of the posterior by choosing one of five different models for each dye, that account for the different spatial and orientational behavior exhibited by the dye molecules due to their local environment. Here we present a detailed protocol for obtaining smFRET data and applying the Fast-NPS. We provide detailed instructions for the acquisition of the three input parameters of Fast-NPS: the smFRET values, as well as the quantum yield and anisotropy of the dye molecules. Recently, the NPS has been used to elucidate the architecture of an archaeal open promotor complex. This data is used to demonstrate the influence of the five different dye models on the posterior distribution.

Microsecond Resolution of Single-Molecule Rotation Catalyzed by Molecular Motors

PubMed Central

Hornung, Tassilo; Martin, James; Spetzler, David; Ishmukhametov, Robert; Frasch, Wayne D.

2017-01-01

Single-molecule measurements of rotation catalyzed by the F1-ATPase or the FoF1 ATP synthase have provided new insights into the molecular mechanisms of the F1 and Fo molecular motors. We recently developed a method to record ATPase-driven rotation of F1 or FoF1 in a manner that solves several technical limitations of earlier approaches that were significantly hampered by time and angular resolution, and restricted the duration of data collection. With our approach it is possible to collect data for hours and obtain statistically significant quantities of data on each molecule examined with a time resolution of up to 5 μs at unprecedented signal-to-noise. PMID:21809213

Single Molecule Study of Metalloregulatory Protein-DNA Interactions

NASA Astrophysics Data System (ADS)

Sarkar, Susanta; Benitez, Jaime; Huang, Zhengxi; Wang, Qi; Chen, Peng

2007-03-01

Control of metal concentrations is essential for living body. Metalloregulatory proteins respond to metal concentrations by regulating transcriptions of metal resistance genes via protein-DNA interactions. It is thus necessary to understand interactions of metalloregulatory proteins with DNA. Ensemble measurements provide average behavior of a vast number of biomolecules. In contrast, single molecule spectroscopy can track single molecules individually and elucidate dynamics of processes of short time scales and intermediate structures not revealed by ensemble measurements. Here we present single molecule study of interactions between PbrR691, a MerR-family metalloregulatory protein and DNA. We presume that the dynamics of protein/DNA conformational changes and interactions are important for the transcription regulation and kinetics of these dynamic processes can provide useful information about the mechanisms of these metalloregulatory proteins.

Tracking single mRNA molecules in live cells

NASA Astrophysics Data System (ADS)

Moon, Hyungseok C.; Lee, Byung Hun; Lim, Kiseong; Son, Jae Seok; Song, Minho S.; Park, Hye Yoon

2016-06-01

mRNAs inside cells interact with numerous RNA-binding proteins, microRNAs, and ribosomes that together compose a highly heterogeneous population of messenger ribonucleoprotein (mRNP) particles. Perhaps one of the best ways to investigate the complex regulation of mRNA is to observe individual molecules. Single molecule imaging allows the collection of quantitative and statistical data on subpopulations and transient states that are otherwise obscured by ensemble averaging. In addition, single particle tracking reveals the sequence of events that occur in the formation and remodeling of mRNPs in real time. Here, we review the current state-of-the-art techniques in tagging, delivery, and imaging to track single mRNAs in live cells. We also discuss how these techniques are applied to extract dynamic information on the transcription, transport, localization, and translation of mRNAs. These studies demonstrate how single molecule tracking is transforming the understanding of mRNA regulation in live cells.

Single-Molecule Electrical Random Resequencing of DNA and RNA

NASA Astrophysics Data System (ADS)

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

2012-07-01

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

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

PubMed Central

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

Protein mechanics: from single molecules to functional biomaterials.

PubMed

Li, Hongbin; Cao, Yi

2010-10-19

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

In situ temperature monitoring in single-molecule FRET experiments

NASA Astrophysics Data System (ADS)

Hartmann, Andreas; Berndt, Frederic; Ollmann, Simon; Krainer, Georg; Schlierf, Michael

2018-03-01

Thermodynamic properties of single molecules including enthalpic and entropic contributions are often determined from experiments by a direct control and precise measurement of the local temperature. However, common temperature monitoring techniques using, for example, ultrafine temperature probes can lead to uncertainties as the probe cannot be placed in the vicinity of the molecule of interest. Here, we devised an approach to measure the local temperature in freely diffusing confocal single-molecule Förster Resonance Energy Transfer (smFRET) experiments in situ by directly adding the temperature-sensitive fluorescent dye Rhodamine B, whose fluorescence lifetime serves as a probe of the local temperature in the confocal volume. We demonstrate that the temperature and FRET efficiencies of static and dynamic molecules can be extracted within one measurement simultaneously, without the need of a reference chamber. We anticipate this technique to be particularly useful in the physicochemical analyses of temperature-dependent biomolecular processes from single-molecule measurements.

Single molecule diffusion and the solution of the spherically symmetric residence time equation.

PubMed

Agmon, Noam

2011-06-16

The residence time of a single dye molecule diffusing within a laser spot is propotional to the total number of photons emitted by it. With this application in mind, we solve the spherically symmetric "residence time equation" (RTE) to obtain the solution for the Laplace transform of the mean residence time (MRT) within a d-dimensional ball, as a function of the initial location of the particle and the observation time. The solutions for initial conditions of potential experimental interest, starting in the center, on the surface or uniformly within the ball, are explicitly presented. Special cases for dimensions 1, 2, and 3 are obtained, which can be Laplace inverted analytically for d = 1 and 3. In addition, the analytic short- and long-time asymptotic behaviors of the MRT are derived and compared with the exact solutions for d = 1, 2, and 3. As a demonstration of the simplification afforded by the RTE, the Appendix obtains the residence time distribution by solving the Feynman-Kac equation, from which the MRT is obtained by differentiation. Single-molecule diffusion experiments could be devised to test the results for the MRT presented in this work. © 2011 American Chemical Society

Synthesis of single-molecule nanocars.

PubMed

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

DOEpatents

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.

Quantum-limited evanescent single molecule sensing.

NASA Astrophysics Data System (ADS)

Bowen, Warwick; Mauranyapin, Nicolas; Madsen, Lars; Taylor, Michael; Waleed, Muhammad

Sensors that are able to detect and track single unlabeled biomolecules are an important tool both to understand biomolecular dynamics and interactions, and for medical diagnostics operating at their ultimate detection limits. Recently, exceptional sensitivity has been achieved using the strongly enhanced evanescent fields provided by optical microcavities and plasmonic resonators. However, at high field intensities photodamage to the biological specimen becomes increasingly problematic. Here, we introduce a new approach that combines dark field illumination and heterodyne detection in an optical nanofibre. This allows operation at the fundamental precision limit introduced by quantisation of light. We achieve state-of-the-art sensitivity with a four order-of-magnitude reduction in optical intensity. This enables quantum noise limited tracking of single biomolecules as small as 3.5 nm and surface-molecule interactions to be montored over extended periods. By achieving quantum noise limited precision, our approach provides a pathway towards quantum-enhanced single-molecule biosensors. We acknkowledge financial support from AFOSR and AOARD.

Fast temporal fluctuations in single-molecule junctions.

PubMed

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.

Scaling from single molecule to macroscopic adhesion at polymer/metal interfaces.

PubMed

Utzig, Thomas; Raman, Sangeetha; Valtiner, Markus

2015-03-10

Understanding the evolution of macroscopic adhesion based on fundamental molecular interactions is crucial to designing strong and smart polymer/metal interfaces that play an important role in many industrial and biomedical applications. Here we show how macroscopic adhesion can be predicted on the basis of single molecular interactions. In particular, we carry out dynamic single molecule-force spectroscopy (SM-AFM) in the framework of Bell-Evans' theory to gain information about the energy barrier between the bound and unbound states of an amine/gold junction. Furthermore, we use Jarzynski's equality to obtain the equilibrium ground-state energy difference of the amine/gold bond from these nonequilibrium force measurements. In addition, we perform surface forces apparatus (SFA) experiments to measure macroscopic adhesion forces at contacts where approximately 10(7) amine/gold bonds are formed simultaneously. The SFA approach provides an amine/gold interaction energy (normalized by the number of interacting molecules) of (36 ± 1)k(B)T, which is in excellent agreement with the interaction free energy of (35 ± 3)k(B)T calculated using Jarzynski's equality and single-molecule AFM experiments. Our results validate Jarzynski's equality for the field of polymer/metal interactions by measuring both sides of the equation. Furthermore, the comparison of SFA and AFM shows how macroscopic interaction energies can be predicted on the basis of single molecular interactions, providing a new strategy to potentially predict adhesive properties of novel glues or coatings as well as bio- and wet adhesion.

Exploring the folding pattern of a polymer chain in a single crystal by combining single-molecule force spectroscopy and steered molecular dynamics simulations.

PubMed

Song, Yu; Feng, Wei; Liu, Kai; Yang, Peng; Zhang, Wenke; Zhang, Xi

2013-03-26

Understanding the folding pattern of a single polymer chain within its single crystal will shed light on the mechanism of crystallization. Here, we use the combined techniques of atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) and steered molecular dynamics (SMD) simulations to study the folding pattern of a polyethylene oxide (PEO) chain in its single crystal. Our results show that the folding pattern of a PEO chain in the crystal formed in dilute solution follows the adjacent re-entry folding model. While in the crystal obtained from the melt, the nonadjacent folding with large and irregular loops contributes to big force fluctuations in the force-extension curves. The method established here can offer a novel strategy to directly unravel the chain-folding pattern of polymer single crystals at single-molecule level.

Homebuilt single-molecule scanning confocal fluorescence microscope studies of single DNA/protein interactions.

PubMed

Zheng, Haocheng; Goldner, Lori S; Leuba, Sanford H

2007-03-01

Many technical improvements in fluorescence microscopy over the years have focused on decreasing background and increasing the signal to noise ratio (SNR). The scanning confocal fluorescence microscope (SCFM) represented a major improvement in these efforts. The SCFM acquires signal from a thin layer of a thick sample, rejecting light whose origin is not in the focal plane thereby dramatically decreasing the background signal. A second major innovation was the advent of high quantum-yield, low noise, single-photon counting detectors. The superior background rejection of SCFM combined with low-noise, high-yield detectors makes it possible to detect the fluorescence from single-dye molecules. By labeling a DNA molecule or a DNA/protein complex with a donor/acceptor dye pair, fluorescence resonance energy transfer (FRET) can be used to track conformational changes in the molecule/complex itself, on a single molecule/complex basis. In this methods paper, we describe the core concepts of SCFM in the context of a study that uses FRET to reveal conformational fluctuations in individual Holliday junction DNA molecules and nucleosomal particles. We also discuss data processing methods for SCFM.

Finding a Single Molecule in a Haystack: Optical Detection and Spectroscopy of Single Absorbers in Solids

DTIC Science & Technology

1989-08-18

CODES 18 SUBJECT TERMS (Continue on reverse if necessary and identify by block number) FIELD GROUP SUB-GROUP Single Molecule Detection Pentacene in p...and 10 additional pentacene molecules. This may be accomplished by- a combination of laser FM spectroscopy and either Stark or ultrasonic double...6099 408-927-2426 ABSTRACT: Single-absorber optical spectroscopy in solids is described for the case of finding a single pentacene molecule in a

PREFACE: Nanoelectronics, sensors and single molecule biophysics Nanoelectronics, sensors and single molecule biophysics

NASA Astrophysics Data System (ADS)

Tao, Nongjian

2012-04-01

This special section of Journal of Physics: Condensed Matter (JPCM) is dedicated to Professor Stuart M Lindsay on the occasion of his 60th birthday and in recognition of his outstanding contributions to multiple research areas, including light scattering spectroscopy, scanning probe microscopy, biophysics, solid-liquid interfaces and molecular and nanoelectronics. It contains a collection of 14 papers in some of these areas, including a feature article by Lindsay. Each paper was subject to the normal rigorous review process of JPCM. In Lindsay's paper, he discusses the next generations of hybrid chemical-CMOS devices for low cost and personalized medical diagnosis. The discussion leads to several papers on nanotechnology for biomedical applications. Kawaguchi et al report on the detection of single pollen allergen particles using electrode embedded microchannels. Stern et al describe a structural study of three-dimensional DNA-nanoparticle assemblies. Hihath et al measure the conductance of methylated DNA, and discuss the possibility of electrical detection DNA methylation. Portillo et al study the electrostatic effects on the aggregation of prion proteins and peptides with atomic force microscopy. In an effort to understand the interactions between nanostructures and cells, Lamprecht et al report on the mapping of the intracellular distribution of carbon nanotubes with a confocal Raman imaging technique, and Wang et al focus on the intracellular delivery of gold nanoparticles using fluorescence microscopy. Park and Kristic provide theoretical analysis of micro- and nano-traps and their biological applications. This section also features several papers on the fundamentals of electron transport in single atomic wires and molecular junctions. The papers by Xu et al and by Wandlowksi et al describe new methods to measure conductance and forces in single molecule junctions and metallic atomic wires. Scullion et al report on the conductance of molecules with similar

Lucky Imaging: Improved Localization Accuracy for Single Molecule Imaging

PubMed Central

Cronin, Bríd; de Wet, Ben; Wallace, Mark I.

2009-01-01

We apply the astronomical data-analysis technique, Lucky imaging, to improve resolution in single molecule fluorescence microscopy. We show that by selectively discarding data points from individual single-molecule trajectories, imaging resolution can be improved by a factor of 1.6 for individual fluorophores and up to 5.6 for more complex images. The method is illustrated using images of fluorescent dye molecules and quantum dots, and the in vivo imaging of fluorescently labeled linker for activation of T cells. PMID:19348772

Contact and Length Dependent Effects in Single-Molecule Electronics

NASA Astrophysics Data System (ADS)

Hines, Thomas

Understanding charge transport in single molecules covalently bonded to electrodes is a fundamental goal in the field of molecular electronics. In the past decade, it has become possible to measure charge transport on the single-molecule level using the STM break junction method. Measurements on the single-molecule level shed light on charge transport phenomena which would otherwise be obfuscated by ensemble measurements of groups of molecules. This thesis will discuss three projects carried out using STM break junction. In the first project, the transition between two different charge transport mechanisms is reported in a set of molecular wires. The shortest wires show highly length dependent and temperature invariant conductance behavior, whereas the longer wires show weakly length dependent and temperature dependent behavior. This trend is consistent with a model whereby conduction occurs by coherent tunneling in the shortest wires and by incoherent hopping in the longer wires. Measurements are supported with calculations and the evolution of the molecular junction during the pulling process is investigated. The second project reports controlling the formation of single-molecule junctions by means of electrochemically reducing two axial-diazonium terminal groups on a molecule, thereby producing direct Au-C covalent bonds in-situ between the molecule and gold electrodes. Step length analysis shows that the molecular junction is significantly more stable, and can be pulled over a longer distance than a comparable junction created with amine anchoring bonds. The stability of the junction is explained by the calculated lower binding energy associated with the direct Au-C bond compared with the Au-N bond. Finally, the third project investigates the role that molecular conformation plays in the conductance of oligothiophene single-molecule junctions. Ethyl substituted oligothiophenes were measured and found to exhibit temperature dependent conductance and transition

Multiplexed single-molecule force spectroscopy using a centrifuge.

PubMed

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.

Multiplexed single-molecule force spectroscopy using a centrifuge

PubMed Central

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

2016-01-01

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. PMID:26984516

Fluorescence Excitation of Single Molecules,

DTIC Science & Technology

localized neighborhoods and to optical addressing of local spots in solids may now be envisioned. The purpose of this presentation is to show that single...molecules can be studied at helium temperatures by means of a fairly simple setup, at least in the very favorable case of pentacene in terphenyl

Drug-DNA interactions at single molecule level: A view with optical tweezers

NASA Astrophysics Data System (ADS)

Paramanathan, Thayaparan

Studies of small molecule--DNA interactions are essential for developing new drugs for challenging diseases like cancer and HIV. The main idea behind developing these molecules is to target and inhibit the reproduction of the tumor cells and infected cells. We mechanically manipulate single DNA molecule using optical tweezers to investigate two molecules that have complex and multiple binding modes. Mononuclear ruthenium complexes have been extensively studied as a test for rational drug design. Potential drug candidates should have high affinity to DNA and slow dissociation kinetics. To achieve this, motifs of the ruthenium complexes are altered. Our collaborators designed a dumb-bell shaped binuclear ruthenium complex that can only intercalate DNA by threading through its bases. Studying the binding properties of this complex in bulk studies took hours. By mechanically manipulating a single DNA molecule held with optical tweezers, we lower the barrier to thread and make it fast compared to the bulk experiments. Stretching single DNA molecules with different concentration of drug molecules and holding it at a constant force allows the binding to reach equilibrium. By this we can obtain the equilibrium fractional ligand binding and length of DNA at saturated binding. Fitting these results yields quantitative measurements of the binding thermodynamics and kinetics of this complex process. The second complex discussed in this study is Actinomycin D (ActD), a well studied anti-cancer agent that is used as a prototype for developing new generations of drugs. However, the biophysical basis of its activity is still unclear. Because ActD is known to intercalate double stranded DNA (dsDNA), it was assumed to block replication by stabilizing dsDNA in front of the replication fork. However, recent studies have shown that ActD binds with even higher affinity to imperfect duplexes and some sequences of single stranded DNA (ssDNA). We directly measure the on and off rates by

Isolated single-molecule magnets on native gold.

PubMed

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.

Single-molecule experiments in biological physics: methods and applications.

PubMed

Ritort, F

2006-08-16

I review single-molecule experiments (SMEs) in biological physics. Recent technological developments have provided the tools to design and build scientific instruments of high enough sensitivity and precision to manipulate and visualize individual molecules and measure microscopic forces. Using SMEs it is possible to manipulate molecules one at a time and measure distributions describing molecular properties, characterize the kinetics of biomolecular reactions and detect molecular intermediates. SMEs provide additional information about thermodynamics and kinetics of biomolecular processes. This complements information obtained in traditional bulk assays. In SMEs it is also possible to measure small energies and detect large Brownian deviations in biomolecular reactions, thereby offering new methods and systems to scrutinize the basic foundations of statistical mechanics. This review is written at a very introductory level, emphasizing the importance of SMEs to scientists interested in knowing the common playground of ideas and the interdisciplinary topics accessible by these techniques. The review discusses SMEs from an experimental perspective, first exposing the most common experimental methodologies and later presenting various molecular systems where such techniques have been applied. I briefly discuss experimental techniques such as atomic-force microscopy (AFM), laser optical tweezers (LOTs), magnetic tweezers (MTs), biomembrane force probes (BFPs) and single-molecule fluorescence (SMF). I then present several applications of SME to the study of nucleic acids (DNA, RNA and DNA condensation) and proteins (protein-protein interactions, protein folding and molecular motors). Finally, I discuss applications of SMEs to the study of the nonequilibrium thermodynamics of small systems and the experimental verification of fluctuation theorems. I conclude with a discussion of open questions and future perspectives.

Monitoring Single-Molecule Protein Dynamics with a Carbon Nanotube Transistor

NASA Astrophysics Data System (ADS)

Collins, Philip G.

2014-03-01

Nanoscale electronic devices like field-effect transistors have long promised to provide sensitive, label-free detection of biomolecules. Single-walled carbon nanotubes press this concept further by not just detecting molecules but also monitoring their dynamics in real time. Recent measurements have demonstrated this premise by monitoring the single-molecule processivity of three different enzymes: lysozyme, protein Kinase A, and the Klenow fragment of DNA polymerase I. With all three enzymes, single molecules tethered to nanotube transistors were electronically monitored for 10 or more minutes, allowing us to directly observe a range of activity including rare transitions to chemically inactive and hyperactive conformations. The high bandwidth of the nanotube transistors further allow every individual chemical event to be clearly resolved, providing excellent statistics from tens of thousands of turnovers by a single enzyme. Initial success with three different enzymes indicates the generality and attractiveness of the nanotube devices as a new tool to complement other single-molecule techniques. Research on transduction mechanisms provides the design rules necessary to further generalize this architecture and apply it to other proteins. The purposeful incorporation of just one amino acid is sufficient to fabricate effective, single molecule sensors from a wide range of enzymes or proteins.

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

PubMed Central

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

Electron transport in single molecules: from benzene to graphene.

PubMed

Chen, F; Tao, N J

2009-03-17

Electron movement within and between molecules--that is, electron transfer--is important in many chemical, electrochemical, and biological processes. Recent advances, particularly in scanning electrochemical microscopy (SECM), scanning-tunneling microscopy (STM), and atomic force microscopy (AFM), permit the study of electron movement within single molecules. In this Account, we describe electron transport at the single-molecule level. We begin by examining the distinction between electron transport (from semiconductor physics) and electron transfer (a more general term referring to electron movement between donor and acceptor). The relation between these phenomena allows us to apply our understanding of single-molecule electron transport between electrodes to a broad range of other electron transfer processes. Electron transport is most efficient when the electron transmission probability via a molecule reaches 100%; the corresponding conductance is then 2e(2)/h (e is the charge of the electron and h is the Planck constant). This ideal conduction has been observed in a single metal atom and a string of metal atoms connected between two electrodes. However, the conductance of a molecule connected to two electrodes is often orders of magnitude less than the ideal and strongly depends on both the intrinsic properties of the molecule and its local environment. Molecular length, means of coupling to the electrodes, the presence of conjugated double bonds, and the inclusion of possible redox centers (for example, ferrocene) within the molecular wire have a pronounced effect on the conductance. This complex behavior is responsible for diverse chemical and biological phenomena and is potentially useful for device applications. Polycyclic aromatic hydrocarbons (PAHs) afford unique insight into electron transport in single molecules. The simplest one, benzene, has a conductance much less than 2e(2)/h due to its large LUMO-HOMO gap. At the other end of the spectrum, graphene

Single-Molecule Analysis for RISC Assembly and Target Cleavage.

PubMed

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.

PubMed

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.

Peering into Cells One Molecule at a Time: Single-molecule and plasmon-enhanced fluorescence super-resolution imaging

NASA Astrophysics Data System (ADS)

Biteen, Julie

2013-03-01

Single-molecule fluorescence brings the resolution of optical microscopy down to the nanometer scale, allowing us to unlock the mysteries of how biomolecules work together to achieve the complexity that is a cell. This high-resolution, non-destructive method for examining subcellular events has opened up an exciting new frontier: the study of macromolecular localization and dynamics in living cells. We have developed methods for single-molecule investigations of live bacterial cells, and have used these techniques to investigate thee important prokaryotic systems: membrane-bound transcription activation in Vibrio cholerae, carbohydrate catabolism in Bacteroides thetaiotaomicron, and DNA mismatch repair in Bacillus subtilis. Each system presents unique challenges, and we will discuss the important methods developed for each system. Furthermore, we use the plasmon modes of bio-compatible metal nanoparticles to enhance the emissivity of single-molecule fluorophores. The resolution of single-molecule imaging in cells is generally limited to 20-40 nm, far worse than the 1.5-nm localization accuracies which have been attained in vitro. We use plasmonics to improve the brightness and stability of single-molecule probes, and in particular fluorescent proteins, which are widely used for bio-imaging. We find that gold-coupled fluorophores demonstrate brighter, longer-lived emission, yielding an overall enhancement in total photons detected. Ultimately, this results in increased localization accuracy for single-molecule imaging. Furthermore, since fluorescence intensity is proportional to local electromagnetic field intensity, these changes in decay intensity and rate serve as a nm-scale read-out of the field intensity. Our work indicates that plasmonic substrates are uniquely advantageous for super-resolution imaging, and that plasmon-enhanced imaging is a promising technique for improving live cell single-molecule microscopy.

Simulation of single-molecule trapping in a nanochannel

PubMed Central

Robinson, William Neil; Davis, Lloyd M.

2010-01-01

The detection and trapping of single fluorescent molecules in solution within a nanochannel is studied using numerical simulations. As optical forces are insufficient for trapping molecules much smaller than the optical wavelength, a means for sensing a molecule’s position along the nanochannel and adjusting electrokinetic motion to compensate diffusion is assessed. Fluorescence excitation is provided by two adjacently focused laser beams containing temporally interleaved laser pulses. Photon detection is time-gated, and the displacement of the molecule from the middle of the two foci alters the count rates collected in the two detection channels. An algorithm for feedback control of the electrokinetic motion in response to the timing of photons, to reposition the molecule back toward the middle for trapping and to rapidly reload the trap after a molecule photobleaches or escapes, is evaluated. While accommodating the limited electrokinetic speed and the finite latency of feedback imposed by experimental hardware, the algorithm is shown to be effective for trapping fast-diffusing single-chromophore molecules within a micron-sized confocal region. Studies show that there is an optimum laser power for which loss of molecules from the trap due to either photobleaching or shot-noise fluctuations is minimized. PMID:20799801

Single molecule studies of flexible polymers under shear and mixed flows

NASA Astrophysics Data System (ADS)

Teixeira, Rodrigo Esquivel

We combine manipulation and single molecule visualization of flexible DNA polymers with the generation of controlled simple shear and planar mixed flows for the investigation of polymer flow physics. With the ability to observe polymer conformation directly and follow its evolution in both dilute and entangled regimes we provide a direct test for molecular models. The coil-stretch transition of polymer extension was investigated in planar mixed flows approaching simple shear. Visualization of individual molecules revealed a sharp coil-stretch transition in the steady-state length of the polymer with increasing strain rate in flows slightly more straining than rotational. In slightly more rotational flows significant transient polymer deformation was observed. Next, dilute polymers were visualized in the flow-gradient plane of a steady shear flow. By exploiting the linear proportionality between polymer mass and image intensity, the radius of gyration tensor elements ( Gij) were measured over time. Then, the Giesekus stress tensor was used to obtain the bulk shear viscosity and first normal stress coefficient, thus performing rheology measurements from single molecule conformations. End-over-end tumbling was discovered for the first time, confirming a long-standing prediction and numerous single-chain computer simulation studies. The tumbling frequency followed Wi0.62, and an equation derived from simple advection and diffusion arguments was able to reproduce these observations. Power spectral densities of chain orientation trajectories were found to be single-peaked around the tumbling frequency, thus suggesting a periodic character for polymer dynamics. Finally, we investigated well-entangled polymer solutions. Identical preparations were used in both rheological characterizations and single molecule observations under a variety of shear flow histories. Polymer extension relaxations after the cessation of a fast shear flow revealed two intrinsic characteristic

Single molecules and single nanoparticles as windows to the nanoscale

NASA Astrophysics Data System (ADS)

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

PubMed Central

Balogh, Zoltán; Makk, Péter

2015-01-01

Summary 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. PMID:26199840

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

PubMed

Balogh, Zoltán; Makk, Péter; Halbritter, András

2015-01-01

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.

Direct Observation of Quantum Coherence in Single-Molecule Magnets

NASA Astrophysics Data System (ADS)

Schlegel, C.; van Slageren, J.; Manoli, M.; Brechin, E. K.; Dressel, M.

2008-10-01

Direct evidence of quantum coherence in a single-molecule magnet in a frozen solution is reported with coherence times as long as T2=630±30ns. We can strongly increase the coherence time by modifying the matrix in which the single-molecule magnets are embedded. The electron spins are coupled to the proton nuclear spins of both the molecule itself and, interestingly, also to those of the solvent. The clear observation of Rabi oscillations indicates that we can manipulate the spin coherently, an essential prerequisite for performing quantum computations.

Using a nanopore for single molecule detection and single cell transfection.

PubMed

Nelson, Edward M; Kurz, Volker; Shim, Jiwook; Timp, Winston; Timp, Gregory

2012-07-07

We assert that it is possible to trap and identify proteins, and even (conceivably) manipulate proteins secreted from a single cell (i.e. the secretome) through transfection via electroporation by exploiting the exquisite control over the electrostatic potential available in a nanopore. These capabilities may be leveraged for single cell analysis and transfection with single molecule resolution, ultimately enabling a careful scrutiny of tissue heterogeneity.

High Pressure EPR for Probing the Magnetic Anisotropy in Single Molecule Magnets

NASA Astrophysics Data System (ADS)

Bhaskaran, Lakshmi; Trociewitz, Bianca; Dubroca, Thierry; Hill, Stephen

Single-molecule magnets (SMM) are potential candidates for nanoscale magnetic information storage, and a platform for studying classical and quantum behaviors at the mesoscopic scale. Varying the structures of these molecules by chemical modification can give rise to changes in their magnetic properties. However, this approach can be unpredictable, leaving very little control via chemical synthesis. An alternate approach is to exert physical pressure. This convenient tool can be used to vary crystal packing, local coordination geometries, as well as inter-ion and intermolecular interactions without changing the chemical composition of a SMM. Moreover, pressure in combination with Electron Paramagnetic Resonance (EPR), can be employed to better understand the factors that control magnetic anisotropy, both at the single-ion level and in exchange-coupled molecules. Here we present a microwave cavity integrated with a diamond anvil cell with a pressure range up to 1.5 GPa. As an example we show results from single crystal high field EPR experiments performed on an exchange coupled system, [Fe8O2(OH)12(tacn)6] Br8.9H2O, better known as Fe8 with a giant spin of S =10. The obtained pressure-dependent results will be discussed. National High Magnetic Field Laboratory.

Mass action at the single-molecule level.

PubMed

Shon, Min Ju; Cohen, Adam E

2012-09-05

We developed a system to reversibly encapsulate small numbers of molecules in an array of nanofabricated "dimples". This system enables highly parallel, long-term, and attachment-free studies of molecular dynamics via single-molecule fluorescence. In studies of bimolecular reactions of small numbers of confined molecules, we see phenomena that, while expected from basic statistical mechanics, are not observed in bulk chemistry. Statistical fluctuations in the occupancy of sealed reaction chambers lead to steady-state fluctuations in reaction equilibria and rates. These phenomena are likely to be important whenever reactions happen in confined geometries.

Force-Manipulation Single-Molecule Spectroscopy Studies of Enzymatic Dynamics

NASA Astrophysics Data System (ADS)

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

2014-03-01

Subtle conformational changes play a crucial role in protein functions, especially in enzymatic reactions involving complex substrate-enzyme interactions and chemical reactions. We applied AFM-enhanced and magnetic tweezers-correlated single-molecule spectroscopy to study the mechanisms and dynamics of enzymatic reactions involved with kinase and lysozyme proteins. Enzymatic reaction turnovers and the associated structure changes of individual protein molecules were observed simultaneously in real-time by single-molecule FRET detections. Our single-molecule spectroscopy measurements of enzymatic conformational dynamics have revealed time bunching effect and intermittent coherence in conformational state change dynamics involving in enzymatic reaction cycles. The coherent conformational state dynamics suggests that the enzymatic catalysis involves a multi-step conformational motion along the coordinates of substrate-enzyme complex formation and product releasing. Our results support a multiple-conformational state model, being consistent with a complementary conformation selection and induced-fit enzymatic loop-gated conformational change mechanism in substrate-enzyme active complex formation.

Quantum transport of the single metallocene molecule

NASA Astrophysics Data System (ADS)

Yu, Jing-Xin; Chang, Jing; Wei, Rong-Kai; Liu, Xiu-Ying; Li, Xiao-Dong

2016-10-01

The Quantum transport of three single metallocene molecule is investigated by performing theoretical calculations using the non-equilibrium Green's function method combined with density functional theory. We find that the three metallocen molecules structure become stretched along the transport direction, the distance between two Cp rings longer than the other theory and experiment results. The lager conductance is found in nickelocene molecule, the main transmission channel is the electron coupling between molecule and the electrodes is through the Ni dxz and dyz orbitals and the s, dxz, dyz of gold. This is also confirmed by the highest occupied molecular orbital resonance at Fermi level. In addition, negative differential resistance effect is found in the ferrocene, cobaltocene molecules, this is also closely related with the evolution of the transmission spectrum under applied bias.

Single molecule data under scrutiny. Comment on "Extracting physics of life at the molecular level: A review of single-molecule data analyses" by W. Colomb & S.K. Sarkar

NASA Astrophysics Data System (ADS)

Wohland, Thorsten

2015-06-01

Single Molecule Detection and Spectroscopy have grown from their first beginnings into mainstream, mature research areas that are widely applied in the biological sciences. However, despite the advances in technology and the application of many single molecule techniques even in in vivo settings, the data analysis of single molecule experiments is complicated by noise, systematic errors, and complex underlying processes that are only incompletely understood. Colomb and Sarkar provide in this issue an overview of single molecule experiments and the accompanying problems in data analysis, which have to be overcome for a proper interpretation of the experiments [1].

Single-Molecule Spectroscopy and Imaging Studies of Protein Dynamics

NASA Astrophysics Data System (ADS)

Lu, H. Peter

2012-04-01

Enzymatic reactions and protein-protein interactions are traditionally studied at the ensemble level, despite significant static and dynamic inhomogeneities. Subtle conformational changes play a crucial role in protein functions, and these protein conformations are highly dynamic rather than being static. We applied AFM-enhanced single-molecule spectroscopy to study the mechanisms and dynamics of enzymatic reactions involved with kinase and lysozyme proteins. Enzymatic reaction turnovers and the associated structure changes of individual protein molecules were observed simultaneously in real-time by single-molecule FRET detections. Our single-molecule spectroscopy measurements of T4 lysozyme and HPPK enzymatic conformational dynamics have revealed time bunching effect and intermittent coherence in conformational state change dynamics involving in enzymatic reaction cycles. The coherent conformational state dynamics suggests that the enzymatic catalysis involves a multi-step conformational motion along the coordinates of substrate-enzyme complex formation and product releasing, presenting as an extreme dynamic behavior intrinsically related to the time bunching effect that we have reported previously. Our results of HPPK interaction with substrate support a multiple-conformational state model, being consistent with a complementary conformation selection and induced-fit enzymatic loop-gated conformational change mechanism in substrate-enzyme active complex formation. Our new approach is applicable to a wide range of single-molecule FRET measurements for protein conformational changes under enzymatic reactions.

Modified atomic force microscope applied to the measurement of elastic modulus for a single peptide molecule

NASA Astrophysics Data System (ADS)

Ptak, Arkadiusz; Takeda, Seiji; Nakamura, Chikashi; Miyake, Jun; Kageshima, Masami; Jarvis, Suzanne P.; Tokumoto, Hiroshi

2001-09-01

A modified atomic force microscopy (AFM) system, based on a force modulation technique, has been used to find an approximate value for the elastic modulus of a single peptide molecule directly from a mechanical test. For this purpose a self-assembled monolayer built from two kinds of peptides, reactive (able to anchor to the AFM tip) and nonreactive, was synthesized. In a typical experiment a single C3K30C (C=cysteine, K=lysine) peptide molecule was stretched between a Au(111) substrate and the gold-coated tip of an AFM cantilever to which it was attached via gold-sulfur bonds. The amplitude of the cantilever oscillations, due to an external force applied via a magnetic particle to the cantilever, was recorded by a lock-in amplifier and recalculated into stiffness of the stretched molecule. A longitudinal Young's modulus for the α-helix of a single peptide molecule and for the elongated state of this molecule has been estimated. The obtained values; 1.2±0.3 and 50±15 GPa, for the peptide α-helix and elongated peptide backbone, respectively, seem to be reasonable comparing them to the Young's modulus of protein crystals and linear organic polymers. We believe this research opens up a means by which scientists can perform quantitative studies of the elastic properties of single molecule, especially of biologically important polymers like peptides or DNA.

Figuration and detection of single molecules

NASA Astrophysics Data System (ADS)

Nevels, R.; Welch, G. R.; Cremer, P. S.; Hemmer, P.; Phillips, T.; Scully, S.; Sokolov, A. V.; Svidzinsky, A. A.; Xia, H.; Zheltikov, A.; Scully, M. O.

2012-08-01

Recent advances in the description of atoms and molecules based on Dimensional scaling analysis, developed by Dudley Herschbach and co-workers, provided new insights into visualization of molecular structure and chemical bonding. Prof. Herschbach is also a giant in the field of single molecule scattering. We here report on the engineering of molecular detectors. Such systems have a wide range of application from medical diagnostics to the monitoring of chemical, biological and environmental hazards. We discuss ways to identify preselected molecules, in particular, mycotoxin contaminants using coherent laser spectroscopy. Mycotoxin contaminants, e.g. aflatoxin B1 which is present in corn and peanuts, are usually analysed by time-consuming microscopic, chemical and biological assays. We present a new approach that derives from recent experiments in which molecules are prepared by one (or more) femtosecond laser(s) and probed by another set. We call this technique FAST CARS (femto second adaptive spectroscopic technique for coherent anti-Stokes Raman spectroscopy). We propose and analyse ways in which FAST CARS can be used to identify preselected molecules, e.g. aflatoxin, rapidly and economically.

Single-molecule spectromicroscopy: a route towards sub-wavelength refractometry.

PubMed

Anikushina, T A; Gladush, M G; Gorshelev, A A; Naumov, A V

2015-01-01

We suggest a novel approach for spatially resolved probing of local fluctuations of the refractive index n in solids by means of single-molecule (SM) spectroscopy. It is based on the dependence T1(n) of the effective radiative lifetime T1 of dye centres in solids on n due to the local-field effects. Detection of SM zero-phonon lines at low temperatures gives the values of the SM natural spectral linewidth (which is inversely proportional to T1) and makes it possible to reveal the distribution of the local n values in solids. Here we demonstrate this possibility on the example of amorphous polyethylene and polycrystalline naphthalene doped with terrylene. In particular, we show that the obtained distributions of lifetime limited spectral linewidths of terrylene molecules embedded into these matrices are due to the spatial fluctuations of the refractive index local values.

Ultrasensitive Laser Spectroscopy in Solids: Single-Molecule Detection

DTIC Science & Technology

1989-10-25

spite of detection intensity constraints necessary to avoid power broadening, the optical absorption spectrum of single molecules of pentacene In p...molecule detection, or SMD) would provide a useful tool for the study of local host-absorber interactions where tihe absorbing ,ontor is essentially at...modulation techniques 7. 8 for the model system composed of pentacene substitutional impurities in p-terphenyl crystals at 1.5K. The pontacene molecules can

Force and Conductance Spectroscopy of Single Molecule Junctions

NASA Astrophysics Data System (ADS)

Frei, Michael

Investigation of mechanical properties of single molecule junctions is crucial to develop an understanding and enable control of single molecular junctions. This work presents an experimental and analytical approach that enables the statistical evaluation of force and simultaneous conductance data of metallic atomic point contacts and molecular junctions. A conductive atomic force microscope based break junction technique is developed to form single molecular junctions and collect conductance and force data simultaneously. Improvements of the optical components have been achieved through the use of a super-luminescent diode, enabling tremendous increases in force resolution. An experimental procedure to collect data for various molecular junctions has been developed and includes deposition, calibration, and analysis methods. For the statistical analysis of force, novel approaches based on two dimensional histograms and a direct force identification method are presented. The two dimensional method allows for an unbiased evaluation of force events that are identified using corresponding conductance signatures. This is not always possible however, and in these situations, the force based identification of junction rearrangement events is an attractive alternative method. This combined experimental and analytical approach is then applied to three studies: First, the impact of molecular backbones to the mechanical behavior of single molecule junctions is investigated and it is found that junctions formed with identical linkers but different backbone structure result in junctions with varying breaking forces. All molecules used show a clear molecular signature and force data can be evaluated using the 2D method. Second, the effects of the linker group used to attach molecules to gold electrodes are investigated. A study of four alkane molecules with different linkers finds a drastic difference in the evolution of donor-acceptor and covalently bonded molecules

The optics inside an automated single molecule array analyzer

NASA Astrophysics Data System (ADS)

McGuigan, William; Fournier, David R.; Watson, Gary W.; Walling, Les; Gigante, Bill; Duffy, David C.; Rissin, David M.; Kan, Cheuk W.; Meyer, Raymond E.; Piech, Tomasz; Fishburn, Matthew W.

2014-02-01

Quanterix and Stratec Biomedical have developed an instrument that enables the automated measurement of multiple proteins at concentration ~1000 times lower than existing immunoassays. The instrument is based on Quanterix's proprietary Single Molecule Array technology (Simoa™ ) that facilitates the detection and quantification of biomarkers previously difficult to measure, thus opening up new applications in life science research and in-vitro diagnostics. Simoa is based on trapping individual beads in arrays of femtoliter-sized wells that, when imaged with sufficient resolution, allows for counting of single molecules associated with each bead. When used to capture and detect proteins, this approach is known as digital ELISA (Enzyme-linked immunosorbent assay). The platform developed is a merger of many science and engineering disciplines. This paper concentrates on the optical technologies that have enabled the development of a fully-automated single molecule analyzer. At the core of the system is a custom, wide field-of-view, fluorescence microscope that images arrays of microwells containing single molecules bound to magnetic beads. A consumable disc containing 24 microstructure arrays was developed previously in collaboration with Sony DADC. The system cadence requirements, array dimensions, and requirement to detect single molecules presented significant optical challenges. Specifically, the wide field-of-view needed to image the entire array resulted in the need for a custom objective lens. Additionally, cost considerations for the system required a custom solution that leveraged the image processing capabilities. This paper will discuss the design considerations and resultant optical architecture that has enabled the development of an automated digital ELISA platform.

Single Molecule Spectral Diffusion in a Solid Detected Via Fluorescence Spectroscopy

DTIC Science & Technology

1991-10-15

other local fields) at the position of the molecule, the spectral jumps may occur because the class II pentacene molecules are coupled to an...and identify by block number) FIELD jGROUP SUB-GROUP_ Single molecule spectroscopy Precision detection Spectral diffusion, Pentacene in p-terphenyl 19...significant increases in detection sensitivity for single pentacene molecules in crystals of p-terphenyl at low temperatures. With the increased signal to

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xie, Xiaoliang Sunney

Single-molecule spectroscopy has made considerable impact on many disciplines including chemistry, physics, and biology. To date, most single-molecule spectroscopy work is accomplished by detecting fluorescence. On the other hand, many naturally occurring chromophores, such as retinal, hemoglobin and cytochromes, do not have detectable fluorescence. There is an emerging need for single-molecule spectroscopy techniques that do not require fluorescence. In the last proposal period, we have successfully demonstrated stimulated emission microscopy, single molecule absorption, and stimulated Raman microscopy based on a high-frequency modulation transfer technique. These first-of-a- kind new spectroscopy/microscopy methods tremendously improved our ability to observe molecules that fluorescence weakly,more » even to the limit of single molecule detection for absorption measurement. All of these methods employ two laser beams: one (pump beam) excites a single molecule to a real or virtual excited state, and the other (probe beam) monitors the absorption/emission property of the single. We extract the intensity change of the probe beam with high sensitivity by implementing a high-frequency phase-sensitive detection scheme, which offers orders of magnitude improvement in detection sensitivity over direct absorption/emission measurement. However, single molecule detection based on fluorescence or absorption is fundamentally limited due to their broad spectral response. It is important to explore other avenues in single molecule detection and imaging which provides higher molecular specificity for studying a wide variety of heterogeneous chemical and biological systems. This proposal aimed to achieve single-molecule detection sensitivity with near resonance stimulated Raman scattering (SRS) microscopy. SRS microscopy was developed in our lab as a powerful technique for imaging heterogeneous samples based on their intrinsic vibrational contrasts, which provides much higher

Optimization of cell morphology measurement via single-molecule tracking PALM.

PubMed

Frost, Nicholas A; Lu, Hsiangmin E; Blanpied, Thomas A

2012-01-01

In neurons, the shape of dendritic spines relates to synapse function, which is rapidly altered during experience-dependent neural plasticity. The small size of spines makes detailed measurement of their morphology in living cells best suited to super-resolution imaging techniques. The distribution of molecular positions mapped via live-cell Photoactivated Localization Microscopy (PALM) is a powerful approach, but molecular motion complicates this analysis and can degrade overall resolution of the morphological reconstruction. Nevertheless, the motion is of additional interest because tracking single molecules provides diffusion coefficients, bound fraction, and other key functional parameters. We used Monte Carlo simulations to examine features of single-molecule tracking of practical utility for the simultaneous determination of cell morphology. We find that the accuracy of determining both distance and angle of motion depend heavily on the precision with which molecules are localized. Strikingly, diffusion within a bounded region resulted in an inward bias of localizations away from the edges, inaccurately reflecting the region structure. This inward bias additionally resulted in a counterintuitive reduction of measured diffusion coefficient for fast-moving molecules; this effect was accentuated by the long camera exposures typically used in single-molecule tracking. Thus, accurate determination of cell morphology from rapidly moving molecules requires the use of short integration times within each image to minimize artifacts caused by motion during image acquisition. Sequential imaging of neuronal processes using excitation pulses of either 2 ms or 10 ms within imaging frames confirmed this: processes appeared erroneously thinner when imaged using the longer excitation pulse. Using this pulsed excitation approach, we show that PALM can be used to image spine and spine neck morphology in living neurons. These results clarify a number of issues involved in

Measurement and statistical analysis of single-molecule current-voltage characteristics, transition voltage spectroscopy, and tunneling barrier height.

PubMed

Guo, Shaoyin; Hihath, Joshua; Díez-Pérez, Ismael; Tao, Nongjian

2011-11-30

We report on the measurement and statistical study of thousands of current-voltage characteristics and transition voltage spectra (TVS) of single-molecule junctions with different contact geometries that are rapidly acquired using a new break junction method at room temperature. This capability allows one to obtain current-voltage, conductance voltage, and transition voltage histograms, thus adding a new dimension to the previous conductance histogram analysis at a fixed low-bias voltage for single molecules. This method confirms the low-bias conductance values of alkanedithiols and biphenyldithiol reported in literature. However, at high biases the current shows large nonlinearity and asymmetry, and TVS allows for the determination of a critically important parameter, the tunneling barrier height or energy level alignment between the molecule and the electrodes of single-molecule junctions. The energy level alignment is found to depend on the molecule and also on the contact geometry, revealing the role of contact geometry in both the contact resistance and energy level alignment of a molecular junction. Detailed statistical analysis further reveals that, despite the dependence of the energy level alignment on contact geometry, the variation in single-molecule conductance is primarily due to contact resistance rather than variations in the energy level alignment.

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

PubMed

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.

Single-molecule imaging in live bacteria cells.

PubMed

Ritchie, Ken; Lill, Yoriko; Sood, Chetan; Lee, Hochan; Zhang, Shunyuan

2013-02-05

Bacteria, such as Escherichia coli and Caulobacter crescentus, are the most studied and perhaps best-understood organisms in biology. The advances in understanding of living systems gained from these organisms are immense. Application of single-molecule techniques in bacteria have presented unique difficulties owing to their small size and highly curved form. The aim of this review is to show advances made in single-molecule imaging in bacteria over the past 10 years, and to look to the future where the combination of implementing such high-precision techniques in well-characterized and controllable model systems such as E. coli could lead to a greater understanding of fundamental biological questions inaccessible through classic ensemble methods.

Probe-based measurement of lateral single-electron transfer between individual molecules

PubMed Central

Steurer, Wolfram; Fatayer, Shadi; Gross, Leo; Meyer, Gerhard

2015-01-01

The field of molecular electronics aims at using single molecules as functional building blocks for electronics components, such as switches, rectifiers or transistors. A key challenge is to perform measurements with atomistic control over the alignment of the molecule and its contacting electrodes. Here we use atomic force microscopy to examine charge transfer between weakly coupled pentacene molecules on insulating films with single-electron sensitivity and control over the atomistic details. We show that, in addition to the imaging capability, the probe tip can be used to control the charge state of individual molecules and to detect charge transfers to/from the tip, as well as between individual molecules. Our approach represents a novel route for molecular charge transfer studies with a host of opportunities, especially in combination with single atom/molecule manipulation and nanopatterning techniques. PMID:26387533

Nanodevices for Single Molecule Studies

NASA Astrophysics Data System (ADS)

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

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

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

PubMed

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.

Deep learning for single-molecule science

NASA Astrophysics Data System (ADS)

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.

Microfluidic mixing for non-equilibrium single-molecule optical spectroscopy

NASA Astrophysics Data System (ADS)

Pfeil, Shawn H.

binds cocaine put a lower bound on its folding rate of 100 s-1. Single-Molecule measurements at equilibrium, in conjunction with ensemble measurements, demonstrate that this system can be described thermodynamically with a two-state model. In addition, standard free energy changes for folding, in the presence and absence of 1 mM cocaine, are obtained. Ensemble chemiluminescence experiments demonstrate the broad applicability of themixing system. The luciferase-luciferin system from Photinus pyralis, the North American firefly, is studied. Kinetically resolved chemiluminescence measurements are performed using only 50 femtomoles of target, and microliters of sample. This compares favorably to stopped-flow, which requires milliliters of sample and picomoles of target.

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

PubMed

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.

Single molecules can operate as primitive biological sensors, switches and oscillators.

PubMed

Hernansaiz-Ballesteros, Rosa D; Cardelli, Luca; Csikász-Nagy, Attila

2018-06-18

Switch-like and oscillatory dynamical systems are widely observed in biology. We investigate the simplest biological switch that is composed of a single molecule that can be autocatalytically converted between two opposing activity forms. We test how this simple network can keep its switching behaviour under perturbations in the system. We show that this molecule can work as a robust bistable system, even for alterations in the reactions that drive the switching between various conformations. We propose that this single molecule system could work as a primitive biological sensor and show by steady state analysis of a mathematical model of the system that it could switch between possible states for changes in environmental signals. Particularly, we show that a single molecule phosphorylation-dephosphorylation switch could work as a nucleotide or energy sensor. We also notice that a given set of reductions in the reaction network can lead to the emergence of oscillatory behaviour. We propose that evolution could have converted this switch into a single molecule oscillator, which could have been used as a primitive timekeeper. We discuss how the structure of the simplest known circadian clock regulatory system, found in cyanobacteria, resembles the proposed single molecule oscillator. Besides, we speculate if such minimal systems could have existed in an RNA world.

Intranucleus Single-Molecule Imaging in Living Cells.

PubMed

Shao, Shipeng; Xue, Boxin; Sun, Yujie

2018-06-01

Many critical processes occurring in mammalian cells are stochastic and can be directly observed at the single-molecule level within their physiological environment, which would otherwise be obscured in an ensemble measurement. There are various fundamental processes in the nucleus, such as transcription, replication, and DNA repair, the study of which can greatly benefit from intranuclear single-molecule imaging. However, the number of such studies is relatively small mainly because of lack of proper labeling and imaging methods. In the past decade, tremendous efforts have been devoted to developing tools for intranuclear imaging. Here, we mainly describe the recent methodological developments of single-molecule imaging and their emerging applications in the live nucleus. We also discuss the remaining issues and provide a perspective on future developments and applications of this field. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Counting numbers of synaptic proteins: absolute quantification and single molecule imaging techniques

PubMed Central

Patrizio, Angela; Specht, Christian G.

2016-01-01

Abstract. The ability to count molecules is essential to elucidating cellular mechanisms, as these often depend on the absolute numbers and concentrations of molecules within specific compartments. Such is the case at chemical synapses, where the transmission of information from presynaptic to postsynaptic terminals requires complex interactions between small sets of molecules. Be it the subunit stoichiometry specifying neurotransmitter receptor properties, the copy numbers of scaffold proteins setting the limit of receptor accumulation at synapses, or protein packing densities shaping the molecular organization and plasticity of the postsynaptic density, all of these depend on exact quantities of components. A variety of proteomic, electrophysiological, and quantitative imaging techniques have yielded insights into the molecular composition of synaptic complexes. In this review, we compare the different quantitative approaches and consider the potential of single molecule imaging techniques for the quantification of synaptic components. We also discuss specific neurobiological data to contextualize the obtained numbers and to explain how they aid our understanding of synaptic structure and function. PMID:27335891

Counting numbers of synaptic proteins: absolute quantification and single molecule imaging techniques.

PubMed

Patrizio, Angela; Specht, Christian G

2016-10-01

The ability to count molecules is essential to elucidating cellular mechanisms, as these often depend on the absolute numbers and concentrations of molecules within specific compartments. Such is the case at chemical synapses, where the transmission of information from presynaptic to postsynaptic terminals requires complex interactions between small sets of molecules. Be it the subunit stoichiometry specifying neurotransmitter receptor properties, the copy numbers of scaffold proteins setting the limit of receptor accumulation at synapses, or protein packing densities shaping the molecular organization and plasticity of the postsynaptic density, all of these depend on exact quantities of components. A variety of proteomic, electrophysiological, and quantitative imaging techniques have yielded insights into the molecular composition of synaptic complexes. In this review, we compare the different quantitative approaches and consider the potential of single molecule imaging techniques for the quantification of synaptic components. We also discuss specific neurobiological data to contextualize the obtained numbers and to explain how they aid our understanding of synaptic structure and function.

Identifying Mechanisms of Interfacial Dynamics Using Single-Molecule Tracking

PubMed Central

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

2012-01-01

The “soft” (i.e. non-covalent) interactions between molecules and surfaces are complex and highly-varied (e.g. hydrophobic, hydrogen bonding, ionic) often leading to heterogeneous interfacial behavior. Heterogeneity can arise either from spatial variation of the surface/interface itself or from molecular configurations (i.e. conformation, orientation, aggregation state, etc.). By observing adsorption, diffusion, and desorption of individual fluorescent molecules, single-molecule tracking can characterize these types of heterogeneous interfacial behavior in ways that are inaccessible to traditional ensemble-averaged methods. Moreover, the fluorescence intensity or emission wavelength (in resonance energy transfer experiments) can be used to simultaneously track molecular configuration and directly relate this to the resulting interfacial mobility or affinity. In this feature article, we review recent advances involving the use of single-molecule tracking to characterize heterogeneous molecule-surface interactions including: multiple modes of diffusion and desorption associated with both internal and external molecular configuration, Arrhenius activated interfacial transport, spatially dependent interactions, and many more. PMID:22716995

Thermal bleaching in single fluorescent molecules under two-photon excitation regime

NASA Astrophysics Data System (ADS)

Chirico, Giuseppe; Cannone, Fabio; Baldini, Giancarlo; Diaspro, Alberto

2003-07-01

Single molecule spectroscopy often requires the immobilization of the molecules onto solid or quasi-solid substrates and the use of relatively high excitation intensity We have studied the fluorescence emission of four common dyes used for bio-imaging studies, rhodamine 6G, fluorescein, pyrene and indo-1 at the single molecule level under two-photon excitation regime. We focus on two-photon excitation thermal effects on the stability of the single molecules, influencing the internal photo-dynamics and the total duration of the fluorescent emission. Single dye molecules, spread on a glass substrate by spin coating, show a constant fluorescence output till a sudden transition to a dark state very close to the background. The bleaching time that is found to vary in the series pyrene, Indo-1, fluorescein and rhodamine 6G from the fastest to the slowest one respectively, has a Gaussian distribution suggesting that bleaching is not due to photo-bleaching. Moreover it shows a correlation to the amount of absorbed power not re-irradiated as fluorescence and to the complexity of the molecule. These observations are interpreted as thermal bleaching where the temperature increase is induced by the two-photon excitation process. This study should be extended to different trapping media of interest in single molecule basic research and applications, such as silica and polyacrylamide gels or nanosctructured polyelectrolyte matrices. We think that the observed behavior and the correlations found to the molecular chemical and physical parameters, may be of some help for the design of molecules with switching on-off behavior of longer duration.

Real-time single-molecule imaging of quantum interference.

PubMed

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.

Real-time single-molecule imaging of quantum interference

NASA Astrophysics Data System (ADS)

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

2012-05-01

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.

Conductance of Single Molecule Junctions: Dependence on Structure and Conformation

NASA Astrophysics Data System (ADS)

Venkataraman, Latha

2007-03-01

We recently demonstrated that the conductance of single molecule junctions formed by breaking Au point contacts in an environment of molecules with amine linkages can be measured reliably and reproducibly^1. We have now studied junctions formed by approximately 30 different amine terminated molecules, allowing systematic study of the correlation between molecular properties and single molecule junction conductance. This talk will focus on the relation between molecular conductance and molecule conformation for the simple case of a biphenyl, two benzene rings linked together by a single C-C bond. Our results from a series of seven biphenyl derivatives show that the molecular junction conductance depends on the twist angle. Specifically, we find that the planar molecule has the highest conductance, and the conductance for the series decreases with increasing twist angle, consistent with a cosine squared relation predicted theoretically^2. 1. L. Venkataraman, J.E. Klare, I.W. Tam, C. Nuckolls, M.S Hybertsen and M. Steigerwald, Nano Letters, vol. 5, pp. 458-462, 2006. 2. L. Venkataraman, J.E. Klare, C. Nuckolls, M.S Hybertsen and M. Steigerwald, Nature, vol. 442, pp. 904-907, 2006.

Single Molecule Approaches in RNA-Protein Interactions.

PubMed

Serebrov, Victor; Moore, Melissa J

RNA-protein interactions govern every aspect of RNA metabolism, and aberrant RNA-binding proteins are the cause of hundreds of genetic diseases. Quantitative measurements of these interactions are necessary in order to understand mechanisms leading to diseases and to develop efficient therapies. Existing methods of RNA-protein interactome capture can afford a comprehensive snapshot of RNA-protein interaction networks but lack the ability to characterize the dynamics of these interactions. As all ensemble methods, their resolution is also limited by statistical averaging. Here we discuss recent advances in single molecule techniques that have the potential to tackle these challenges. We also provide a thorough overview of single molecule colocalization microscopy and the essential protein and RNA tagging and detection techniques.

Photoinduced electron transfer of oxazine 1/TiO2 nanoparticles at single molecule level by using confocal fluorescence microscopy.

PubMed

Chen, Yi-Ju; Tzeng, Hsin-Yu; Fan, Hsiu-Fang; Chen, Ming-Shiang; Huang, Jer-Shing; Lin, King-Chuen

2010-06-01

Kinetics of photoinduced electron transfer (ET) from oxazine 1 dye to TiO(2) nanoparticles (NPs) surface is studied at a single molecule level by using confocal fluorescence microscopy. Upon irradiation with a pulsed laser at 630 nm, the fluorescence lifetimes sampled among 100 different dye molecules are determined to yield an average lifetime of 2.9 +/- 0.3 ns, which is close to the value of 3.0 +/- 0.6 ns measured on the bare coverslip. The lifetime proximity suggests that most interfacial electron transfer (IFET) processes for the current system are inefficient, probably caused by physisorption between dye and the TiO(2) film. However, there might exist some molecules which are quenched before fluorescing and fail to be detected. With the aid of autocorrelation analysis under a three-level energy system, the IFET kinetics of single dye molecules in the conduction band of TiO(2) NPs is evaluated to be (1.0 +/- 0.1) x 10(4) s(-1) averaged over 100 single molecules and the back ET rate constant is 4.7 +/- 0.9 s(-1). When a thicker TiO(2) film is substituted, the resultant kinetic data do not make a significant difference. The trend of IFET efficacy agrees with the method of fluorescence lifetime measurements. The obtained forward ET rate constants are about ten times smaller than the photovoltage response measured in an assembled dye-sensitized solar cell. The discrepancy is discussed. The inhomogeneous and fluctuation characters for the IFET process are attributed to microenvironment variation for each single molecule. The obtained ET rates are much slower than the fluorescence relaxation. Such a small ET quantum yield is yet feasibly detectable at a single molecule level.

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

NASA Astrophysics Data System (ADS)

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

Atomic-Scale Control of Electron Transport through Single Molecules

NASA Astrophysics Data System (ADS)

Wang, Y. F.; Kröger, J.; Berndt, R.; Vázquez, H.; Brandbyge, M.; Paulsson, M.

2010-04-01

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 of the surface electrode. Nonequilibrium Green’s function calculations reproduce the trend of the conductance and visualize the current flow through the junction, which is guided through molecule-electrode chemical bonds.

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

PubMed

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

2013-02-05

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.

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

PubMed Central

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

Nanosecond to submillisecond dynamics in dye-labeled single-stranded DNA, as revealed by ensemble measurements and photon statistics at single-molecule level.

PubMed

Kaji, Takahiro; Ito, Syoji; Iwai, Shigenori; Miyasaka, Hiroshi

2009-10-22

Single-molecule and ensemble time-resolved fluorescence measurements were applied for the investigation of the conformational dynamics of single-stranded DNA, ssDNA, connected with a fluorescein dye by a C6 linker, where the motions both of DNA and the C6 linker affect the geometry of the system. From the ensemble measurement of the fluorescence quenching via photoinduced electron transfer with a guanine base in the DNA sequence, three main conformations were found in aqueous solution: a conformation unaffected by the guanine base in the excited state lifetime of fluorescein, a conformation in which the fluorescence is dynamically quenched in the excited-state lifetime, and a conformation leading to rapid quenching via nonfluorescent complex. The analysis by using the parameters acquired from the ensemble measurements for interphoton time distribution histograms and FCS autocorrelations by the single-molecule measurement revealed that interconversion in these three conformations took place with two characteristic time constants of several hundreds of nanoseconds and tens of microseconds. The advantage of the combination use of the ensemble measurements with the single-molecule detections for rather complex dynamic motions is discussed by integrating the experimental results with those obtained by molecular dynamics simulation.

Molecular spintronics using single-molecule magnets

NASA Astrophysics Data System (ADS)

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.

Single cell and single molecule techniques for the analysis of the epigenome

NASA Astrophysics Data System (ADS)

Wallin, Christopher Benjamin

Epigenetic regulation is a critical biological process for the health and development of a cell. Epigenetic regulation is facilitated by covalent modifications to the underlying DNA and chromatin proteins. A fundamental understanding of these epigenetic modifications and their associated interactions at the molecular scale is necessary to explain phenomena including cellular identity, stem cell plasticity, and neoplastic transformation. It is widely known that abnormal epigenetic profiles have been linked to many diseases, most notably cancer. While the field of epigenetics has progressed rapidly with conventional techniques, significant advances remain to be made with respect to combinatoric analysis of epigenetic marks and single cell epigenetics. Therefore, in this dissertation, I will discuss our development of devices and methodologies to address these pertinent issues. First, we designed a preparatory polydimethylsiloxane (PDMS) microdevice for the extraction, purification, and stretching of human chromosomal DNA and chromatin from small cell populations down to a single cell. The valveless device captures cells by size exclusion within the micropillars, entraps the DNA or chromatin in the micropillars after cell lysis, purifies away the cellular debris, and fluorescently labels the DNA and/or chromatin all within a single reaction chamber. With the device, we achieve nearly 100% extraction efficiency of the DNA. The device is also used for in-channel immunostaining of chromatin followed by downstream single molecule chromatin analysis in nanochannels (SCAN). Second, using multi-color, time-correlated single molecule measurements in nanochannels, simultaneous coincidence detection of 2 epigenetic marks is demonstrated. Coincidence detection of 3 epigenetic marks is also established using a pulsed interleaved excitation scheme. With these two promising results, genome-wide quantification of epigenetic marks was pursued. Unfortunately, quantitative SCAN never

High sensitivity fluorescent single particle and single molecule detection apparatus and method

DOEpatents

Mathies, Richard A.; Peck, Konan; Stryer, Lubert

1990-01-01

Apparatus is described for ultrasensitive detection of single fluorescent particles down to the single fluorescent molecule limit in a fluid or on a substrate comprising means for illuminating a predetermined volume of the fluid or area of the substrate whereby to emit light including background light from the fluid and burst of photons from particles residing in the area. The photon burst is detected in real time to generate output representative signal. The signal is received and the burst of energy from the fluorescent particles is distinguished from the background energy to provide an indication of the number, location or concentration of the particles or molecules.

Ultrasensitive Laser Spectroscopy in Solids: Statistical Fine Structure and Single-Molecule Detection

DTIC Science & Technology

1990-03-28

observation, detection of the optical absorption of a single pentacene molecule in a p-terphenyl crystal, opens the door to new studies of single local ...produce appreciable quadratic Stark shifting. Such effects would greatly perturb the local field around the pentacene molecule, making detection of the...of the local surroundings of pentacene molecules with single injected charge carriers nearby may become an interesting field; however, for the

Giant light-harvesting nanoantenna for single-molecule detection in ambient light

PubMed Central

Trofymchuk, Kateryna; Reisch, Andreas; Didier, Pascal; Fras, François; Gilliot, Pierre; Mely, Yves; Klymchenko, Andrey S.

2017-01-01

Here, we explore the enhancement of single molecule emission by polymeric nano-antenna that can harvest energy from thousands of donor dyes to a single acceptor. In this nano-antenna, the cationic dyes are brought together in very close proximity using bulky counterions, thus enabling ultrafast diffusion of excitation energy (≤30 fs) with minimal losses. Our 60-nm nanoparticles containing >10,000 rhodamine-based donor dyes can efficiently transfer energy to 1-2 acceptors resulting in an antenna effect of ~1,000. Therefore, single Cy5-based acceptors become 25-fold brighter than quantum dots QD655. This unprecedented amplification of the acceptor dye emission enables observation of single molecules at illumination powers (1-10 mW cm-2) that are >10,000-fold lower than typically required in single-molecule measurements. Finally, using a basic setup, which includes a 20X air objective and a sCMOS camera, we could detect single Cy5 molecules by simply shining divergent light on the sample at powers equivalent to sunlight. PMID:28983324

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

PubMed Central

Shashkova, Sviatlana

2017-01-01

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. PMID:28694303

Single-molecule dilution and multiple displacement amplification for molecular haplotyping.

PubMed

Paul, Philip; Apgar, Josh

2005-04-01

Separate haploid analysis is frequently required for heterozygous genotyping to resolve phase ambiguity or confirm allelic sequence. We demonstrate a technique of single-molecule dilution followed by multiple strand displacement amplification to haplotype polymorphic alleles. Dilution of DNA to haploid equivalency, or a single molecule, is a simple method for separating di-allelic DNA. Strand displacement amplification is a robust method for non-specific DNA expansion that employs random hexamers and phage polymerase Phi29 for double-stranded DNA displacement and primer extension, resulting in high processivity and exceptional product length. Single-molecule dilution was followed by strand displacement amplification to expand separated alleles to microgram quantities of DNA for more efficient haplotype analysis of heterozygous genes.

Single molecule sequencing of the M13 virus genome without amplification.

PubMed

Zhao, Luyang; Deng, Liwei; Li, Gailing; Jin, Huan; Cai, Jinsen; Shang, Huan; Li, Yan; Wu, Haomin; Xu, Weibin; Zeng, Lidong; Zhang, Renli; Zhao, Huan; Wu, Ping; Zhou, Zhiliang; Zheng, Jiao; Ezanno, Pierre; Yang, Andrew X; Yan, Qin; Deem, Michael W; He, Jiankui

2017-01-01

Next generation sequencing (NGS) has revolutionized life sciences research. However, GC bias and costly, time-intensive library preparation make NGS an ill fit for increasing sequencing demands in the clinic. A new class of third-generation sequencing platforms has arrived to meet this need, capable of directly measuring DNA and RNA sequences at the single-molecule level without amplification. Here, we use the new GenoCare single-molecule sequencing platform from Direct Genomics to sequence the genome of the M13 virus. Our platform detects single-molecule fluorescence by total internal reflection microscopy, with sequencing-by-synthesis chemistry. We sequenced the genome of M13 to a depth of 316x, with 100% coverage. We determined a consensus sequence accuracy of 100%. In contrast to GC bias inherent to NGS results, we demonstrated that our single-molecule sequencing method yields minimal GC bias.

Single-Molecule Resolution of Antimicrobial Peptide Interactions with Supported Lipid A Bilayers.

PubMed

Nelson, Nathaniel; Schwartz, Daniel K

2018-06-05

The molecular interactions between antimicrobial peptides (AMPs) and lipid A-containing supported lipid bilayers were probed using single-molecule total internal reflection fluorescence microscopy. Hybrid supported lipid bilayers with lipid A outer leaflets and phospholipid (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)) inner leaflets were prepared and characterized, and the spatiotemporal trajectories of individual fluorescently labeled LL37 and Melittin AMPs were determined as they interacted with the bilayer surfaces comprising either monophosphoryl or diphosphoryl lipid A (from Escherichia coli) to determine the impact of electrostatic interactions. Large numbers of trajectories were obtained and analyzed to obtain the distributions of surface residence times and the statistics of the spatial trajectories. Interestingly, the AMP species were sensitive to subtle differences in the charge of the lipid, with both peptides diffusing more slowly and residing longer on the diphosphoryl lipid A. Furthermore, the single-molecule dynamics indicated a qualitative difference between the behavior of AMPs on hybrid Lipid A bilayers and on those composed entirely of DOPE. Whereas AMPs interacting with a DOPE bilayer exhibited two-dimensional Brownian diffusion with a diffusion coefficient of ∼1.7 μm 2 /s, AMPs adsorbed to the lipid A surface exhibited much slower apparent diffusion (on the order of ∼0.1 μm 2 /s) and executed intermittent trajectories that alternated between two-dimensional Brownian diffusion and desorption-mediated three-dimensional flights. Overall, these findings suggested that bilayers with lipid A in the outer leaflet, as it is in bacterial outer membranes, are valuable model systems for the study of the initial stage of AMP-bacterium interactions. Furthermore, single-molecule dynamics was sensitive to subtle differences in electrostatic interactions between cationic AMPs and monovalent or divalent anionic lipid A moieties. Copyright �

Biology and polymer physics at the single-molecule level.

PubMed

Chu, Steven

2003-04-15

The ability to look at individual molecules has given us new insights into molecular processes. Examples of our recent work are given to illustrate how behaviour that may otherwise be hidden from view can be clearly seen in single-molecule experiments.

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

PubMed

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.

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

PubMed Central

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

PubMed Central

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

Single molecule sequencing of the M13 virus genome without amplification

PubMed Central

Zhao, Luyang; Deng, Liwei; Li, Gailing; Jin, Huan; Cai, Jinsen; Shang, Huan; Li, Yan; Wu, Haomin; Xu, Weibin; Zeng, Lidong; Zhang, Renli; Zhao, Huan; Wu, Ping; Zhou, Zhiliang; Zheng, Jiao; Ezanno, Pierre; Yang, Andrew X.; Yan, Qin; Deem, Michael W.; He, Jiankui

2017-01-01

Next generation sequencing (NGS) has revolutionized life sciences research. However, GC bias and costly, time-intensive library preparation make NGS an ill fit for increasing sequencing demands in the clinic. A new class of third-generation sequencing platforms has arrived to meet this need, capable of directly measuring DNA and RNA sequences at the single-molecule level without amplification. Here, we use the new GenoCare single-molecule sequencing platform from Direct Genomics to sequence the genome of the M13 virus. Our platform detects single-molecule fluorescence by total internal reflection microscopy, with sequencing-by-synthesis chemistry. We sequenced the genome of M13 to a depth of 316x, with 100% coverage. We determined a consensus sequence accuracy of 100%. In contrast to GC bias inherent to NGS results, we demonstrated that our single-molecule sequencing method yields minimal GC bias. PMID:29253901

Single Lipid Molecule Dynamics on Supported Lipid Bilayers with Membrane Curvature.

PubMed

Cheney, Philip P; Weisgerber, Alan W; Feuerbach, Alec M; Knowles, Michelle K

2017-03-15

The plasma membrane is a highly compartmentalized, dynamic material and this organization is essential for a wide variety of cellular processes. Nanoscale domains allow proteins to organize for cell signaling, endo- and exocytosis, and other essential processes. Even in the absence of proteins, lipids have the ability to organize into domains as a result of a variety of chemical and physical interactions. One feature of membranes that affects lipid domain formation is membrane curvature. To directly test the role of curvature in lipid sorting, we measured the accumulation of two similar lipids, 1,2-Dihexadecanoyl- sn -glycero-3-phosphoethanolamine (DHPE) and hexadecanoic acid (HDA), using a supported lipid bilayer that was assembled over a nanopatterned surface to obtain regions of membrane curvature. Both lipids studied contain 16 carbon, saturated tails and a head group tag for fluorescence microscopy measurements. The accumulation of lipids at curvatures ranging from 28 nm to 55 nm radii was measured and fluorescein labeled DHPE accumulated more than fluorescein labeled HDA at regions of membrane curvature. We then tested whether single biotinylated DHPE molecules sense curvature using single particle tracking methods. Similar to groups of fluorescein labeled DHPE accumulating at curvature, the dynamics of single molecules of biotinylated DHPE was also affected by membrane curvature and highly confined motion was observed.

In situ single molecule imaging of cell membranes: linking basic nanotechniques to cell biology, immunology and medicine.

PubMed

Pi, Jiang; Jin, Hua; Yang, Fen; Chen, Zheng W; Cai, Jiye

2014-11-07

The cell membrane, which consists of a viscous phospholipid bilayer, different kinds of proteins and various nano/micrometer-sized domains, plays a very important role in ensuring the stability of the intracellular environment and the order of cellular signal transductions. Exploring the precise cell membrane structure and detailed functions of the biomolecules in a cell membrane would be helpful to understand the underlying mechanisms involved in cell membrane signal transductions, which could further benefit research into cell biology, immunology and medicine. The detection of membrane biomolecules at the single molecule level can provide some subtle information about the molecular structure and the functions of the cell membrane. In particular, information obtained about the molecular mechanisms and other information at the single molecule level are significantly different from that detected from a large amount of biomolecules at the large-scale through traditional techniques, and can thus provide a novel perspective for the study of cell membrane structures and functions. However, the precise investigations of membrane biomolecules prompts researchers to explore cell membranes at the single molecule level by the use of in situ imaging methods, as the exact conformation and functions of biomolecules are highly controlled by the native cellular environment. Recently, the in situ single molecule imaging of cell membranes has attracted increasing attention from cell biologists and immunologists. The size of biomolecules and their clusters on the cell surface are set at the nanoscale, which makes it mandatory to use high- and super-resolution imaging techniques to realize the in situ single molecule imaging of cell membranes. In the past few decades, some amazing imaging techniques and instruments with super resolution have been widely developed for molecule imaging, which can also be further employed for the in situ single molecule imaging of cell membranes. In

In situ single molecule imaging of cell membranes: linking basic nanotechniques to cell biology, immunology and medicine

NASA Astrophysics Data System (ADS)

Pi, Jiang; Jin, Hua; Yang, Fen; Chen, Zheng W.; Cai, Jiye

2014-10-01

The cell membrane, which consists of a viscous phospholipid bilayer, different kinds of proteins and various nano/micrometer-sized domains, plays a very important role in ensuring the stability of the intracellular environment and the order of cellular signal transductions. Exploring the precise cell membrane structure and detailed functions of the biomolecules in a cell membrane would be helpful to understand the underlying mechanisms involved in cell membrane signal transductions, which could further benefit research into cell biology, immunology and medicine. The detection of membrane biomolecules at the single molecule level can provide some subtle information about the molecular structure and the functions of the cell membrane. In particular, information obtained about the molecular mechanisms and other information at the single molecule level are significantly different from that detected from a large amount of biomolecules at the large-scale through traditional techniques, and can thus provide a novel perspective for the study of cell membrane structures and functions. However, the precise investigations of membrane biomolecules prompts researchers to explore cell membranes at the single molecule level by the use of in situ imaging methods, as the exact conformation and functions of biomolecules are highly controlled by the native cellular environment. Recently, the in situ single molecule imaging of cell membranes has attracted increasing attention from cell biologists and immunologists. The size of biomolecules and their clusters on the cell surface are set at the nanoscale, which makes it mandatory to use high- and super-resolution imaging techniques to realize the in situ single molecule imaging of cell membranes. In the past few decades, some amazing imaging techniques and instruments with super resolution have been widely developed for molecule imaging, which can also be further employed for the in situ single molecule imaging of cell membranes. In

Detection of Single Molecules Illuminated by a Light-Emitting Diode

PubMed Central

Gerhardt, Ilja; Mai, Lijian; Lamas-Linares, Antía; Kurtsiefer, Christian

2011-01-01

Optical detection and spectroscopy of single molecules has become an indispensable tool in biological imaging and sensing. Its success is based on fluorescence of organic dye molecules under carefully engineered laser illumination. In this paper we demonstrate optical detection of single molecules on a wide-field microscope with an illumination based on a commercially available, green light-emitting diode. The results are directly compared with laser illumination in the same experimental configuration. The setup and the limiting factors, such as light transfer to the sample, spectral filtering and the resulting signal-to-noise ratio are discussed. A theoretical and an experimental approach to estimate these parameters are presented. The results can be adapted to other single emitter and illumination schemes. PMID:22346610

Nanogap Electrodes towards Solid State Single-Molecule Transistors.

PubMed

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.

Single-Molecule Detection in Micron-Sized Capillaries

NASA Astrophysics Data System (ADS)

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

2004-11-01

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

Using Multiorder Time-Correlation Functions (TCFs) To Elucidate Biomolecular Reaction Pathways from Microsecond Single-Molecule Fluorescence Experiments.

PubMed

Phelps, Carey; Israels, Brett; Marsh, Morgan C; von Hippel, Peter H; Marcus, Andrew H

2016-12-29

Recent advances in single-molecule fluorescence imaging have made it possible to perform measurements on microsecond time scales. Such experiments have the potential to reveal detailed information about the conformational changes in biological macromolecules, including the reaction pathways and dynamics of the rearrangements involved in processes, such as sequence-specific DNA "breathing" and the assembly of protein-nucleic acid complexes. Because microsecond-resolved single-molecule trajectories often involve "sparse" data, that is, they contain relatively few data points per unit time, they cannot be easily analyzed using the standard protocols that were developed for single-molecule experiments carried out with tens-of-millisecond time resolution and high "data density." Here, we describe a generalized approach, based on time-correlation functions, to obtain kinetic information from microsecond-resolved single-molecule fluorescence measurements. This approach can be used to identify short-lived intermediates that lie on reaction pathways connecting relatively long-lived reactant and product states. As a concrete illustration of the potential of this methodology for analyzing specific macromolecular systems, we accompany the theoretical presentation with the description of a specific biologically relevant example drawn from studies of reaction mechanisms of the assembly of the single-stranded DNA binding protein of the T4 bacteriophage replication complex onto a model DNA replication fork.

Manipulating, Reacting, and Constructing Single Molecules with a Scanning Tunneling Microscope Tip

NASA Astrophysics Data System (ADS)

Hla, S.-W.

The fascinating advances in atom and molecule manipulation with the scanning tunneling microscope (STM) tip allow scientists to fabricate artificial atomic scale structures, to study local quantum phenomena, or to probe physical and chemical properties of single atoms and molecules on surfaces. Recent achievements in individual synthesis of single molecules with the STM tip further open up an entirely new opportunities in nanoscience and technology. The STM manipulation techniques usef ul in the molecular construction are reviewed and prospects for future opportunities of single molecule chemical engineering and their possible implications to nano-scale science and technology are discussed.

Rapid and efficient detection of single chromophore molecules in aqueous solution

NASA Astrophysics Data System (ADS)

Li, Li-Qiang; Davis, Lloyd M.

1995-06-01

The first experiments on the detection of single fluorescent molecules in a flowing stream of an aqueous solution with high total efficiency are reported. A capillary injection system for sample delivery causes all the dye molecules to pass in a diffusion-broadened stream within a fast-moving sheath flow, through the center of the tightly focused laser excitation beam. Single-molecule detection with a transit time of approximately 1 ms is accomplished with a high-quantum-efficiency single-photon avalanche diode and a low dead-time time-gating circuit for discrimination of Raman-scattered light from the solvent.

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

PubMed

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.

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

NASA Astrophysics Data System (ADS)

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

Theoretical Investigation of Single-Molecule Sensing Using Nanotube-Enhanced Circular Dichroism.

PubMed

Silva, Jaime; Milne, Bruce F; Nogueira, Fernando

2018-06-19

First-principles calculations have been used to investigate the potential use of circular dichroism (CD) spectroscopy in single-molecule sensing. Using a real-space implementation of time-dependent density functional theory (TDDFT), several systems involving single-walled carbon nanotubes (SWCNT) and small molecules have been studied to evaluate their CD response. Large induced CD (ICD) effects, differing for each test molecule, were observed in all SWCNT-molecule complexes. As the SWCNT used in this study shows no intrinsic CD response, the ICD spectra are the result of interaction with the small molecules. This finding is general and independent of the (a)chiral nature of the adsorbed molecule. Our results indicate that it is possible to design a system that uses SWCNT for detection of molecules using the change in CD spectrum of the system induced by adsorption of the molecule onto the SWCNT surface.

Single molecule microscopy in 3D cell cultures and tissues.

PubMed

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.

Electron transfer dynamics of bistable single-molecule junctions.

PubMed

Danilov, Andrey V; Kubatkin, Sergey E; Kafanov, Sergey G; Flensberg, Karsten; Bjørnholm, Thomas

2006-10-01

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.

Quantum-Sequencing: Fast electronic single DNA molecule sequencing

NASA Astrophysics Data System (ADS)

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.

Superconducting molybdenum-rhenium electrodes for single-molecule transport studies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gaudenzi, R.; Island, J. O.; Bruijckere, J. de

2015-06-01

We demonstrate that electronic transport through single molecules or molecular ensembles, commonly based on gold (Au) electrodes, can be extended to superconducting electrodes by combining gold with molybdenum-rhenium (MoRe). This combination induces proximity-effect superconductivity in the gold to temperatures of at least 4.6 K and magnetic fields of 6 T, improving on previously reported aluminum based superconducting nanojunctions. As a proof of concept, we show three-terminal superconductive transport measurements through an individual Fe{sub 4} single-molecule magnet.

New Antifouling Platform Characterized by Single-Molecule Imaging

PubMed Central

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.

PubMed

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.

Towards monitoring conformational changes of the GPCR neurotensin receptor 1 by single-molecule FRET

NASA Astrophysics Data System (ADS)

Heitkamp, Thomas; Grisshammer, Reinhard; Börsch, Michael

2018-02-01

Neurotensin receptor 1 (NTSR1) is a G protein-coupled receptor that is important for signaling in the brain and the gut. Its agonist ligand neurotensin (NTS), a 13-amino-acid peptide, binds with nanomolar affinity from the extracellular side to NTSR1 and induces conformational changes that trigger intracellular signaling processes. Our goal is to monitor the conformational dynamics of single fluorescently labeled NTSR1. For this, we fused the fluorescent protein mNeonGreen to the C terminus of NTSR1, purified the receptor fusion protein from E. coli membranes, and reconstituted NTSR1 into liposomes with E. coli polar lipids. Using single-molecule anisotropy measurements, NTSR1 was found to be monomeric in liposomes, with a small fraction being dimeric and oligomeric, showing homoFRET. Similar results were obtained for NTSR1 in detergent solution. Furthermore, we demonstrated agonist binding to NTSR1 by time-resolved single-molecule Förster resonance energy transfer (smFRET), using neurotensin labeled with the fluorophore ATTO594.

Detailed analysis of complex single molecule FRET data with the software MASH

NASA Astrophysics Data System (ADS)

Hadzic, Mélodie C. A. S.; Kowerko, Danny; Börner, Richard; Zelger-Paulus, Susann; Sigel, Roland K. O.

2016-04-01

The processing and analysis of surface-immobilized single molecule FRET (Förster resonance energy transfer) data follows systematic steps (e.g. single molecule localization, clearance of different sources of noise, selection of the conformational and kinetic model, etc.) that require a solid knowledge in optics, photophysics, signal processing and statistics. The present proceeding aims at standardizing and facilitating procedures for single molecule detection by guiding the reader through an optimization protocol for a particular experimental data set. Relevant features were determined from single molecule movies (SMM) imaging Cy3- and Cy5-labeled Sc.ai5γ group II intron molecules synthetically recreated, to test the performances of four different detection algorithms. Up to 120 different parameterizations per method were routinely evaluated to finally establish an optimum detection procedure. The present protocol is adaptable to any movie displaying surface-immobilized molecules, and can be easily reproduced with our home-written software MASH (multifunctional analysis software for heterogeneous data) and script routines (both available in the download section of www.chem.uzh.ch/rna).

Distinguishing Lead and Molecule States in Graphene-Based Single-Electron Transistors

PubMed Central

2017-01-01

Graphene provides a two-dimensional platform for contacting individual molecules, which enables transport spectroscopy of molecular orbital, spin, and vibrational states. Here we report single-electron tunneling through a molecule that has been anchored to two graphene leads. Quantum interference within the graphene leads gives rise to an energy-dependent transmission and fluctuations in the sequential tunnel-rates. The lead states are electrostatically tuned by a global back-gate, resulting in a distinct pattern of varying intensity in the measured conductance maps. This pattern could potentially obscure transport features that are intrinsic to the molecule under investigation. Using ensemble averaged magneto-conductance measurements, lead and molecule states are disentangled, enabling spectroscopic investigation of the single molecule. PMID:28423272

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

PubMed

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.

Single-molecule imaging of cytoplasmic dynein in vivo.

PubMed

Ananthanarayanan, Vaishnavi; Tolić, Iva M

2015-01-01

While early fluorescence microscopy experiments employing fluorescent probes afforded snapshots of the cell, the power of live-cell microscopy is required to understand complex dynamics in biological processes. The first successful cloning of green fluorescent protein in the 1990s paved the way for development of approaches that we now utilize for visualization in a living cell. In this chapter, we discuss a technique to observe fluorescently tagged single molecules in fission yeast. With a few simple modifications to the established total internal reflection fluorescence microscopy, cytoplasmic dynein molecules in the cytoplasm and on the microtubules can be visualized and their intracellular dynamics can be studied. We illustrate a technique to study motor behavior, which is not apparent in conventional ensemble studies of motors. In general, this technique can be employed to study single-molecule dynamics of fluorescently tagged proteins in the cell interior. Copyright © 2015 Elsevier Inc. All rights reserved.

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

NASA Astrophysics Data System (ADS)

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.

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

PubMed Central

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

2017-01-01

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. PMID:28382928

Single molecule photobleaching (SMPB) technology for counting of RNA, DNA, protein and other molecules in nanoparticles and biological complexes by TIRF instrumentation.

PubMed

Zhang, Hui; Guo, Peixuan

2014-05-15

Direct counting of biomolecules within biological complexes or nanomachines is demanding. Single molecule counting using optical microscopy is challenging due to the diffraction limit. The single molecule photobleaching (SMPB) technology for direct counting developed by our team (Shu et al., 2007 [18]; Zhang et al., 2007 [19]) offers a simple and straightforward method to determine the stoichiometry of molecules or subunits within biocomplexes or nanomachines at nanometer scales. Stoichiometry is determined by real-time observation of the number of descending steps resulted from the photobleaching of individual fluorophore. This technology has now been used extensively for single molecule counting of protein, RNA, and other macromolecules in a variety of complexes or nanostructures. Here, we elucidate the SMPB technology, using the counting of RNA molecules within a bacteriophage phi29 DNA-packaging biomotor as an example. The method described here can be applied to the single molecule counting of other molecules in other systems. The construction of a concise, simple and economical single molecule total internal reflection fluorescence (TIRF) microscope combining prism-type and objective-type TIRF is described. The imaging system contains a deep-cooled sensitive EMCCD camera with single fluorophore detection sensitivity, a laser combiner for simultaneous dual-color excitation, and a Dual-View™ imager to split the multiple outcome signals to different detector channels based on their wavelengths. Methodology of the single molecule photobleaching assay used to elucidate the stoichiometry of RNA on phi29 DNA packaging motor and the mechanism of protein/RNA interaction are described. Different methods for single fluorophore labeling of RNA molecules are reviewed. The process of statistical modeling to reveal the true copy number of the biomolecules based on binomial distribution is also described. Copyright © 2014 Elsevier Inc. All rights reserved.

Single-molecule fluorescence detection: autocorrelation criterion and experimental realization with phycoerythrin.

PubMed Central

Peck, K; Stryer, L; Glazer, A N; Mathies, R A

1989-01-01

A theory for single-molecule fluorescence detection is developed and then used to analyze data from subpicomolar solutions of B-phycoerythrin (PE). The distribution of detected counts is the convolution of a Poissonian continuous background with bursts arising from the passage of individual fluorophores through the focused laser beam. The autocorrelation function reveals single-molecule events and provides a criterion for optimizing experimental parameters. The transit time of fluorescent molecules through the 120-fl imaged volume was 800 microseconds. The optimal laser power (32 mW at 514.5 nm) gave an incident intensity of 1.8 x 10(23) photons.cm-2.s-1, corresponding to a mean time of 1.1 ns between absorptions. The mean incremental count rate was 1.5 per 100 microseconds for PE monomers and 3.0 for PE dimers above a background count rate of 1.0. The distribution of counts and the autocorrelation function for 200 fM monomer and 100 fM dimer demonstrate that single-molecule detection was achieved. At this concentration, the mean occupancy was 0.014 monomer molecules in the probed volume. A hard-wired version of this detection system was used to measure the concentration of PE down to 1 fM. This single-molecule counter is 3 orders of magnitude more sensitive than conventional fluorescence detection systems. PMID:2726766

Magnetic relaxation pathways in lanthanide single-molecule magnets.

PubMed

Blagg, Robin J; Ungur, Liviu; Tuna, Floriana; Speak, James; Comar, Priyanka; Collison, David; Wernsdorfer, Wolfgang; McInnes, Eric J L; Chibotaru, Liviu F; Winpenny, Richard E P

2013-08-01

Single-molecule magnets are compounds that exhibit magnetic bistability caused by an energy barrier for the reversal of magnetization (relaxation). Lanthanide compounds are proving promising as single-molecule magnets: recent studies show that terbium phthalocyanine complexes possess large energy barriers, and dysprosium and terbium complexes bridged by an N2(3-) radical ligand exhibit magnetic hysteresis up to 13 K. Magnetic relaxation is typically controlled by single-ion factors rather than magnetic exchange (whether one or more 4f ions are present) and proceeds through thermal relaxation of the lowest excited states. Here we report polylanthanide alkoxide cage complexes, and their doped diamagnetic yttrium analogues, in which competing relaxation pathways are observed and relaxation through the first excited state can be quenched. This leads to energy barriers for relaxation of magnetization that exceed 800 K. We investigated the factors at the lanthanide sites that govern this behaviour.

Single-molecule quantum dot as a Kondo simulator

NASA Astrophysics Data System (ADS)

Hiraoka, R.; Minamitani, E.; Arafune, R.; Tsukahara, N.; Watanabe, S.; Kawai, M.; Takagi, N.

2017-06-01

Structural flexibility of molecule-based systems is key to realizing the novel functionalities. Tuning the structure in the atomic scale enables us to manipulate the quantum state in the molecule-based system. Here we present the reversible Hamiltonian manipulation in a single-molecule quantum dot consisting of an iron phthalocyanine molecule attached to an Au electrode and a scanning tunnelling microscope tip. We precisely controlled the position of Fe2+ ion in the molecular cage by using the tip, and tuned the Kondo coupling between the molecular spins and the Au electrode. Then, we realized the crossover between the strong-coupling Kondo regime and the weak-coupling regime governed by spin-orbit interaction in the molecule. The results open an avenue to simulate low-energy quantum many-body physics and quantum phase transition through the molecular flexibility.

Simple horizontal magnetic tweezers for micromanipulation of single DNA molecules and DNA–protein complexes

PubMed Central

McAndrew, Christopher P.; Tyson, Christopher; Zischkau, Joseph; Mehl, Patrick; Tuma, Pamela L.; Pegg, Ian L.; Sarkar, Abhijit

2016-01-01

We report the development of a simple-to-implement magnetic force transducer that can apply a wide range of piconewton (pN) scale forces on single DNA molecules and DNA–protein complexes in the horizontal plane. The resulting low-noise force-extension data enable very high-resolution detection of changes in the DNA tether’s extension: ~0.05 pN in force and <10 nm change in extension. We have also verified that we can manipulate DNA in near equilibrium conditions through the wide range of forces by ramping the force from low to high and back again, and observing minimal hysteresis in the molecule’s force response. Using a calibration technique based on Stokes’ drag law, we have confirmed our force measurements from DNA force-extension experiments obtained using the fluctuation-dissipation theorem applied to transverse fluctuations of the magnetic microsphere. We present data on the force-distance characteristics of a DNA molecule complexed with histones. The results illustrate how the tweezers can be used to study DNA binding proteins at the single molecule level. PMID:26757808

A Single-Molecule Barcoding System using Nanoslits for DNA Analysis

NASA Astrophysics Data System (ADS)

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

Porous materials with pre-designed single-molecule traps for CO2 selective adsorption

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, JR; Yu, JM; Lu, WG

2013-02-26

Despite tremendous efforts, precise control in the synthesis of porous materials with pre-designed pore properties for desired applications remains challenging. Newly emerged porous metal-organic materials, such as metal-organic polyhedra and metal-organic frameworks, are amenable to design and property tuning, enabling precise control of functionality by accurate design of structures at the molecular level. Here we propose and validate, both experimentally and computationally, a precisely designed cavity, termed a 'single-molecule trap', with the desired size and properties suitable for trapping target CO2 molecules. Such a single-molecule trap can strengthen CO2-host interactions without evoking chemical bonding, thus showing potential for CO2 capture.more » Molecular single-molecule traps in the form of metal-organic polyhedra are designed, synthesised and tested for selective adsorption of CO2 over N-2 and CH4, demonstrating the trapping effect. Building these pre-designed single-molecule traps into extended frameworks yields metal-organic frameworks with efficient mass transfer, whereas the CO2 selective adsorption nature of single-molecule traps is preserved.« less

The bright future of single-molecule fluorescence imaging

PubMed Central

Juette, Manuel F.; Terry, Daniel S.; Wasserman, Michael R.; Zhou, Zhou; Altman, Roger B.; Zheng, Qinsi; Blanchard, Scott C.

2014-01-01

Single-molecule Förster resonance energy transfer (smFRET) is an essential and maturing tool to probe biomolecular interactions and conformational dynamics in vitro and, increasingly, in living cells. Multi-color smFRET enables the correlation of multiple such events and the precise dissection of their order and timing. However, the requirements for good spectral separation, high time resolution, and extended observation times place extraordinary demands on the fluorescent labels used in such experiments. Together with advanced experimental designs and data analysis, the development of long-lasting, non-fluctuating fluorophores is therefore proving key to progress in the field. Recently developed strategies for obtaining ultra-stable organic fluorophores spanning the visible spectrum are underway that will enable multi-color smFRET studies to deliver on their promise of previously unachievable biological insights. PMID:24956235

Single-Molecule Tracking and Its Application in Biomolecular Binding Detection

PubMed Central

Liu, Cong; Liu, Yen-Liang; Perillo, Evan P.; Dunn, Andrew K.; Yeh, Hsin-Chih

2016-01-01

In the past two decades significant advances have been made in single-molecule detection, which enables the direct observation of single biomolecules at work in real time and under physiological conditions. In particular, the development of single-molecule tracking (SMT) microscopy allows us to monitor the motion paths of individual biomolecules in living systems, unveiling the localization dynamics and transport modalities of the biomolecules that support the development of life. Beyond the capabilities of traditional camera-based tracking techniques, state-of-the-art SMT microscopies developed in recent years can record fluorescence lifetime while tracking a single molecule in the 3D space. This multiparameter detection capability can open the door to a wide range of investigations at the cellular or tissue level, including identification of molecular interaction hotspots and characterization of association/dissociation kinetics between molecules. In this review, we discuss various SMT techniques developed to date, with an emphasis on our recent development of the next generation 3D tracking system that not only achieves ultrahigh spatiotemporal resolution but also provides sufficient working depth suitable for live animal imaging. We also discuss the challenges that current SMT techniques are facing and the potential strategies to tackle those challenges. PMID:27660404

Single-Molecule Tracking and Its Application in Biomolecular Binding Detection.

PubMed

Liu, Cong; Liu, Yen-Liang; Perillo, Evan P; Dunn, Andrew K; Yeh, Hsin-Chih

2016-01-01

In the past two decades significant advances have been made in single-molecule detection, which enables the direct observation of single biomolecules at work in real time and under physiological conditions. In particular, the development of single-molecule tracking (SMT) microscopy allows us to monitor the motion paths of individual biomolecules in living systems, unveiling the localization dynamics and transport modalities of the biomolecules that support the development of life. Beyond the capabilities of traditional camera-based tracking techniques, state-of-the-art SMT microscopies developed in recent years can record fluorescence lifetime while tracking a single molecule in the 3D space. This multiparameter detection capability can open the door to a wide range of investigations at the cellular or tissue level, including identification of molecular interaction hotspots and characterization of association/dissociation kinetics between molecules. In this review, we discuss various SMT techniques developed to date, with an emphasis on our recent development of the next generation 3D tracking system that not only achieves ultrahigh spatiotemporal resolution but also provides sufficient working depth suitable for live animal imaging. We also discuss the challenges that current SMT techniques are facing and the potential strategies to tackle those challenges.

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

PubMed

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.

Reversible gating of smart plasmonic molecular traps using thermoresponsive polymers for single-molecule detection

PubMed Central

Zheng, Yuanhui; Soeriyadi, Alexander H.; Rosa, Lorenzo; Ng, Soon Hock; Bach, Udo; Justin Gooding, J.

2015-01-01

Single-molecule surface-enhanced Raman spectroscopy (SERS) has attracted increasing interest for chemical and biochemical sensing. Many conventional substrates have a broad distribution of SERS enhancements, which compromise reproducibility and result in slow response times for single-molecule detection. Here we report a smart plasmonic sensor that can reversibly trap a single molecule at hotspots for rapid single-molecule detection. The sensor was fabricated through electrostatic self-assembly of gold nanoparticles onto a gold/silica-coated silicon substrate, producing a high yield of uniformly distributed hotspots on the surface. The hotspots were isolated with a monolayer of a thermoresponsive polymer (poly(N-isopropylacrylamide)), which act as gates for molecular trapping at the hotspots. The sensor shows not only a good SERS reproducibility but also a capability to repetitively trap and release molecules for single-molecular sensing. The single-molecule sensitivity is experimentally verified using SERS spectral blinking and bianalyte methods. PMID:26549539

Single-molecule fluorescence study of the inhibition of the oncogenic functionality of STAT3

NASA Astrophysics Data System (ADS)

Liu, Baoxu; Badali, Daniel; Fletcher, Steven; Avadisian, Miriam; Gunning, Patrick; Gradinaru, Claudiu

2009-06-01

Signal-Transducer-and-Activator-of-Transcription 3 (STAT3) protein plays an important role in the onset of cancers such as leukemia and lymphoma. In this study, we aim to test the effectiveness of a novel peptide drug designed to tether STAT3 to the phospholipid bilayer of the cell membrane and thus inhibit unwanted transcription. As a first step, STAT3 proteins were successfully labelled with tetramethylrhodamine (TMR), a fluorescent dye with suitable photostability for single molecule studies. The effectiveness of labelling was determined using fluorescence correlation spectroscopy in a custom built confocal microscope, from which diffusion times and hydrodynamic radii of individual proteins were determined. A newly developed fluorescein derivative label (F-NAc) has been designed to be incorporated into the structure of the peptide drug so that peptide-STAT3 interactions can be examined. This dye is spectrally characterized and is found to be well suited for its application to this project, as well as other single-molecule studies. The membrane localization via high-affinity cholesterol-bound small-molecule binding agents can be demonstrated by encapsulating TMR-labeled STAT3 and inhibitors within a vesicle model cell system. To this end, unilaminar lipid vesicles were examined for size and encapsulation ability. Preliminary results of the efficiency and stability of the STAT3 anchoring in lipid membranes obtained via quantitative confocal imaging and single-molecule spectroscopy using a custom-built multiparameter fluorescence microscope are reported here.

Kinetic Characterization of Nonmuscle Myosin IIB at the Single Molecule Level*

PubMed Central

Nagy, Attila; Takagi, Yasuharu; Billington, Neil; Sun, Sara A.; Hong, Davin K. T.; Homsher, Earl; Wang, Aibing; Sellers, James R.

2013-01-01

Nonmuscle myosin IIB (NMIIB) is a cytoplasmic myosin, which plays an important role in cell motility by maintaining cortical tension. It forms bipolar thick filaments with ∼14 myosin molecule dimers on each side of the bare zone. Our previous studies showed that the NMIIB is a moderately high duty ratio (∼20–25%) motor. The ADP release step (∼0.35 s−1) of NMIIB is only ∼3 times faster than the rate-limiting phosphate release (0.13 ± 0.01 s−1). The aim of this study was to relate the known in vitro kinetic parameters to the results of single molecule experiments and to compare the kinetic and mechanical properties of single- and double-headed myosin fragments and nonmuscle IIB thick filaments. Examination of the kinetics of NMIIB interaction with actin at the single molecule level was accomplished using total internal reflection fluorescence (TIRF) with fluorescence imaging with 1-nm accuracy (FIONA) and dual-beam optical trapping. At a physiological ATP concentration (1 mm), the rate of detachment of the single-headed and double-headed molecules was similar (∼0.4 s−1). Using optical tweezers we found that the power stroke sizes of single- and double-headed heavy meromyosin (HMM) were each ∼6 nm. No signs of processive stepping at the single molecule level were observed in the case of NMIIB-HMM in optical tweezers or TIRF/in vitro motility experiments. In contrast, robust motility of individual fluorescently labeled thick filaments of full-length NMIIB was observed on actin filaments. Our results are in good agreement with the previous steady-state and transient kinetic studies and show that the individual nonprocessive nonmuscle myosin IIB molecules form a highly processive unit when polymerized into filaments. PMID:23148220

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

PubMed

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

2018-03-25

The nanopore can generate an electrochemical confinement for single-molecule sensing that 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 article, 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

PubMed Central

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. PMID:25283876

Biocompatible fluorescent silicon nanocrystals for single-molecule tracking and fluorescence imaging

PubMed Central

Nishimura, Hirohito; Ritchie, Ken; Kasai, Rinshi S.; Goto, Miki; Morone, Nobuhiro; Sugimura, Hiroyuki; Tanaka, Koichiro; Sase, Ichiro; Yoshimura, Akihiko; Nakano, Yoshitaro; Fujiwara, Takahiro K.

2013-01-01

Fluorescence microscopy is used extensively in cell-biological and biomedical research, but it is often plagued by three major problems with the presently available fluorescent probes: photobleaching, blinking, and large size. We have addressed these problems, with special attention to single-molecule imaging, by developing biocompatible, red-emitting silicon nanocrystals (SiNCs) with a 4.1-nm hydrodynamic diameter. Methods for producing SiNCs by simple chemical etching, for hydrophilically coating them, and for conjugating them to biomolecules precisely at a 1:1 ratio have been developed. Single SiNCs neither blinked nor photobleached during a 300-min overall period observed at video rate. Single receptor molecules in the plasma membrane of living cells (using transferrin receptor) were imaged for ≥10 times longer than with other probes, making it possible for the first time to observe the internalization process of receptor molecules at the single-molecule level. Spatial variations of molecular diffusivity in the scale of 1–2 µm, i.e., a higher level of domain mosaicism in the plasma membrane, were revealed. PMID:24043702

TOPICAL REVIEW: Single-molecule experiments in biological physics: methods and applications

NASA Astrophysics Data System (ADS)

Ritort, F.

2006-08-01

I review single-molecule experiments (SMEs) in biological physics. Recent technological developments have provided the tools to design and build scientific instruments of high enough sensitivity and precision to manipulate and visualize individual molecules and measure microscopic forces. Using SMEs it is possible to manipulate molecules one at a time and measure distributions describing molecular properties, characterize the kinetics of biomolecular reactions and detect molecular intermediates. SMEs provide additional information about thermodynamics and kinetics of biomolecular processes. This complements information obtained in traditional bulk assays. In SMEs it is also possible to measure small energies and detect large Brownian deviations in biomolecular reactions, thereby offering new methods and systems to scrutinize the basic foundations of statistical mechanics. This review is written at a very introductory level, emphasizing the importance of SMEs to scientists interested in knowing the common playground of ideas and the interdisciplinary topics accessible by these techniques. The review discusses SMEs from an experimental perspective, first exposing the most common experimental methodologies and later presenting various molecular systems where such techniques have been applied. I briefly discuss experimental techniques such as atomic-force microscopy (AFM), laser optical tweezers (LOTs), magnetic tweezers (MTs), biomembrane force probes (BFPs) and single-molecule fluorescence (SMF). I then present several applications of SME to the study of nucleic acids (DNA, RNA and DNA condensation) and proteins (protein-protein interactions, protein folding and molecular motors). Finally, I discuss applications of SMEs to the study of the nonequilibrium thermodynamics of small systems and the experimental verification of fluctuation theorems. I conclude with a discussion of open questions and future perspectives.

PhotoGate microscopy: tracking single molecules in a cytoplasm (Conference Presentation)

NASA Astrophysics Data System (ADS)

Yildiz, Ahmet

2016-02-01

Tracking single molecules inside cells reveals the dynamics of biological processes, including receptor trafficking, signaling and cargo transport. However, individual molecules often cannot be resolved inside cells due to their high density in the cellular environment. We developed a photobleaching gate assay, which controls the number of fluorescent particles in a region of interest by repeatedly photobleaching its boundary. Using this method, we tracked single particles at surface densities two orders of magnitude higher than the single-molecule detection limit. We observed ligand-induced dimerization of epidermal growth factor receptors (EGFR) on a live cell membrane. In addition, we tracked individual intraflagellar transport (IFT) trains along the length of a cilium and observed their remodeling at the ciliary tip.

Hydrophobic Hydration of Stimulus-Responsive Polyproteins Measured by Single Molecule Force Spectroscopy

NASA Astrophysics Data System (ADS)

Zauscher, Stefan

2007-03-01

We present a new procedure to reduce and analyze force-extension data obtained by single molecule force spectroscopy (SMFS). This approach allows, for the first time, to infer effects of solvent quality and minor changes in molecular architecture on molecular-elasticity of individual (bio)macromolecules. Specifically, we show how changes in the effective Kuhn segment length can be used to interpret the hydrophobic hydration behavior of elastin-like polypeptides (ELPs).Our results are intriguing as they suggest that SMFS in combination with our analysis procedure can be used to study the subtleties of polypeptide-water interactions on the single molecule level. We also report on the force-induced cis-trans isomerization of prolines, which are repeated every fifth residue in the main chain of ELPs. We present evidence for this mechanism by Monte Carlo simulations of the force-extension curves using an elastically coupled two-state system. Our results suggest that SMFS could be used to assay proline cis-trans isomerization in proteins and may thus have significant potential diagnostic utility.

Single-Molecule Counting of Point Mutations by Transient DNA Binding

NASA Astrophysics Data System (ADS)

Su, Xin; Li, Lidan; Wang, Shanshan; Hao, Dandan; Wang, Lei; Yu, Changyuan

2017-03-01

High-confidence detection of point mutations is important for disease diagnosis and clinical practice. Hybridization probes are extensively used, but are hindered by their poor single-nucleotide selectivity. Shortening the length of DNA hybridization probes weakens the stability of the probe-target duplex, leading to transient binding between complementary sequences. The kinetics of probe-target binding events are highly dependent on the number of complementary base pairs. Here, we present a single-molecule assay for point mutation detection based on transient DNA binding and use of total internal reflection fluorescence microscopy. Statistical analysis of single-molecule kinetics enabled us to effectively discriminate between wild type DNA sequences and single-nucleotide variants at the single-molecule level. A higher single-nucleotide discrimination is achieved than in our previous work by optimizing the assay conditions, which is guided by statistical modeling of kinetics with a gamma distribution. The KRAS c.34 A mutation can be clearly differentiated from the wild type sequence (KRAS c.34 G) at a relative abundance as low as 0.01% mutant to WT. To demonstrate the feasibility of this method for analysis of clinically relevant biological samples, we used this technology to detect mutations in single-stranded DNA generated from asymmetric RT-PCR of mRNA from two cancer cell lines.

Single-Molecule and Superresolution Imaging in Live Bacteria Cells

PubMed Central

Biteen, Julie S.; Moerner, W.E.

2010-01-01

Single-molecule imaging enables biophysical measurements devoid of ensemble averaging, gives enhanced spatial resolution beyond the diffraction limit, and permits superresolution reconstructions. Here, single-molecule and superresolution imaging are applied to the study of proteins in live Caulobacter crescentus cells to illustrate the power of these methods in bacterial imaging. Based on these techniques, the diffusion coefficient and dynamics of the histidine protein kinase PleC, the localization behavior of the polar protein PopZ, and the treadmilling behavior and protein superstructure of the structural protein MreB are investigated with sub-40-nm spatial resolution, all in live cells. PMID:20300204

Distinguishing between protein dynamics and dye photophysics in single-molecule FRET experiments.

PubMed

Chung, Hoi Sung; Louis, John M; Eaton, William A

2010-02-17

Förster resonance energy transfer (FRET) efficiency distributions in single-molecule experiments contain both structural and dynamical information. Extraction of this information from these distributions requires a careful analysis of contributions from dye photophysics. To investigate how mechanisms other than FRET affect the distributions obtained by counting donor and acceptor photons, we have measured single-molecule fluorescence trajectories of a small alpha/beta protein, i.e., protein GB1, undergoing two-state, folding/unfolding transitions. Alexa 488 donor and Alexa 594 acceptor dyes were attached to cysteines at positions 10 and 57 to yield two isomers-donor(10)/acceptor(57) and donor(57)/acceptor(10)-which could not be separated in the purification. The protein was immobilized via binding of a histidine tag added to a linker sequence at the N-terminus to cupric ions embedded in a polyethylene-glycol-coated glass surface. The distribution of FRET efficiencies assembled from the trajectories is complex with widths for the individual peaks in large excess of that caused by shot noise. Most of this complexity can be explained by two interfering photophysical effects-a photoinduced red shift of the donor dye and differences in the quantum yield of the acceptor dye for the two isomers resulting from differences in quenching rate by the cupric ion. Measurements of steady-state polarization, calculation of the donor-acceptor cross-correlation function from photon trajectories, and comparison of the single molecule and ensemble kinetics all indicate that conformational distributions and dynamics do not contribute to the complexity. Copyright 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Detecting single DNA molecule interactions with optical microcavities (Presentation Recording)

NASA Astrophysics Data System (ADS)

Vollmer, Frank

2015-09-01

Detecting molecules and their interactions lies at the heart of all biosensor devices, which have important applications in health, environmental monitoring and biomedicine. Achieving biosensing capability at the single molecule level is, moreover, a particularly important goal since single molecule biosensors would not only operate at the ultimate detection limit by resolving individual molecular interactions, but they could also monitor biomolecular properties which are otherwise obscured in ensemble measurements. For example, a single molecule biosensor could resolve the fleeting interaction kinetics between a molecule and its receptor, with immediate applications in clinical diagnostics. We have now developed a label-free biosensing platform that is capable of monitoring single DNA molecules and their interaction kinetics[1], hence achieving an unprecedented sensitivity in the optical domain, Figure 1. We resolve the specific contacts between complementary oligonucleotides, thereby detecting DNA strands with less than 2.4 kDa molecular weight. Furthermore we can discern strands with single nucleotide mismatches by monitoring their interaction kinetics. Our device utilizes small glass microspheres as optical transducers[1,2, 3], which are capable of increasing the number of interactions between a light beam and analyte molecules. A prism is used to couple the light beam into the microsphere. Ourr biosensing approach resolves the specific interaction kinetics between single DNA fragments. The optical transducer is assembled in a simple three-step protocol, and consists of a gold nanorod attached to a glass microsphere, where the surface of the nanorod is further modified with oligonucleotide receptors. The interaction kinetics of an oligonucleotide receptor with DNA fragments in the surrounding aqueous solution is monitored at the single molecule level[1]. The light remains confined inside the sphere where it is guided by total internal reflections along a

Classification of Dynamical Diffusion States in Single Molecule Tracking Microscopy

PubMed Central

Bosch, Peter J.; Kanger, Johannes S.; Subramaniam, Vinod

2014-01-01

Single molecule tracking of membrane proteins by fluorescence microscopy is a promising method to investigate dynamic processes in live cells. Translating the trajectories of proteins to biological implications, such as protein interactions, requires the classification of protein motion within the trajectories. Spatial information of protein motion may reveal where the protein interacts with cellular structures, because binding of proteins to such structures often alters their diffusion speed. For dynamic diffusion systems, we provide an analytical framework to determine in which diffusion state a molecule is residing during the course of its trajectory. We compare different methods for the quantification of motion to utilize this framework for the classification of two diffusion states (two populations with different diffusion speed). We found that a gyration quantification method and a Bayesian statistics-based method are the most accurate in diffusion-state classification for realistic experimentally obtained datasets, of which the gyration method is much less computationally demanding. After classification of the diffusion, the lifetime of the states can be determined, and images of the diffusion states can be reconstructed at high resolution. Simulations validate these applications. We apply the classification and its applications to experimental data to demonstrate the potential of this approach to obtain further insights into the dynamics of cell membrane proteins. PMID:25099798

Desolvation-Driven 100-Fold Slow-down of Tunneling Relaxation Rate in Co(II)-Dy(III) Single-Molecule Magnets through a Single-Crystal-to-Single-Crystal Process

NASA Astrophysics Data System (ADS)

Liu, Jun-Liang; Wu, Jie-Yi; Huang, Guo-Zhang; Chen, Yan-Cong; Jia, Jian-Hua; Ungur, Liviu; Chibotaru, Liviu F.; Chen, Xiao-Ming; Tong, Ming-Liang

2015-11-01

Single-molecule magnets (SMMs) are regarded as a class of promising materials for spintronic and ultrahigh-density storage devices. Tuning the magnetic dynamics of single-molecule magnets is a crucial challenge for chemists. Lanthanide ions are not only highly magnetically anisotropic but also highly sensitive to the changes in the coordination environments. We developed a feasible approach to understand parts of the magneto-structure correlations and propose to regulate the relaxation behaviors via rational design. A series of Co(II)-Dy(III)-Co(II) complexes were obtained using in situ synthesis; in this system of complexes, the relaxation dynamics can be greatly improved, accompanied with desolvation, via single-crystal to single-crystal transformation. The effective energy barrier can be increased from 293 cm-1 (422 K) to 416 cm-1 (600 K), and the tunneling relaxation time can be grown from 8.5 × 10-4 s to 7.4 × 10-2 s. These remarkable improvements are due to the change in the coordination environments of Dy(III) and Co(II). Ab initio calculations were performed to better understand the magnetic dynamics.

Plasmonic tunnel junctions for single-molecule redox chemistry.

PubMed

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.

Single-Molecule Optical Spectroscopy and Imaging: From Early Steps to Recent Advances

NASA Astrophysics Data System (ADS)

Moerner, William E.

The initial steps toward optical detection and spectroscopy of single molecules arose out of the study of spectral hole-burning in inhomogeneously broadened optical absorption profiles of molecular impurities in solids at low temperatures. Spectral signatures relating to the fluctuations of the number of molecules in resonance led to the attainment of the single-molecule limit in 1989. In the early 1990s, many fascinating physical effects were observed for individual molecules such as spectral diffusion, optical switching, vibrational spectra, and magnetic resonance of a single molecular spin. Since the mid-1990s when experiments moved to room temperature, a wide variety of biophysical effects may be explored, and a number of physical phenomena from the low temperature studies have analogs at high temperature. Recent advances worldwide cover a huge range, from in vitro studies of enzymes, proteins, and oligonucleotides, to observations in real time of a single protein performing a specific function inside a living cell. Because each single fluorophore acts a light source roughly 1 nm in size, microscopic observation of individual fluorophores leads naturally to localization beyond the optical diffraction limit. Combining this with active optical control of the number of emitting molecules leads to superresolution imaging, a new frontier for optical microscopy beyond the optical diffraction limit and for chemical design of photoswitchable fluorescent labels. Finally, to study one molecule in aqueous solution without surface perturbations, a new electrokinetic trap is described (the ABEL trap) which can trap single small biomolecules without the need for large dielectric beads.

Single Molecule Visualization of Protein-DNA Complexes: Watching Machines at Work

NASA Astrophysics Data System (ADS)

Kowalczykowski, Stephen

2013-03-01

We can now watch individual proteins acting on single molecules of DNA. Such imaging provides unprecedented interrogation of fundamental biophysical processes. Visualization is achieved through the application of two complementary procedures. In one, single DNA molecules are attached to a polystyrene bead and are then captured by an optical trap. The DNA, a worm-like coil, is extended either by the force of solution flow in a micro-fabricated channel, or by capturing the opposite DNA end in a second optical trap. In the second procedure, DNA is attached by one end to a glass surface. The coiled DNA is elongated either by continuous solution flow or by subsequently tethering the opposite end to the surface. Protein action is visualized by fluorescent reporters: fluorescent dyes that bind double-stranded DNA (dsDNA), fluorescent biosensors for single-stranded DNA (ssDNA), or fluorescently-tagged proteins. Individual molecules are imaged using either epifluorescence microscopy or total internal reflection fluorescence (TIRF) microscopy. Using these approaches, we imaged the search for DNA sequence homology conducted by the RecA-ssDNA filament. The manner by which RecA protein finds a single homologous sequence in the genome had remained undefined for almost 30 years. Single-molecule imaging revealed that the search occurs through a mechanism termed ``intersegmental contact sampling,'' in which the randomly coiled structure of DNA is essential for reiterative sampling of DNA sequence identity: an example of parallel processing. In addition, the assembly of RecA filaments on single molecules of single-stranded DNA was visualized. Filament assembly requires nucleation of a protein dimer on DNA, and subsequent growth occurs via monomer addition. Furthermore, we discovered a class of proteins that catalyzed both nucleation and growth of filaments, revealing how the cell controls assembly of this protein-DNA complex.

Inferring subunit stoichiometry from single molecule photobleaching

PubMed Central

2013-01-01

Single molecule photobleaching is a powerful tool for determining the stoichiometry of protein complexes. By attaching fluorophores to proteins of interest, the number of associated subunits in a complex can be deduced by imaging single molecules and counting fluorophore photobleaching steps. Because some bleaching steps might be unobserved, the ensemble of steps will be binomially distributed. In this work, it is shown that inferring the true composition of a complex from such data is nontrivial because binomially distributed observations present an ill-posed inference problem. That is, a unique and optimal estimate of the relevant parameters cannot be extracted from the observations. Because of this, a method has not been firmly established to quantify confidence when using this technique. This paper presents a general inference model for interpreting such data and provides methods for accurately estimating parameter confidence. The formalization and methods presented here provide a rigorous analytical basis for this pervasive experimental tool. PMID:23712552

Probing Enzyme-Surface Interactions via Protein Engineering and Single-Molecule Techniques

DTIC Science & Technology

2017-06-26

SECURITY CLASSIFICATION OF: The overall objective of this research was to exploit protein engineering and fluorescence single-molecule methods to... Engineering and Single-Molecule Techniques The views, opinions and/or findings contained in this report are those of the author(s) and should not...Status: Technology Transfer: Report Date: 1 FINAL REPORT Project Title: Probing Enzyme-Surface Interactions via Protein Engineering and

Interaction of dihydrofolate reductase with methotrexate: Ensemble and single-molecule kinetics

NASA Astrophysics Data System (ADS)

Rajagopalan, P. T. Ravi; Zhang, Zhiquan; McCourt, Lynn; Dwyer, Mary; Benkovic, Stephen J.; Hammes, Gordon G.

2002-10-01

The thermodynamics and kinetics of the interaction of dihydrofolate reductase (DHFR) with methotrexate have been studied by using fluorescence, stopped-flow, and single-molecule methods. DHFR was modified to permit the covalent addition of a fluorescent molecule, Alexa 488, and a biotin at the N terminus of the molecule. The fluorescent molecule was placed on a protein loop that closes over methotrexate when binding occurs, thus causing a quenching of the fluorescence. The biotin was used to attach the enzyme in an active form to a glass surface for single-molecule studies. The equilibrium dissociation constant for the binding of methotrexate to the enzyme is 9.5 nM. The stopped-flow studies revealed that methotrexate binds to two different conformations of the enzyme, and the association and dissociation rate constants were determined. The single-molecule investigation revealed a conformational change in the enzyme-methotrexate complex that was not observed in the stopped-flow studies. The ensemble averaged rate constants for this conformation change in both directions is about 2-4 s1 and is attributed to the opening and closing of the enzyme loop over the bound methotrexate. Thus the mechanism of methotrexate binding to DHFR involves multiple steps and protein conformational changes.

Viruses and tetraspanins: lessons from single molecule approaches.

PubMed

Dahmane, Selma; Rubinstein, Eric; Milhiet, Pierre-Emmanuel

2014-05-05

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.

Transport mirages in single-molecule devices

NASA Astrophysics Data System (ADS)

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.

High-throughput single-molecule telomere characterization.

PubMed

McCaffrey, Jennifer; Young, Eleanor; Lassahn, Katy; Sibert, Justin; Pastor, Steven; Riethman, Harold; Xiao, Ming

2017-11-01

We have developed a novel method that enables global subtelomere and haplotype-resolved analysis of telomere lengths at the single-molecule level. An in vitro CRISPR/Cas9 RNA-directed nickase system directs the specific labeling of human (TTAGGG)n DNA tracts in genomes that have also been barcoded using a separate nickase enzyme that recognizes a 7-bp motif genome-wide. High-throughput imaging and analysis of large DNA single molecules from genomes labeled in this fashion using a nanochannel array system permits mapping through subtelomere repeat element (SRE) regions to unique chromosomal DNA while simultaneously measuring the (TTAGGG)n tract length at the end of each large telomere-terminal DNA segment. The methodology also permits subtelomere and haplotype-resolved analyses of SRE organization and variation, providing a window into the population dynamics and potential functions of these complex and structurally variant telomere-adjacent DNA regions. At its current stage of development, the assay can be used to identify and characterize telomere length distributions of 30-35 discrete telomeres simultaneously and accurately. The assay's utility is demonstrated using early versus late passage and senescent human diploid fibroblasts, documenting the anticipated telomere attrition on a global telomere-by-telomere basis as well as identifying subtelomere-specific biases for critically short telomeres. Similarly, we present the first global single-telomere-resolved analyses of two cancer cell lines. © 2017 McCaffrey et al.; Published by Cold Spring Harbor Laboratory Press.

Single Molecule Electrochemical Detection in Aqueous Solutions and Ionic Liquids.

PubMed

Byers, Joshua C; Paulose Nadappuram, Binoy; Perry, David; McKelvey, Kim; Colburn, Alex W; Unwin, Patrick R

2015-10-20

Single molecule electrochemical detection (SMED) is an extremely challenging aspect of electroanalytical chemistry, requiring unconventional electrochemical cells and measurements. Here, SMED is reported using a "quad-probe" (four-channel probe) pipet cell, fabricated by depositing carbon pyrolytically into two diagonally opposite barrels of a laser-pulled quartz quadruple-barreled pipet and filling the open channels with electrolyte solution, and quasi-reference counter electrodes. A meniscus forms at the end of the probe covering the two working electrodes and is brought into contact with a substrate working electrode surface. In this way, a nanogap cell is produced whereby the two carbon electrodes in the pipet can be used to promote redox cycling of an individual molecule with the substrate. Anticorrelated currents generated at the substrate and tip electrodes, at particular distances (typically tens of nanometers), are consistent with the detection of single molecules. The low background noise realized in this droplet format opens up new opportunities in single molecule electrochemistry, including the use of ionic liquids, as well as aqueous solution, and the quantitative assessment and analysis of factors influencing redox cycling currents, due to a precisely known gap size.

Single-molecule studies of oligomer extraction and uptake of dyes in poly(dimethylsiloxane) films.

PubMed

Lange, Jeffrey J; Collinson, Maryanne M; Culbertson, Christopher T; Higgins, Daniel A

2009-12-15

Single-molecule microscopic methods were used to probe the uptake, mobility, and entrapment of dye molecules in cured poly(dimethylsiloxane) (PDMS) films as a function of oligomer extraction. The results are relevant to the use of PDMS in microfluidic separations, pervaporation, solid-phase microextraction, and nanofiltration. PDMS films were prepared by spin-casting dilute solutions of Sylgard 184 onto glass coverslips, yielding approximately 1.4 microm thick films after curing. Residual oligomers were subsequently extracted from the films by "spin extraction". In this procedure, 200 microL aliquots of isopropyl alcohol were repeatedly dropped onto the film surface and spun off at 2000 rpm. Samples extracted 5, 10, 20, and 40 times were investigated. Dye molecules were loaded into these films by spin-casting nanomolar dye solutions onto the films. Both neutral perylene diimide (N,N'-bis(butoxypropyl)perylene-3,4,9,10-tetracarboxylic diimide) and cationic rhodamine 6G (R6G) dyes were employed. The films were imaged by confocal fluorescence microscopy. The images obtained depict nonzero populations of fixed and mobile molecules in all films. Cross-correlation methods were used to quantitatively determine the population of fixed molecules in a given region, while a Bayesian burst analysis was used to obtain the total population of molecules. The results show that the total amount of dye loaded increases with increased oligomer extraction, while the relative populations of fixed and mobile molecules decrease and increase, respectively. Bulk R6G data also show greater dye loading with increased oligomer extraction.

Experimental demonstration of a single-molecule electric motor.

PubMed

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.

Click strategies for single-molecule protein fluorescence.

PubMed

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.

Single-molecule designs for electric switches and rectifiers.

PubMed

Kornilovitch, Pavel; Bratkovsky, Alexander; Williams, Stanley

2003-12-01

A design for molecular rectifiers is proposed. Current rectification is based on the spatial asymmetry of a molecule and requires only one resonant conducting molecular orbital. Rectification is caused by asymmetric coupling of the orbital to the electrodes, which results in asymmetric movement of the two Fermi levels with respect to the orbital under external bias. Results from numerical studies of the family of suggested molecular rectifiers, HS-(CH(2))(n)-C(6)H(4)(CH(2))(m)SH, are presented. Current rectification ratios in excess of 100 are achievable for n = 2 and m > 6. A class of bistable stator-rotor molecules is proposed. The stationary part connects the two electrodes and facilitates electron transport between them. The rotary part, which has a large dipole moment, is attached to an atom of the stator via a single sigma bond. Electrostatic bonds formed between the oxygen atom of the rotor and hydrogen atoms of the stator make the symmetric orientation of the dipole unstable. The rotor has two potential minima with equal energy for rotation about the sigma bond. The dipole can be flipped between the two states by an external electric field. Both rotor-orientation states have asymmetric current-voltage characteristics that are the reverse of each other, so they are distinguishable electrically. Theoretical results on conformation, energy barriers, retention times, switching voltages, and current-voltage characteristics are presented for a particular stator-rotor molecule. Such molecules could be the base for single-molecule switches, reversible diodes, and other molecular electronic devices.

Precision analysis for standard deviation measurements of immobile single fluorescent molecule images.

PubMed

DeSantis, Michael C; DeCenzo, Shawn H; Li, Je-Luen; Wang, Y M

2010-03-29

Standard deviation measurements of intensity profiles of stationary single fluorescent molecules are useful for studying axial localization, molecular orientation, and a fluorescence imaging system's spatial resolution. Here we report on the analysis of the precision of standard deviation measurements of intensity profiles of single fluorescent molecules imaged using an EMCCD camera.We have developed an analytical expression for the standard deviation measurement error of a single image which is a function of the total number of detected photons, the background photon noise, and the camera pixel size. The theoretical results agree well with the experimental, simulation, and numerical integration results. Using this expression, we show that single-molecule standard deviation measurements offer nanometer precision for a large range of experimental parameters.

Stiffness, working stroke, and force of single-myosin molecules in skeletal muscle: elucidation of these mechanical properties via nonlinear elasticity evaluation.

PubMed

Kaya, Motoshi; Higuchi, Hideo

2013-11-01

In muscles, the arrays of skeletal myosin molecules interact with actin filaments and continuously generate force at various contraction speeds. Therefore, it is crucial for myosin molecules to generate force collectively and minimize the interference between individual myosin molecules. Knowledge of the elasticity of myosin molecules is crucial for understanding the molecular mechanisms of muscle contractions because elasticity directly affects the working and drag (resistance) force generation when myosin molecules are positively or negatively strained. The working stroke distance is also an important mechanical property necessary for elucidation of the thermodynamic efficiency of muscle contractions at the molecular level. In this review, we focus on these mechanical properties obtained from single-fiber and single-molecule studies and discuss recent findings associated with these mechanical properties. We also discuss the potential molecular mechanisms associated with reduction of the drag effect caused by negatively strained myosin molecules.

Reducing assembly complexity of microbial genomes with single-molecule sequencing.

PubMed

Koren, Sergey; Harhay, Gregory P; Smith, Timothy P L; Bono, James L; Harhay, Dayna M; Mcvey, Scott D; Radune, Diana; Bergman, Nicholas H; Phillippy, Adam M

2013-01-01

The short reads output by first- and second-generation DNA sequencing instruments cannot completely reconstruct microbial chromosomes. Therefore, most genomes have been left unfinished due to the significant resources required to manually close gaps in draft assemblies. Third-generation, single-molecule sequencing addresses this problem by greatly increasing sequencing read length, which simplifies the assembly problem. To measure the benefit of single-molecule sequencing on microbial genome assembly, we sequenced and assembled the genomes of six bacteria and analyzed the repeat complexity of 2,267 complete bacteria and archaea. Our results indicate that the majority of known bacterial and archaeal genomes can be assembled without gaps, at finished-grade quality, using a single PacBio RS sequencing library. These single-library assemblies are also more accurate than typical short-read assemblies and hybrid assemblies of short and long reads. Automated assembly of long, single-molecule sequencing data reduces the cost of microbial finishing to $1,000 for most genomes, and future advances in this technology are expected to drive the cost lower. This is expected to increase the number of completed genomes, improve the quality of microbial genome databases, and enable high-fidelity, population-scale studies of pan-genomes and chromosomal organization.

Single-molecule optomechanics in "picocavities".

PubMed

Benz, Felix; Schmidt, Mikolaj K; Dreismann, Alexander; Chikkaraddy, Rohit; Zhang, Yao; Demetriadou, Angela; Carnegie, Cloudy; Ohadi, Hamid; de Nijs, Bart; Esteban, Ruben; Aizpurua, Javier; Baumberg, Jeremy J

2016-11-11

Trapping light with noble metal nanostructures overcomes the diffraction limit and can confine light to volumes typically on the order of 30 cubic nanometers. We found that individual atomic features inside the gap of a plasmonic nanoassembly can localize light to volumes well below 1 cubic nanometer ("picocavities"), enabling optical experiments on the atomic scale. These atomic features are dynamically formed and disassembled by laser irradiation. Although unstable at room temperature, picocavities can be stabilized at cryogenic temperatures, allowing single atomic cavities to be probed for many minutes. Unlike traditional optomechanical resonators, such extreme optical confinement yields a factor of 10 6 enhancement of optomechanical coupling between the picocavity field and vibrations of individual molecular bonds. This work sets the basis for developing nanoscale nonlinear quantum optics on the single-molecule level. Copyright © 2016, American Association for the Advancement of Science.

Single-molecule pull-down (SiMPull) for new-age biochemistry: methodology and biochemical applications of single-molecule pull-down (SiMPull) for probing biomolecular interactions in crude cell extracts.

PubMed

Aggarwal, Vasudha; Ha, Taekjip

2014-11-01

Macromolecular interactions play a central role in many biological processes. Protein-protein interactions have mostly been studied by co-immunoprecipitation, which cannot provide quantitative information on all possible molecular connections present in the complex. We will review a new approach that allows cellular proteins and biomolecular complexes to be studied in real-time at the single-molecule level. This technique is called single-molecule pull-down (SiMPull), because it integrates principles of conventional immunoprecipitation with the powerful single-molecule fluorescence microscopy. SiMPull is used to count how many of each protein is present in the physiological complexes found in cytosol and membranes. Concurrently, it serves as a single-molecule biochemical tool to perform functional studies on the pulled-down proteins. In this review, we will focus on the detailed methodology of SiMPull, its salient features and a wide range of biological applications in comparison with other biosensing tools. © 2014 WILEY Periodicals, Inc.

Single-Molecule Probing the Energy Landscape of Enzymatic Reaction and Non-Covalent Interactions

NASA Astrophysics Data System (ADS)

Lu, H. Peter; Hu, Dehong; Chen, Yu; Vorpagel, Erich R.

2002-03-01

We have applied single-molecule spectroscopy under physiological conditions to study the mechanisms and dynamics of T4 lysozyme enzymatic reactions, characterizing mode-specific protein conformational dynamics. Enzymatic reaction turnovers and the associated structure changes of individual protein molecules were observed simultaneously in real-time. The overall reaction rates were found to vary widely from molecule-to-molecule, and the initial non-specific binding of the enzyme to the substrate was seen to dominate this inhomogeneity. The reaction steps subsequent to the initial binding were found to have homogeneous rates. Molecular dynamics simulation has been applied to elucidate the mechanism and intermediate states of the single-molecule enzymatic reaction. Combining the analysis of single-molecule experimental trajectories, MD simulation trajectories, and statistical modeling, we have revealed the nature of multiple intermediate states involved in the active enzyme-substrate complex formation and the associated conformational change mechanism and dynamics.

Identification of vibrational signatures from short chains of interlinked molecule-nanoparticle junctions obtained by inelastic electron tunnelling spectroscopy

NASA Astrophysics Data System (ADS)

Jafri, S. H. M.; Löfås, H.; Fransson, J.; Blom, T.; Grigoriev, A.; Wallner, A.; Ahuja, R.; Ottosson, H.; Leifer, K.

2013-05-01

Short chains containing a series of metal-molecule-nanoparticle nanojunctions are a nano-material system with the potential to give electrical signatures close to those from single molecule experiments while enabling us to build portable devices on a chip. Inelastic electron tunnelling spectroscopy (IETS) measurements provide one of the most characteristic electrical signals of single and few molecules. In interlinked molecule-nanoparticle (NP) chains containing typically 5-7 molecules in a chain, the spectrum is expected to be a superposition of the vibrational signatures of individual molecules. We have established a stable and reproducible molecule-AuNP multi-junction by placing a few 1,8-octanedithiol (ODT) molecules onto a versatile and portable nanoparticle-nanoelectrode platform and measured for the first time vibrational molecular signatures at complex and coupled few-molecule-NP junctions. From quantum transport calculations, we model the IETS spectra and identify vibrational modes as well as the number of molecules contributing to the electron transport in the measured spectra.Short chains containing a series of metal-molecule-nanoparticle nanojunctions are a nano-material system with the potential to give electrical signatures close to those from single molecule experiments while enabling us to build portable devices on a chip. Inelastic electron tunnelling spectroscopy (IETS) measurements provide one of the most characteristic electrical signals of single and few molecules. In interlinked molecule-nanoparticle (NP) chains containing typically 5-7 molecules in a chain, the spectrum is expected to be a superposition of the vibrational signatures of individual molecules. We have established a stable and reproducible molecule-AuNP multi-junction by placing a few 1,8-octanedithiol (ODT) molecules onto a versatile and portable nanoparticle-nanoelectrode platform and measured for the first time vibrational molecular signatures at complex and coupled few-molecule

Roles of vacuum tunnelling and contact mechanics in single-molecule thermopower

NASA Astrophysics Data System (ADS)

Tsutsui, Makusu; Yokota, Kazumichi; Morikawa, Takanori; Taniguchi, Masateru

2017-03-01

Molecular junction is a chemically-defined nanostructure whose discrete electronic states are expected to render enhanced thermoelectric figure of merit suitable for energy-harvesting applications. Here, we report on geometrical dependence of thermoelectricity in metal-molecule-metal structures. We performed simultaneous measurements of the electrical conductance and thermovoltage of aromatic molecules having different anchoring groups at room temperature in vacuum. We elucidated the mutual contributions of vacuum tunnelling on thermoelectricity in the short molecular bridges. We also found stretching-induced thermoelectric voltage enhancement in thiol-linked single-molecule bridges along with absence of the pulling effects in diamine counterparts, thereby suggested that the electromechanical effect would be a rather universal phenomenon in Au-S anchored molecular junctions that undergo substantial metal-molecule contact elongation upon stretching. The present results provide a novel concept for molecular design to achieve high thermopower with single-molecule junctions.

SNSMIL, a real-time single molecule identification and localization algorithm for super-resolution fluorescence microscopy

PubMed Central

Tang, Yunqing; Dai, Luru; Zhang, Xiaoming; Li, Junbai; Hendriks, Johnny; Fan, Xiaoming; Gruteser, Nadine; Meisenberg, Annika; Baumann, Arnd; Katranidis, Alexandros; Gensch, Thomas

2015-01-01

Single molecule localization based super-resolution fluorescence microscopy offers significantly higher spatial resolution than predicted by Abbe’s resolution limit for far field optical microscopy. Such super-resolution images are reconstructed from wide-field or total internal reflection single molecule fluorescence recordings. Discrimination between emission of single fluorescent molecules and background noise fluctuations remains a great challenge in current data analysis. Here we present a real-time, and robust single molecule identification and localization algorithm, SNSMIL (Shot Noise based Single Molecule Identification and Localization). This algorithm is based on the intrinsic nature of noise, i.e., its Poisson or shot noise characteristics and a new identification criterion, QSNSMIL, is defined. SNSMIL improves the identification accuracy of single fluorescent molecules in experimental or simulated datasets with high and inhomogeneous background. The implementation of SNSMIL relies on a graphics processing unit (GPU), making real-time analysis feasible as shown for real experimental and simulated datasets. PMID:26098742

The scanning tunnelling microscope as an operative tool: doing physics and chemistry with single atoms and molecules.

PubMed

Rieder, Karl-Heinz; Meyer, Gerhard; Hla, Saw-Wai; Moresco, Francesca; Braun, Kai F; Morgenstern, Karina; Repp, Jascha; Foelsch, Stefan; Bartels, Ludwig

2004-06-15

The scanning tunnelling microscope, initially invented to image surfaces down to the atomic scale, has been further developed in the last few years to an operative tool, with which atoms and molecules can be manipulated at will at low substrate temperatures in different manners to create and investigate artificial structures, whose properties can be investigated employing spectroscopic dI/dV measurements. The tunnelling current can be used to selectively break chemical bonds, but also to induce chemical association. These possibilities give rise to startling new opportunities for physical and chemical experiments on the single atom and single molecule level. Here we provide a short overview on recent results obtained with these techniques.

Blinking effect and the use of quantum dots in single molecule spectroscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rombach-Riegraf, Verena; Oswald, Peter; Bienert, Roland

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 singlemore » 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.« less

Single-molecule strong coupling at room temperature in plasmonic nanocavities

NASA Astrophysics Data System (ADS)

Chikkaraddy, Rohit; de Nijs, Bart; Benz, Felix; Barrow, Steven J.; Scherman, Oren A.; Rosta, Edina; Demetriadou, Angela; Fox, Peter; Hess, Ortwin; Baumberg, Jeremy J.

2016-07-01

Photon emitters placed in an optical cavity experience an environment that changes how they are coupled to the surrounding light field. In the weak-coupling regime, the extraction of light from the emitter is enhanced. But more profound effects emerge when single-emitter strong coupling occurs: mixed states are produced that are part light, part matter, forming building blocks for quantum information systems and for ultralow-power switches and lasers. Such cavity quantum electrodynamics has until now been the preserve of low temperatures and complicated fabrication methods, compromising its use. Here, by scaling the cavity volume to less than 40 cubic nanometres and using host-guest chemistry to align one to ten protectively isolated methylene-blue molecules, we reach the strong-coupling regime at room temperature and in ambient conditions. Dispersion curves from more than 50 such plasmonic nanocavities display characteristic light-matter mixing, with Rabi frequencies of 300 millielectronvolts for ten methylene-blue molecules, decreasing to 90 millielectronvolts for single molecules—matching quantitative models. Statistical analysis of vibrational spectroscopy time series and dark-field scattering spectra provides evidence of single-molecule strong coupling. This dressing of molecules with light can modify photochemistry, opening up the exploration of complex natural processes such as photosynthesis and the possibility of manipulating chemical bonds.

Solvent dependence of the activation energy of attachment determined by single molecule observations of surfactant adsorption.

PubMed

Honciuc, Andrei; Baptiste, Denver Jn; Campbell, Ian P; Schwartz, Daniel K

2009-07-07

Single-molecule total internal reflection fluorescence microscopy was used to obtain real-time images of fluorescently labeled hexadecanoic (palmitic) acid molecules as they adsorbed at the interface between fused silica and three different solvents: hexadecane (HD), tetrahydrofuran (THF), and water. These solvents were chosen to explore the effect of solvent polarity on the activation energy associated with the attachment rate, i.e., the rate at which molecules were transferred to the surface from the near-surface layer. Direct counting of single-molecule events, made under steady-state conditions at extremely low coverage, provided direct, model-independent measurements of this attachment rate, in contrast with conventional ensemble-averaged methods, which are influenced by bulk transport and competing detachment processes. We found that the attachment rate increased with increasing temperature for all solvents. Arrhenius analyses gave activation energies of 5+/-2 kJ/mol for adsorption from HD, 10+/-2 kJ/mol for adsorption from THF, and 19+/-2 kJ/mol for adsorption from water. These energies increased systematically with the solvent polarity and, therefore, with the expected strength of the solvent-substrate interaction. We hypothesize that the adsorption of amphiphilic solute molecules from solution can be regarded as a competitive exchange between solute molecules and surface-bound solvent. In this scenario, adsorption is an activated process, and the activation energy for attachment is associated with the solvent-substrate interaction energy.

Rotation of a Single Acetylene Molecule on Cu(001) by Tunneling Electrons in STM

NASA Astrophysics Data System (ADS)

Shchadilova, Yulia E.; Tikhodeev, Sergei G.; Paulsson, Magnus; Ueba, Hiromu

2013-11-01

We study the elementary processes behind one of the pioneering works on scanning tunneling microscope controlled reactions of single molecules [Stipe et al., Phys. Rev. Lett. 81, 1263 (1998)]. Using the Keldysh-Green function approach for the vibrational generation rate in combination with density functional theory calculations to obtain realistic parameters we reproduce the experimental rotation rate of an acetylene molecule on a Cu(100) surface as a function of bias voltage and tunneling current. This combined approach allows us to identify the reaction coordinate mode of the acetylene rotation and its anharmonic coupling with the C-H stretch mode. We show that three different elementary processes, the excitation of C-H stretch, the overtone ladder climbing of the hindered rotational mode, and the combination band excitation together explain the rotation of the acetylene molecule on Cu(100).

Current rectification in a single molecule diode: the role of electrode coupling.

PubMed

Sherif, Siya; Rubio-Bollinger, Gabino; Pinilla-Cienfuegos, Elena; Coronado, Eugenio; Cuevas, Juan Carlos; Agraït, Nicolás

2015-07-24

We demonstrate large rectification ratios (> 100) in single-molecule junctions based on a metal-oxide cluster (polyoxometalate), using a scanning tunneling microscope (STM) both at ambient conditions and at low temperature. These rectification ratios are the largest ever observed in a single-molecule junction, and in addition these junctions sustain current densities larger than 10(5) A cm(-2). By following the variation of the I-V characteristics with tip-molecule separation we demonstrate unambiguously that rectification is due to asymmetric coupling to the electrodes of a molecule with an asymmetric level structure. This mechanism can be implemented in other type of molecular junctions using both organic and inorganic molecules and provides a simple strategy for the rational design of molecular diodes.

Current rectification in a single molecule diode: the role of electrode coupling

NASA Astrophysics Data System (ADS)

Sherif, Siya; Rubio-Bollinger, Gabino; Pinilla-Cienfuegos, Elena; Coronado, Eugenio; Cuevas, Juan Carlos; Agraït, Nicolás

2015-07-01

We demonstrate large rectification ratios (\\gt 100) in single-molecule junctions based on a metal-oxide cluster (polyoxometalate), using a scanning tunneling microscope (STM) both at ambient conditions and at low temperature. These rectification ratios are the largest ever observed in a single-molecule junction, and in addition these junctions sustain current densities larger than 105 A cm-2. By following the variation of the I-V characteristics with tip-molecule separation we demonstrate unambiguously that rectification is due to asymmetric coupling to the electrodes of a molecule with an asymmetric level structure. This mechanism can be implemented in other type of molecular junctions using both organic and inorganic molecules and provides a simple strategy for the rational design of molecular diodes.

Screening by imaging: scaling up single-DNA-molecule analysis with a novel parabolic VA-TIRF reflector and noise-reduction techniques.

PubMed

van 't Hoff, Marcel; Reuter, Marcel; Dryden, David T F; Oheim, Martin

2009-09-21

Bacteriophage lambda-DNA molecules are frequently used as a scaffold to characterize the action of single proteins unwinding, translocating, digesting or repairing DNA. However, scaling up such single-DNA-molecule experiments under identical conditions to attain statistically relevant sample sizes remains challenging. Additionally the movies obtained are frequently noisy and difficult to analyse with any precision. We address these two problems here using, firstly, a novel variable-angle total internal reflection fluorescence (VA-TIRF) reflector composed of a minimal set of optical reflective elements, and secondly, using single value decomposition (SVD) to improve the signal-to-noise ratio prior to analysing time-lapse image stacks. As an example, we visualize under identical optical conditions hundreds of surface-tethered single lambda-DNA molecules, stained with the intercalating dye YOYO-1 iodide, and stretched out in a microcapillary flow. Another novelty of our approach is that we arrange on a mechanically driven stage several capillaries containing saline, calibration buffer and lambda-DNA, respectively, thus extending the approach to high-content, high-throughput screening of single molecules. Our length measurements of individual DNA molecules from noise-reduced kymograph images using SVD display a 6-fold enhanced precision compared to raw-data analysis, reaching approximately 1 kbp resolution. Combining these two methods, our approach provides a straightforward yet powerful way of collecting statistically relevant amounts of data in a semi-automated manner. We believe that our conceptually simple technique should be of interest for a broader range of single-molecule studies, well beyond the specific example of lambda-DNA shown here.

Axial Colocalization of Single Molecules with Nanometer Accuracy Using Metal-Induced Energy Transfer.

PubMed

Isbaner, Sebastian; Karedla, Narain; Kaminska, Izabela; Ruhlandt, Daja; Raab, Mario; Bohlen, Johann; Chizhik, Alexey; Gregor, Ingo; Tinnefeld, Philip; Enderlein, Jörg; Tsukanov, Roman

2018-04-11

Single-molecule localization based super-resolution microscopy has revolutionized optical microscopy and routinely allows for resolving structural details down to a few nanometers. However, there exists a rather large discrepancy between lateral and axial localization accuracy, the latter typically three to five times worse than the former. Here, we use single-molecule metal-induced energy transfer (smMIET) to localize single molecules along the optical axis, and to measure their axial distance with an accuracy of 5 nm. smMIET relies only on fluorescence lifetime measurements and does not require additional complex optical setups.

Nanopore arrays in a silicon membrane for parallel single-molecule detection: fabrication

NASA Astrophysics Data System (ADS)

Schmidt, Torsten; Zhang, Miao; Sychugov, Ilya; Roxhed, Niclas; Linnros, Jan

2015-08-01

Solid state nanopores enable translocation and detection of single bio-molecules such as DNA in buffer solutions. Here, sub-10 nm nanopore arrays in silicon membranes were fabricated by using electron-beam lithography to define etch pits and by using a subsequent electrochemical etching step. This approach effectively decouples positioning of the pores and the control of their size, where the pore size essentially results from the anodizing current and time in the etching cell. Nanopores with diameters as small as 7 nm, fully penetrating 300 nm thick membranes, were obtained. The presented fabrication scheme to form large arrays of nanopores is attractive for parallel bio-molecule sensing and DNA sequencing using optical techniques. In particular the signal-to-noise ratio is improved compared to other alternatives such as nitride membranes suffering from a high-luminescence background.

Nanopore arrays in a silicon membrane for parallel single-molecule detection: fabrication.

PubMed

Schmidt, Torsten; Zhang, Miao; Sychugov, Ilya; Roxhed, Niclas; Linnros, Jan

2015-08-07

Solid state nanopores enable translocation and detection of single bio-molecules such as DNA in buffer solutions. Here, sub-10 nm nanopore arrays in silicon membranes were fabricated by using electron-beam lithography to define etch pits and by using a subsequent electrochemical etching step. This approach effectively decouples positioning of the pores and the control of their size, where the pore size essentially results from the anodizing current and time in the etching cell. Nanopores with diameters as small as 7 nm, fully penetrating 300 nm thick membranes, were obtained. The presented fabrication scheme to form large arrays of nanopores is attractive for parallel bio-molecule sensing and DNA sequencing using optical techniques. In particular the signal-to-noise ratio is improved compared to other alternatives such as nitride membranes suffering from a high-luminescence background.

Data mining for materials design: A computational study of single molecule magnet

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dam, Hieu Chi; Faculty of Physics, Vietnam National University, 334 Nguyen Trai, Hanoi; Pham, Tien Lam

2014-01-28

We develop a method that combines data mining and first principles calculation to guide the designing of distorted cubane Mn{sup 4+} Mn {sub 3}{sup 3+} single molecule magnets. The essential idea of the method is a process consisting of sparse regressions and cross-validation for analyzing calculated data of the materials. The method allows us to demonstrate that the exchange coupling between Mn{sup 4+} and Mn{sup 3+} ions can be predicted from the electronegativities of constituent ligands and the structural features of the molecule by a linear regression model with high accuracy. The relations between the structural features and magnetic propertiesmore » of the materials are quantitatively and consistently evaluated and presented by a graph. We also discuss the properties of the materials and guide the material design basing on the obtained results.« less

Identification of vibrational signatures from short chains of interlinked molecule-nanoparticle junctions obtained by inelastic electron tunnelling spectroscopy.

PubMed

Jafri, S H M; Löfås, H; Fransson, J; Blom, T; Grigoriev, A; Wallner, A; Ahuja, R; Ottosson, H; Leifer, K

2013-06-07

Short chains containing a series of metal-molecule-nanoparticle nanojunctions are a nano-material system with the potential to give electrical signatures close to those from single molecule experiments while enabling us to build portable devices on a chip. Inelastic electron tunnelling spectroscopy (IETS) measurements provide one of the most characteristic electrical signals of single and few molecules. In interlinked molecule-nanoparticle (NP) chains containing typically 5-7 molecules in a chain, the spectrum is expected to be a superposition of the vibrational signatures of individual molecules. We have established a stable and reproducible molecule-AuNP multi-junction by placing a few 1,8-octanedithiol (ODT) molecules onto a versatile and portable nanoparticle-nanoelectrode platform and measured for the first time vibrational molecular signatures at complex and coupled few-molecule-NP junctions. From quantum transport calculations, we model the IETS spectra and identify vibrational modes as well as the number of molecules contributing to the electron transport in the measured spectra.

Dynamic fluctuations in single-molecule biophysics experiments. Comment on "Extracting physics of life at the molecular level: A review of single-molecule data analyses" by W. Colomb and S.K. Sarkar

NASA Astrophysics Data System (ADS)

Krapf, Diego

2015-06-01

Single-molecule biophysics includes the study of isolated molecules and that of individual molecules within living cells. In both cases, dynamic fluctuations at the nanoscale play a critical role. Colomb and Sarkar emphasize how different noise sources affect the analysis of single molecule data [1]. Fluctuations in biomolecular systems arise from two very different mechanisms. On one hand thermal fluctuations are a predominant feature in the behavior of individual molecules. On the other hand, non-Gaussian fluctuations can arise from inter- and intramolecular interactions [2], spatial heterogeneities [3], non-Poisson external perturbations [4] and complex non-linear dynamics in general [5,6].

Single-molecule DNA detection with an engineered MspA protein nanopore

PubMed Central

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

2008-01-01

Nanopores hold great promise as single-molecule analytical devices and biophysical model systems because the ionic current blockades they produce contain information about the identity, concentration, structure, and dynamics of target molecules. The porin MspA of Mycobacterium smegmatis has remarkable stability against environmental stresses and can be rationally modified based on its crystal structure. Further, MspA has a short and narrow channel constriction that is promising for DNA sequencing because it may enable improved characterization of short segments of a ssDNA molecule that is threaded through the pore. By eliminating the negative charge in the channel constriction, we designed and constructed an MspA mutant capable of electronically detecting and characterizing single molecules of ssDNA as they are electrophoretically driven through the pore. A second mutant with additional exchanges of negatively-charged residues for positively-charged residues in the vestibule region exhibited a factor of ≈20 higher interaction rates, required only half as much voltage to observe interaction, and allowed ssDNA to reside in the vestibule ≈100 times longer than the first mutant. Our results introduce MspA as a nanopore for nucleic acid analysis and highlight its potential as an engineerable platform for single-molecule detection and characterization applications. PMID:19098105

From single molecule to single tubules

NASA Astrophysics Data System (ADS)

Guo, Chin-Lin

2012-02-01

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

Random walk on a leash: a simple single-molecule diffusion model for surface-tethered redox molecules with flexible linkers.

PubMed

Huang, Kuan-Chun; White, Ryan J

2013-08-28

We develop a random walk model to simulate the Brownian motion and the electrochemical response of a single molecule confined to an electrode surface via a flexible molecular tether. We use our simple model, which requires no prior knowledge of the physics of the molecular tether, to predict and better understand the voltammetric response of surface-confined redox molecules when motion of the redox molecule becomes important. The single molecule is confined to a hemispherical volume with a maximum radius determined by the flexible molecular tether (5-20 nm) and is allowed to undergo true three-dimensional diffusion. Distance- and potential-dependent electron transfer probabilities are evaluated throughout the simulations to generate cyclic voltammograms of the model system. We find that at sufficiently slow cyclic voltammetric scan rates the electrochemical reaction behaves like an adsorbed redox molecule with no mass transfer limitation; thus, the peak current is proportional to the scan rate. Conversely, at faster scan rates the diffusional motion of the molecule limits the simulated peak current, which exhibits a linear dependence on the square root of the scan rate. The switch between these two limiting regimes occurs when the diffusion layer thickness, (2Dt)(1/2), is ~10 times the tether length. Finally, we find that our model predicts the voltammetric behavior of a redox-active methylene blue tethered to an electrode surface via short flexible single-stranded, polythymine DNAs, allowing the estimation of diffusion coefficients for the end-tethered molecule.

Single Molecule Stepping and Structural Dynamics of Myosin X

PubMed Central

Sun, Yujie; Sato, Osamu; Ruhnow, Felix; Arsenault, Mark E.; Ikebe, Mitsuo; Goldman, Yale E.

2010-01-01

Myosin X is an unconventional myosin with puzzling motility properties. We studied the motility of dimerized myosin X using single molecule fluorescence techniques – polTIRF, FIONA, and Parallax to measure rotation angles and 3-dimensional position of the molecule during its walk. It was found that Myosin X steps processively in a hand-over-hand manner following a left-handed helical path along both single actin filaments and bundles. Its step size and velocity are smaller on actin bundles than individual filaments, suggesting myosin X often steps onto neighboring filaments in a bundle. The data suggest that a previously postulated single α-helical domain mechanically extends the 3-IQ motif lever arm and either the neck-tail hinge or the tail is flexible. These structural features, in conjunction with the membrane and microtubule binding domains, enable myosin X to perform multiple functions on varied actin structures in cells. PMID:20364131

Electrochemical detection of single molecules using abiotic nanopores having electrically tunable dimensions

DOEpatents

Sansinena, Jose-Maria [Los Alamos, NM; Redondo, Antonio [Los Alamos, NM; Olazabal, Virginia [Los Alamos, NM; Hoffbauer, Mark A [Los Alamos, NM; Akhadov, Elshan A [Los Alamos, NM

2009-12-29

A barrier structure for use in an electrochemical stochastic membrane sensor for single molecule detection. The sensor is based upon inorganic nanopores having electrically tunable dimensions. The inorganic nanopores are formed from inorganic materials and an electrically conductive polymer. Methods of making the barrier structure and sensing single molecules using the barrier structure are also described.

Electrochemical detection of single molecules using abiotic nanopores having electrically tunable dimensions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sansinena, Jose-Maria; Redondo, Antonio; Olazabal, Virginia

2017-09-12

A barrier structure for use in an electrochemical stochastic membrane sensor for single molecule detection. The sensor is based upon inorganic nanopores having electrically tunable dimensions. The inorganic nanopores are formed from inorganic materials and an electrically conductive polymer. Methods of making the barrier structure and sensing single molecules using the barrier structure are also described.

Electrochemical detection of single molecules using abiotic nanopores having electrically tunable dimensions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sansinena, Jose-Maria; Redondo, Antonio; Olazabal, Virginia

2017-07-18

A barrier structure for use in an electrochemical stochastic membrane sensor for single molecule detection. The sensor is based upon inorganic nanopores having electrically tunable dimensions. The inorganic nanopores are formed from inorganic materials and an electrically conductive polymer. Methods of making the barrier structure and sensing single molecules using the barrier structure are also described.

Electrochemical detection of single molecules using abiotic nanopores having electrically tunable dimensions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sansinena, Jose-Maria; Redondo, Antonio; Olazabal, Virginia

A barrier structure for use in an electrochemical stochastic membrane sensor for single molecule detection. The sensor is based upon inorganic nanopores having electrically tunable dimensions. The inorganic nanopores are formed from inorganic materials and an electrically conductive polymer. Methods of making the barrier structure and sensing single molecules using the barrier structure are also described.

Linker Dependent Bond Rupture Force Measurements in Single-Molecule Junctions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Frei M.; Hybertsen M.; Aradhya S.V.

We use a modified conducting atomic force microscope to simultaneously probe the conductance of a single-molecule junction and the force required to rupture the junction formed by alkanes terminated with four different chemical link groups which vary in binding strength and mechanism to the gold electrodes. Molecular junctions with amine, methylsulfide, and diphenylphosphine terminated molecules show clear conductance signatures and rupture at a force that is significantly smaller than the measured 1.4 nN force required to rupture the single-atomic gold contact. In contrast, measurements with a thiol terminated alkane which can bind covalently to the gold electrode show conductance andmore » force features unlike those of the other molecules studied. Specifically, the strong Au-S bond can cause structural rearrangements in the electrodes, which are accompanied by substantial conductance changes. Despite the strong Au-S bond and the evidence for disruption of the Au structure, the experiments show that on average these junctions also rupture at a smaller force than that measured for pristine single-atom gold contacts.« less

Dissecting single-molecule signal transduction in carbon nanotube circuits with protein engineering

PubMed Central

Choi, Yongki; Olsen, Tivoli J.; Sims, Patrick C.; Moody, Issa S.; Corso, Brad L.; Dang, Mytrang N.; Weiss, Gregory A.; Collins, Philip G.

2013-01-01

Single molecule experimental methods have provided new insights into biomolecular function, dynamic disorder, and transient states that are all invisible to conventional measurements. A novel, non-fluorescent single molecule technique involves attaching single molecules to single-walled carbon nanotube field-effective transistors (SWNT FETs). These ultrasensitive electronic devices provide long-duration, label-free monitoring of biomolecules and their dynamic motions. However, generalization of the SWNT FET technique first requires design rules that can predict the success and applicability of these devices. Here, we report on the transduction mechanism linking enzymatic processivity to electrical signal generation by a SWNT FET. The interaction between SWNT FETs and the enzyme lysozyme was systematically dissected using eight different lysozyme variants synthesized by protein engineering. The data prove that effective signal generation can be accomplished using a single charged amino acid, when appropriately located, providing a foundation to widely apply SWNT FET sensitivity to other biomolecular systems. PMID:23323846

Distinguishing between Protein Dynamics and Dye Photophysics in Single-Molecule FRET Experiments

PubMed Central

Chung, Hoi Sung; Louis, John M.; Eaton, William A.

2010-01-01

Abstract Förster resonance energy transfer (FRET) efficiency distributions in single-molecule experiments contain both structural and dynamical information. Extraction of this information from these distributions requires a careful analysis of contributions from dye photophysics. To investigate how mechanisms other than FRET affect the distributions obtained by counting donor and acceptor photons, we have measured single-molecule fluorescence trajectories of a small α/β protein, i.e., protein GB1, undergoing two-state, folding/unfolding transitions. Alexa 488 donor and Alexa 594 acceptor dyes were attached to cysteines at positions 10 and 57 to yield two isomers—donor10/acceptor57 and donor57/acceptor10—which could not be separated in the purification. The protein was immobilized via binding of a histidine tag added to a linker sequence at the N-terminus to cupric ions embedded in a polyethylene-glycol–coated glass surface. The distribution of FRET efficiencies assembled from the trajectories is complex with widths for the individual peaks in large excess of that caused by shot noise. Most of this complexity can be explained by two interfering photophysical effects—a photoinduced red shift of the donor dye and differences in the quantum yield of the acceptor dye for the two isomers resulting from differences in quenching rate by the cupric ion. Measurements of steady-state polarization, calculation of the donor-acceptor cross-correlation function from photon trajectories, and comparison of the single molecule and ensemble kinetics all indicate that conformational distributions and dynamics do not contribute to the complexity. PMID:20159166

Conducting single-molecule magnet materials.

PubMed

Cosquer, Goulven; Shen, Yongbing; Almeida, Manuel; Yamashita, Masahiro

2018-05-11

Multifunctional molecular materials exhibiting electrical conductivity and single-molecule magnet (SMM) behaviour are particularly attractive for electronic devices and related applications owing to the interaction between electronic conduction and magnetization of unimolecular units. The preparation of such materials remains a challenge that has been pursued by a bi-component approach of combination of SMM cationic (or anionic) units with conducting networks made of partially oxidized (or reduced) donor (or acceptor) molecules. The present status of the research concerning the preparation of molecular materials exhibiting SMM behaviour and electrical conductivity is reviewed, describing the few molecular compounds where both SMM properties and electrical conductivity have been observed. The evolution of this research field through the years is discussed. The first reported compounds are semiconductors in spite being able to present relatively high electrical conductivity, and the SMM behaviour is observed at low temperatures where the electrical conductivity of the materials is similar to that of an insulator. During the recent years, a breakthrough has been achieved with the coexistence of high electrical conductivity and SMM behaviour in a molecular compound at the same temperature range, but so far without evidence of a synergy between these properties. The combination of high electrical conductivity with SMM behaviour requires not only SMM units but also the regular and as far as possible uniform packing of partially oxidized molecules, which are able to provide a conducting network.

Entangled photons from single atoms and molecules

NASA Astrophysics Data System (ADS)

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.

Colloidal lenses allow high-temperature single-molecule imaging and improve fluorophore photostability

NASA Astrophysics Data System (ADS)

Schwartz, Jerrod J.; Stavrakis, Stavros; Quake, Stephen R.

2010-02-01

Although single-molecule fluorescence spectroscopy was first demonstrated at near-absolute zero temperatures (1.8 K), the field has since advanced to include room-temperature observations, largely owing to the use of objective lenses with high numerical aperture, brighter fluorophores and more sensitive detectors. This has opened the door for many chemical and biological systems to be studied at native temperatures at the single-molecule level both in vitro and in vivo. However, it is difficult to study systems and phenomena at temperatures above 37 °C, because the index-matching fluids used with high-numerical-aperture objective lenses can conduct heat from the sample to the lens, and sustained exposure to high temperatures can cause the lens to fail. Here, we report that TiO2 colloids with diameters of 2 µm and a high refractive index can act as lenses that are capable of single-molecule imaging at 70 °C when placed in immediate proximity to an emitting molecule. The optical system is completed by a low-numerical-aperture optic that can have a long working distance and an air interface, which allows the sample to be independently heated. Colloidal lenses were used for parallel imaging of surface-immobilized single fluorophores and for real-time single-molecule measurements of mesophilic and thermophilic enzymes at 70 °C. Fluorophores in close proximity to TiO2 also showed a 40% increase in photostability due to a reduction of the excited-state lifetime.

Single Molecule and Collective Dynamics of Motor Protein Coupled with Mechano-Sensitive Chemical Reaction

NASA Astrophysics Data System (ADS)

Iwaki, Mitsuhiro; Marcucci, Lorenzo; Togashi, Yuichi; Yanagida, Toshio

2013-12-01

Motor proteins such as myosin and kinesin hydrolyze ATP into ADP and Pi to convert chemical energy into mechanical work. This resultsin various motile processes like muscle contraction, vesicle transport and cell division. Recent single molecule experiments have revealed that external load applied to these motor proteins perturb not only the mechanical motion, but the ATP hydrolysis cycle as well, making these molecules mechano-enzymes. Here, we describe our single molecule detection techniques to reveal the mechano-enzymatic properties of myosin and introduce recent progress from both experimental and theoretical approaches at the single- and multiple-molecule level.

Assembly and diploid architecture of an individual human genome via single-molecule technologies

PubMed Central

Pendleton, Matthew; Sebra, Robert; Pang, Andy Wing Chun; Ummat, Ajay; Franzen, Oscar; Rausch, Tobias; Stütz, Adrian M; Stedman, William; Anantharaman, Thomas; Hastie, Alex; Dai, Heng; Fritz, Markus Hsi-Yang; Cao, Han; Cohain, Ariella; Deikus, Gintaras; Durrett, Russell E; Blanchard, Scott C; Altman, Roger; Chin, Chen-Shan; Guo, Yan; Paxinos, Ellen E; Korbel, Jan O; Darnell, Robert B; McCombie, W Richard; Kwok, Pui-Yan; Mason, Christopher E; Schadt, Eric E; Bashir, Ali

2015-01-01

We present the first comprehensive analysis of a diploid human genome that combines single-molecule sequencing with single-molecule genome maps. Our hybrid assembly markedly improves upon the contiguity observed from traditional shotgun sequencing approaches, with scaffold N50 values approaching 30 Mb, and we identified complex structural variants (SVs) missed by other high-throughput approaches. Furthermore, by combining Illumina short-read data with long reads, we phased both single-nucleotide variants and SVs, generating haplotypes with over 99% consistency with previous trio-based studies. Our work shows that it is now possible to integrate single-molecule and high-throughput sequence data to generate de novo assembled genomes that approach reference quality. PMID:26121404

Assembly and diploid architecture of an individual human genome via single-molecule technologies.

PubMed

Pendleton, Matthew; Sebra, Robert; Pang, Andy Wing Chun; Ummat, Ajay; Franzen, Oscar; Rausch, Tobias; Stütz, Adrian M; Stedman, William; Anantharaman, Thomas; Hastie, Alex; Dai, Heng; Fritz, Markus Hsi-Yang; Cao, Han; Cohain, Ariella; Deikus, Gintaras; Durrett, Russell E; Blanchard, Scott C; Altman, Roger; Chin, Chen-Shan; Guo, Yan; Paxinos, Ellen E; Korbel, Jan O; Darnell, Robert B; McCombie, W Richard; Kwok, Pui-Yan; Mason, Christopher E; Schadt, Eric E; Bashir, Ali

2015-08-01

We present the first comprehensive analysis of a diploid human genome that combines single-molecule sequencing with single-molecule genome maps. Our hybrid assembly markedly improves upon the contiguity observed from traditional shotgun sequencing approaches, with scaffold N50 values approaching 30 Mb, and we identified complex structural variants (SVs) missed by other high-throughput approaches. Furthermore, by combining Illumina short-read data with long reads, we phased both single-nucleotide variants and SVs, generating haplotypes with over 99% consistency with previous trio-based studies. Our work shows that it is now possible to integrate single-molecule and high-throughput sequence data to generate de novo assembled genomes that approach reference quality.

An integrated optics microfluidic device for detecting single DNA molecules.

PubMed

Krogmeier, Jeffrey R; Schaefer, Ian; Seward, George; Yantz, Gregory R; Larson, Jonathan W

2007-12-01

A fluorescence-based integrated optics microfluidic device is presented, capable of detecting single DNA molecules in a high throughput and reproducible manner. The device integrates microfluidics for DNA stretching with two optical elements for single molecule detection (SMD): a plano-aspheric refractive lens for fluorescence excitation (illuminator) and a solid parabolic reflective mirror for fluorescence collection (collector). Although miniaturized in size, both optical components were produced and assembled onto the microfluidic device by readily manufacturable fabrication techniques. The optical resolution of the device is determined by the small and relatively low numerical aperture (NA) illuminator lens (0.10 effective NA, 4.0 mm diameter) that delivers excitation light to a diffraction limited 2.0 microm diameter spot at full width half maximum within the microfluidic channel. The collector (0.82 annular NA, 15 mm diameter) reflects the fluorescence over a large collection angle, representing 71% of a hemisphere, toward a single photon counting module in an infinity-corrected scheme. As a proof-of-principle experiment for this simple integrated device, individual intercalated lambda-phage DNA molecules (48.5 kb) were stretched in a mixed elongational-shear microflow, detected, and sized with a fluorescence signal to noise ratio of 9.9 +/-1.0. We have demonstrated that SMD does not require traditional high numerical aperture objective lenses and sub-micron positioning systems conventionally used in many applications. Rather, standard manufacturing processes can be combined in a novel way that promises greater accessibility and affordability for microfluidic-based single molecule applications.

Single-molecule analysis of DNA uncoiling by a type II topoisomerase

NASA Astrophysics Data System (ADS)

Strick, Terence R.; Croquette, Vincent; Bensimon, David

2000-04-01

Type II DNA topoisomerases are ubiquitous ATP-dependent enzymes capable of transporting a DNA through a transient double-strand break in a second DNA segment. This enables them to untangle DNA and relax the interwound supercoils (plectonemes) that arise in twisted DNA. In vivo, they are responsible for untangling replicated chromosomes and their absence at mitosis or meiosis ultimately causes cell death. Here we describe a micromanipulation experiment in which we follow in real time a single Drosophila melanogaster topoisomerase II acting on a linear DNA molecule which is mechanically stretched and supercoiled. By monitoring the DNA's extension in the presence of ATP, we directly observe the relaxation of two supercoils during a single catalytic turnover. By controlling the force pulling on the molecule, we determine the variation of the reaction rate with the applied stress. Finally, in the absence of ATP, we observe the clamping of a DNA crossover by a single topoisomerase on at least two different timescales (configurations). These results show that single molecule experiments are a powerful new tool for the study of topoisomerases.

Isolation and purification of an early pregnancy factor-like molecule from culture supernatants obtained from lymphocytes of pregnant women.

PubMed

Aranha, C; Natraj, U; Iyer, K S; Shahani, S

1998-03-01

Our purpose was to determine whether lymphocytes synthesize proteins during pregnancy, to observe whether one of the proteins synthesized has early pregnancy factor (EPF)-like activity and to isolate and purify this molecule from culture supernatants obtained from stimulated lymphocytes of pregnant women. Lymphocyte proliferation assay and 35S-methionine labeling were done to study de novo synthesis of proteins followed by autoradiography to confirm synthesis of proteins. The rosette inhibition assay was used for detection of the EPF-like molecule. Gel filtration on Sephadex G-100 and RPHPLC were used for purification of the EPF-like molecule. The rate of incorporation of 35S-methionine was significantly higher in the lymphocytes of pregnant women compared to those of the control, and autoradiography confirmed the synthesis of proteins during pregnancy. There is a total protein enhancement trend observed during the first trimester that declines toward term. The EPF-like molecule is observed to be synthesized during all the trimesters of pregnancy. This molecule, when purified, showed a single homogeneous biologically active peak. The results indicated that there is an enhancement of existing protein or synthesis of new proteins during pregnancy. The EPF-like molecule is one of the many proteins synthesized and secreted by lymphocytes during pregnancy that, when purified, is biologically active.

Single-molecule toroics in Ising-type lanthanide molecular clusters.

PubMed

Ungur, Liviu; Lin, Shuang-Yan; Tang, Jinkui; Chibotaru, Liviu F

2014-01-01

Single-molecule toroics (SMTs) are defined, by analogy with single-molecule magnets, as bistable molecules with a toroidal magnetic state, and seem to be most promising for future applications in quantum computing and information storage and use as multiferroic materials with magnetoelectric effect. As an interdisciplinary research area that spans chemistry, physics and material sciences, synthetic chemists have produced systems suitable for detailed study by physicists and materials scientists, while ab initio calculations have been playing a major role in the detection of toroidal magnetization and the advancement of this field. In this tutorial review, we demonstrate the research developed in the fascinating and challenging field of molecular-based SMTs with particular focus on how recent studies tend to address the issue of toroidal arrangement of the magnetic moment in these systems. Herein, nine typical SMTs are summarized, showing that the assembly of wheel-shaped complexes with the high symmetry of the molecule unit and strong intra-molecular dipolar interactions using strong anisotropy metal ions represents the most promising route toward the design of a toroidal moment. Furthermore, the linkage of such robust toroidal moment units with ferromagnetic type through appropriate bridging ligands enhances the toroidal magnetic moment per unit cell.

DNA Origami Directed Au Nanostar Dimers for Single-Molecule Surface-Enhanced Raman Scattering.

PubMed

Tanwar, Swati; Haldar, Krishna Kanta; Sen, Tapasi

2017-12-06

We demonstrate the synthesis of Au nanostar dimers with tunable interparticle gap and controlled stoichiometry assembled on DNA origami. Au nanostars with uniform and sharp tips were immobilized on rectangular DNA origami dimerized structures to create nanoantennas containing monomeric and dimeric Au nanostars. Single Texas red (TR) dye was specifically attached in the junction of the dimerized origami to act as a Raman reporter molecule. The SERS enhancement factors of single TR dye molecules located in the conjunction region in dimer structures having interparticle gaps of 7 and 13 nm are 2 × 10 10 and 8 × 10 9 , respectively, which are strong enough for single analyte detection. The highly enhanced electromagnetic field generated by the plasmon coupling between sharp tips and cores of two Au nanostars in the wide conjunction region allows the accommodation and specific detection of large biomolecules. Such DNA-directed assembled nanoantennas with controlled interparticle separation distance and stoichiometry, and well-defined geometry, can be used as excellent substrates in single-molecule SERS spectroscopy and will have potential applications as a reproducible platform in single-molecule sensing.

Video-Rate Confocal Microscopy for Single-Molecule Imaging in Live Cells and Superresolution Fluorescence Imaging

PubMed Central

Lee, Jinwoo; Miyanaga, Yukihiro; Ueda, Masahiro; Hohng, Sungchul

2012-01-01

There is no confocal microscope optimized for single-molecule imaging in live cells and superresolution fluorescence imaging. By combining the swiftness of the line-scanning method and the high sensitivity of wide-field detection, we have developed a, to our knowledge, novel confocal fluorescence microscope with a good optical-sectioning capability (1.0 μm), fast frame rates (<33 fps), and superior fluorescence detection efficiency. Full compatibility of the microscope with conventional cell-imaging techniques allowed us to do single-molecule imaging with a great ease at arbitrary depths of live cells. With the new microscope, we monitored diffusion motion of fluorescently labeled cAMP receptors of Dictyostelium discoideum at both the basal and apical surfaces and obtained superresolution fluorescence images of microtubules of COS-7 cells at depths in the range 0–85 μm from the surface of a coverglass. PMID:23083712

Basic concepts of quantum interference and electron transport in single-molecule electronics.

PubMed

Lambert, C J

2015-02-21

This tutorial outlines the basic theoretical concepts and tools which underpin the fundamentals of phase-coherent electron transport through single molecules. The key quantity of interest is the transmission coefficient T(E), which yields the electrical conductance, current-voltage relations, the thermopower S and the thermoelectric figure of merit ZT of single-molecule devices. Since T(E) is strongly affected by quantum interference (QI), three manifestations of QI in single-molecules are discussed, namely Mach-Zehnder interferometry, Breit-Wigner resonances and Fano resonances. A simple MATLAB code is provided, which allows the novice reader to explore QI in multi-branched structures described by a tight-binding (Hückel) Hamiltonian. More generally, the strengths and limitations of materials-specific transport modelling based on density functional theory are discussed.

Tilted Light Sheet Microscopy with 3D Point Spread Functions for Single-Molecule Super-Resolution Imaging in Mammalian Cells.

PubMed

Gustavsson, Anna-Karin; Petrov, Petar N; Lee, Maurice Y; Shechtman, Yoav; Moerner, W E

2018-02-01

To obtain a complete picture of subcellular nanostructures, cells must be imaged with high resolution in all three dimensions (3D). Here, we present tilted light sheet microscopy with 3D point spread functions (TILT3D), an imaging platform that combines a novel, tilted light sheet illumination strategy with engineered long axial range point spread functions (PSFs) for low-background, 3D super localization of single molecules as well as 3D super-resolution imaging in thick cells. TILT3D is built upon a standard inverted microscope and has minimal custom parts. The axial positions of the single molecules are encoded in the shape of the PSF rather than in the position or thickness of the light sheet, and the light sheet can therefore be formed using simple optics. The result is flexible and user-friendly 3D super-resolution imaging with tens of nm localization precision throughout thick mammalian cells. We validated TILT3D for 3D super-resolution imaging in mammalian cells by imaging mitochondria and the full nuclear lamina using the double-helix PSF for single-molecule detection and the recently developed Tetrapod PSF for fiducial bead tracking and live axial drift correction. We envision TILT3D to become an important tool not only for 3D super-resolution imaging, but also for live whole-cell single-particle and single-molecule tracking.

Tilted light sheet microscopy with 3D point spread functions for single-molecule super-resolution imaging in mammalian cells

NASA Astrophysics Data System (ADS)

Gustavsson, Anna-Karin; Petrov, Petar N.; Lee, Maurice Y.; Shechtman, Yoav; Moerner, W. E.

2018-02-01

To obtain a complete picture of subcellular nanostructures, cells must be imaged with high resolution in all three dimensions (3D). Here, we present tilted light sheet microscopy with 3D point spread functions (TILT3D), an imaging platform that combines a novel, tilted light sheet illumination strategy with engineered long axial range point spread functions (PSFs) for low-background, 3D super localization of single molecules as well as 3D super-resolution imaging in thick cells. TILT3D is built upon a standard inverted microscope and has minimal custom parts. The axial positions of the single molecules are encoded in the shape of the PSF rather than in the position or thickness of the light sheet, and the light sheet can therefore be formed using simple optics. The result is flexible and user-friendly 3D super-resolution imaging with tens of nm localization precision throughout thick mammalian cells. We validated TILT3D for 3D superresolution imaging in mammalian cells by imaging mitochondria and the full nuclear lamina using the double-helix PSF for single-molecule detection and the recently developed Tetrapod PSF for fiducial bead tracking and live axial drift correction. We envision TILT3D to become an important tool not only for 3D super-resolution imaging, but also for live whole-cell single-particle and single-molecule tracking.

Nonequilibrium Chemical Effects in Single-Molecule SERS Revealed by Ab Initio Molecular Dynamics Simulations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fischer, Sean A.; Aprà, Edoardo; Govind, Niranjan

2017-02-03

Recent developments in nanophotonics have paved the way for achieving significant advances in the realm of single molecule chemical detection, imaging, and dynamics. In particular, surface-enhanced Raman scattering (SERS) is a powerful analytical technique that is now routinely used to identify the chemical identity of single molecules. Understanding how nanoscale physical and chemical processes affect single molecule SERS spectra and selection rules is a challenging task, and is still actively debated. Herein, we explore underappreciated chemical phenomena in ultrasensitive SERS. We observe a fluctuating excited electronic state manifold, governed by the conformational dynamics of a molecule (4,4’-dimercaptostilbene, DMS) interacting withmore » a metallic cluster (Ag20). This affects our simulated single molecule SERS spectra; the time trajectories of a molecule interacting with its unique local environment dictates the relative intensities of the observable Raman-active vibrational states. Ab initio molecular dynamics of a model Ag20-DMS system are used to illustrate both concepts in light of recent experimental results.« less

Single molecule experimentation in biological physics: exploring the living component of soft condensed matter one molecule at a time.

PubMed

Harriman, O L J; Leake, M C

2011-12-21

The soft matter of biological systems consists of mesoscopic length scale building blocks, composed of a variety of different types of biological molecules. Most single biological molecules are so small that 1 billion would fit on the full-stop at the end of this sentence, but collectively they carry out the vital activities in living cells whose length scale is at least three orders of magnitude greater. Typically, the number of molecules involved in any given cellular process at any one time is relatively small, and so real physiological events may often be dominated by stochastics and fluctuation behaviour at levels comparable to thermal noise, and are generally heterogeneous in nature. This challenging combination of heterogeneity and stochasticity is best investigated experimentally at the level of single molecules, as opposed to more conventional bulk ensemble-average techniques. In recent years, the use of such molecular experimental approaches has become significantly more widespread in research laboratories around the world. In this review we discuss recent experimental approaches in biological physics which can be applied to investigate the living component of soft condensed matter to a precision of a single molecule. © 2011 IOP Publishing Ltd Printed in the UK & the USA

New photon-counting detectors for single-molecule fluorescence spectroscopy and imaging

PubMed Central

Michalet, X.; Colyer, R. A.; Scalia, G.; Weiss, S.; Siegmund, Oswald H. W.; Tremsin, Anton S.; Vallerga, John V.; Villa, F.; Guerrieri, F.; Rech, I.; Gulinatti, A.; Tisa, S.; Zappa, F.; Ghioni, M.; Cova, S.

2013-01-01

Solution-based single-molecule fluorescence spectroscopy is a powerful new experimental approach with applications in all fields of natural sciences. Two typical geometries can be used for these experiments: point-like and widefield excitation and detection. In point-like geometries, the basic concept is to excite and collect light from a very small volume (typically femtoliter) and work in a concentration regime resulting in rare burst-like events corresponding to the transit of a single-molecule. Those events are accumulated over time to achieve proper statistical accuracy. Therefore the advantage of extreme sensitivity is somewhat counterbalanced by a very long acquisition time. One way to speed up data acquisition is parallelization. Here we will discuss a general approach to address this issue, using a multispot excitation and detection geometry that can accommodate different types of novel highly-parallel detector arrays. We will illustrate the potential of this approach with fluorescence correlation spectroscopy (FCS) and single-molecule fluorescence measurements. In widefield geometries, the same issues of background reduction and single-molecule concentration apply, but the duration of the experiment is fixed by the time scale of the process studied and the survival time of the fluorescent probe. Temporal resolution on the other hand, is limited by signal-to-noise and/or detector resolution, which calls for new detector concepts. We will briefly present our recent results in this domain. PMID:24729836

New photon-counting detectors for single-molecule fluorescence spectroscopy and imaging.

PubMed

Michalet, X; Colyer, R A; Scalia, G; Weiss, S; Siegmund, Oswald H W; Tremsin, Anton S; Vallerga, John V; Villa, F; Guerrieri, F; Rech, I; Gulinatti, A; Tisa, S; Zappa, F; Ghioni, M; Cova, S

2011-05-13

Solution-based single-molecule fluorescence spectroscopy is a powerful new experimental approach with applications in all fields of natural sciences. Two typical geometries can be used for these experiments: point-like and widefield excitation and detection. In point-like geometries, the basic concept is to excite and collect light from a very small volume (typically femtoliter) and work in a concentration regime resulting in rare burst-like events corresponding to the transit of a single-molecule. Those events are accumulated over time to achieve proper statistical accuracy. Therefore the advantage of extreme sensitivity is somewhat counterbalanced by a very long acquisition time. One way to speed up data acquisition is parallelization. Here we will discuss a general approach to address this issue, using a multispot excitation and detection geometry that can accommodate different types of novel highly-parallel detector arrays. We will illustrate the potential of this approach with fluorescence correlation spectroscopy (FCS) and single-molecule fluorescence measurements. In widefield geometries, the same issues of background reduction and single-molecule concentration apply, but the duration of the experiment is fixed by the time scale of the process studied and the survival time of the fluorescent probe. Temporal resolution on the other hand, is limited by signal-to-noise and/or detector resolution, which calls for new detector concepts. We will briefly present our recent results in this domain.

Quantitative Connection between Ensemble Thermodynamics and Single-Molecule Kinetics: A Case Study Using Cryogenic Electron Microscopy and Single-Molecule Fluorescence Resonance Energy Transfer Investigations of the Ribosome.

PubMed

Thompson, Colin D Kinz; Sharma, Ajeet K; Frank, Joachim; Gonzalez, Ruben L; Chowdhury, Debashish

2015-08-27

At equilibrium, thermodynamic and kinetic information can be extracted from biomolecular energy landscapes by many techniques. However, while static, ensemble techniques yield thermodynamic data, often only dynamic, single-molecule techniques can yield the kinetic data that describe transition-state energy barriers. Here we present a generalized framework based upon dwell-time distributions that can be used to connect such static, ensemble techniques with dynamic, single-molecule techniques, and thus characterize energy landscapes to greater resolutions. We demonstrate the utility of this framework by applying it to cryogenic electron microscopy (cryo-EM) and single-molecule fluorescence resonance energy transfer (smFRET) studies of the bacterial ribosomal pre-translocation complex. Among other benefits, application of this framework to these data explains why two transient, intermediate conformations of the pre-translocation complex, which are observed in a cryo-EM study, may not be observed in several smFRET studies.

Single molecule tools for enzymology, structural biology, systems biology and nanotechnology: an update

PubMed Central

Widom, Julia R.; Dhakal, Soma; Heinicke, Laurie A.; Walter, Nils G.

2015-01-01

Toxicology is the highly interdisciplinary field studying the adverse effects of chemicals on living organisms. It requires sensitive tools to detect such effects. After their initial implementation during the 1990s, single-molecule fluorescence detection tools were quickly recognized for their potential to contribute greatly to many different areas of scientific inquiry. In the intervening time, technical advances in the field have generated ever-improving spatial and temporal resolution, and have enabled the application of single-molecule fluorescence to increasingly complex systems, such as live cells. In this review, we give an overview of the optical components necessary to implement the most common versions of single-molecule fluorescence detection. We then discuss current applications to enzymology and structural studies, systems biology, and nanotechnology, presenting the technical considerations that are unique to each area of study, along with noteworthy recent results. We also highlight future directions that have the potential to revolutionize these areas of study by further exploiting the capabilities of single-molecule fluorescence microscopy. PMID:25212907

Revealing the Raft Domain Organization in the Plasma Membrane by Single-Molecule Imaging of Fluorescent Ganglioside Analogs.

PubMed

Suzuki, Kenichi G N; Ando, Hiromune; Komura, Naoko; Konishi, Miku; Imamura, Akihiro; Ishida, Hideharu; Kiso, Makoto; Fujiwara, Takahiro K; Kusumi, Akihiro

2018-01-01

Gangliosides have been implicated in a variety of physiological processes, particularly in the formation and function of raft domains in the plasma membrane. However, the scarcity of suitable fluorescent ganglioside analogs had long prevented us from determining exactly how gangliosides perform their functions in the live-cell plasma membrane. With the development of new fluorescent ganglioside analogs, as described by Komura et al. (2017), this barrier has been broken. We can now address the dynamic behaviors of gangliosides in the live-cell plasma membrane, using fluorescence microscopy, particularly by single-fluorescent molecule imaging and tracking. Single-molecule tracking of fluorescent GM1 and GM3 revealed that these molecules are transiently and dynamically recruited to monomers (monomer-associated rafts) and homodimer rafts of the raftophilic GPI-anchored protein CD59 in quiescent cells, with exponential residency times of 12 and 40ms, respectively, in a manner dependent on raft-lipid interactions. Upon CD59 stimulation, which induces CD59-cluster signaling rafts, the fluorescent GM1 and GM3 analogs were recruited to the signaling rafts, with a lifetime of 48ms. These results represent the first direct evidence that GPI-anchored receptors and gangliosides interact in a cholesterol-dependent manner. Furthermore, they show that gangliosides continually move in and out of rafts that contain CD59 in an extremely dynamic manner, with much higher frequency than expected previously. Such studies would not have been possible without fluorescent ganglioside probes, which exhibit native-like behavior and single-molecule tracking. In this chapter, we review the methods for single-molecule tracking of fluorescent ganglioside analogs and the results obtained by applying these methods. © 2018 Elsevier Inc. All rights reserved.

From single-molecule spectroscopy to super-resolution imaging of the neuron: a review

PubMed Central

Laine, Romain F; Kaminski Schierle, Gabriele S; van de Linde, Sebastian; Kaminski, Clemens F

2016-01-01

Abstract For more than 20 years, single-molecule spectroscopy has been providing invaluable insights into nature at the molecular level. The field has received a powerful boost with the development of the technique into super-resolution imaging methods, ca. 10 years ago, which overcome the limitations imposed by optical diffraction. Today, single molecule super-resolution imaging is routinely used in the study of macromolecular function and structure in the cell. Concomitantly, computational methods have been developed that provide information on numbers and positions of molecules at the nanometer-scale. In this overview, we outline the technical developments that have led to the emergence of localization microscopy techniques from single-molecule spectroscopy. We then provide a comprehensive review on the application of the technique in the field of neuroscience research. PMID:28809165

Developing DNA nanotechnology using single-molecule fluorescence.

PubMed

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

Extracting physics of life at the molecular level: A review of single-molecule data analyses.

PubMed

Colomb, Warren; Sarkar, Susanta K

2015-06-01

Studying individual biomolecules at the single-molecule level has proved very insightful recently. Single-molecule experiments allow us to probe both the equilibrium and nonequilibrium properties as well as make quantitative connections with ensemble experiments and equilibrium thermodynamics. However, it is important to be careful about the analysis of single-molecule data because of the noise present and the lack of theoretical framework for processes far away from equilibrium. Biomolecular motion, whether it is free in solution, on a substrate, or under force, involves thermal fluctuations in varying degrees, which makes the motion noisy. In addition, the noise from the experimental setup makes it even more complex. The details of biologically relevant interactions, conformational dynamics, and activities are hidden in the noisy single-molecule data. As such, extracting biological insights from noisy data is still an active area of research. In this review, we will focus on analyzing both fluorescence-based and force-based single-molecule experiments and gaining biological insights at the single-molecule level. Inherently nonequilibrium nature of biological processes will be highlighted. Simulated trajectories of biomolecular diffusion will be used to compare and validate various analysis techniques. Copyright © 2015 Elsevier B.V. All rights reserved.

Probing Electronic and Thermoelectric Properties of Single Molecule Junctions

NASA Astrophysics Data System (ADS)

Widawsky, Jonathan R.

In an effort to further understand electronic and thermoelectric phenomenon at the nanometer scale, we have studied the transport properties of single molecule junctions. To carry out these transport measurements, we use the scanning tunneling microscope-break junction (STM-BJ) technique, which involves the repeated formation and breakage of a metal point contact in an environment of the target molecule. Using this technique, we are able to create gaps that can trap the molecules, allowing us to sequentially and reproducibly create a large number of junctions. By applying a small bias across the junction, we can measure its conductance and learn about the transport mechanisms at the nanoscale. The experimental work presented here directly probes the transmission properties of single molecules through the systematic measurement of junction conductance (at low and high bias) and thermopower. We present measurements on a variety of molecular families and study how conductance depends on the character of the linkage (metal-molecule bond) and the nature of the molecular backbone. We start by describing a novel way to construct single molecule junctions by covalently connecting the molecular backbone to the electrodes. This eliminates the use of linking substituents, and as a result, the junction conductance increases substantially. Then, we compare transport across silicon chains (silanes) and saturated carbon chains (alkanes) while keeping the linkers the same and find a stark difference in their electronic transport properties. We extend our studies of molecular junctions by looking at two additional aspects of quantum transport -- molecular thermopower and molecular current-voltage characteristics. Each of these additional parameters gives us further insight into transport properties at the nanoscale. Evaluating the junction thermopower allows us to determine the nature of charge carriers in the system and we demonstrate this by contrasting the measurement of amine

Nanopore sensing at ultra-low concentrations using single-molecule dielectrophoretic trapping

NASA Astrophysics Data System (ADS)

Freedman, Kevin J.; Otto, Lauren M.; Ivanov, Aleksandar P.; Barik, Avijit; Oh, Sang-Hyun; Edel, Joshua B.

2016-01-01

Single-molecule techniques are being developed with the exciting prospect of revolutionizing the healthcare industry by generating vast amounts of genetic and proteomic data. One exceptionally promising route is in the use of nanopore sensors. However, a well-known complexity is that detection and capture is predominantly diffusion limited. This problem is compounded when taking into account the capture volume of a nanopore, typically 108-1010 times smaller than the sample volume. To rectify this disproportionate ratio, we demonstrate a simple, yet powerful, method based on coupling single-molecule dielectrophoretic trapping to nanopore sensing. We show that DNA can be captured from a controllable, but typically much larger, volume and concentrated at the tip of a metallic nanopore. This enables the detection of single molecules at concentrations as low as 5 fM, which is approximately a 103 reduction in the limit of detection compared with existing methods, while still maintaining efficient throughput.

Magnetic field dependent electronic transport of Mn4 single-molecule magnet.

NASA Astrophysics Data System (ADS)

Haque, F.; Langhirt, M.; Henderson, J. J.; Del Barco, E.; Taguchi, T.; Christou, G.

2010-03-01

We have performed single-electron transport measurements on a Mn4 single-molecule magnet (SMM) in where amino groups were added to electrically protect the magnetic core and to increase the stability of the molecule when deposited on the single-electron transistor (SET) chip. A three-terminal SET with nano-gap electro-migrated gold electrodes and a naturally oxidized Aluminum back gate. Experiments were conducted at temperatures down to 230mK in the presence of high magnetic fields generated by a superconducting vector magnet. Mn4 molecules were deposited from solution to form a mono-layer. The optimum deposition time was determined by AFM analysis on atomically flat gold surfaces. We have observed Coulomb blockade an electronic excitations that curve with the magnetic field and present zero-field splitting, which represents evidence of magnetic anisotropy. Level anticrossings and large excitations slopes are associated with the behavior of molecular states with high spin values (S ˜ 9), as expected from Mn4.

Nanopore sensing at ultra-low concentrations using single-molecule dielectrophoretic trapping

PubMed Central

Freedman, Kevin J.; Otto, Lauren M.; Ivanov, Aleksandar P.; Barik, Avijit; Oh, Sang-Hyun; Edel, Joshua B.

2016-01-01

Single-molecule techniques are being developed with the exciting prospect of revolutionizing the healthcare industry by generating vast amounts of genetic and proteomic data. One exceptionally promising route is in the use of nanopore sensors. However, a well-known complexity is that detection and capture is predominantly diffusion limited. This problem is compounded when taking into account the capture volume of a nanopore, typically 108–1010 times smaller than the sample volume. To rectify this disproportionate ratio, we demonstrate a simple, yet powerful, method based on coupling single-molecule dielectrophoretic trapping to nanopore sensing. We show that DNA can be captured from a controllable, but typically much larger, volume and concentrated at the tip of a metallic nanopore. This enables the detection of single molecules at concentrations as low as 5 fM, which is approximately a 103 reduction in the limit of detection compared with existing methods, while still maintaining efficient throughput. PMID:26732171

Arrhenius plot for a reaction catalyzed by a single molecule of β-galactosidase.

PubMed

Craig, Douglas B; Chase, Linden N

2012-02-21

The activity of a single enzyme molecule of Escherichia coli β-galactosidase was measured using a capillary electrophoresis continuous flow assay. As the enzyme molecule traversed the capillary the incubation temperature was increased from 27 to 37 °C, providing a continuous record of the change in rate with temperature. This data was used to develop a single enzyme molecule Arrhenius plot, from which the activation energy of the reaction was determined to be 31 kJ mol(-1).

Unfolding single RNA molecules: bridging the gap between equilibrium and non-equilibrium statistical thermodynamics.

PubMed

Bustamante, Carlos

2005-11-01

During the last 15 years, scientists have developed methods that permit the direct mechanical manipulation of individual molecules. Using this approach, they have begun to investigate the effect of force and torque in chemical and biochemical reactions. These studies span from the study of the mechanical properties of macromolecules, to the characterization of molecular motors, to the mechanical unfolding of individual proteins and RNA. Here I present a review of some of our most recent results using mechanical force to unfold individual molecules of RNA. These studies make it possible to follow in real time the trajectory of each molecule as it unfolds and characterize the various intermediates of the reaction. Moreover, if the process takes place reversibly it is possible to extract both kinetic and thermodynamic information from these experiments at the same time that we characterize the forces that maintain the three-dimensional structure of the molecule in solution. These studies bring us closer to the biological unfolding processes in the cell as they simulate in vitro, the mechanical unfolding of RNAs carried out in the cell by helicases. If the unfolding process occurs irreversibly, I show here that single-molecule experiments can still provide equilibrium, thermodynamic information from non-equilibrium data by using recently discovered fluctuation theorems. Such theorems represent a bridge between equilibrium and non-equilibrium statistical mechanics. In fact, first derived in 1997, the first experimental demonstration of the validity of fluctuation theorems was obtained by unfolding mechanically a single molecule of RNA. It is perhaps a sign of the times that important physical results are these days used to extract information about biological systems and that biological systems are being used to test and confirm fundamental new laws in physics.

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

PubMed

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.

Video-rate nanoscopy enabled by sCMOS camera-specific single-molecule localization algorithms

PubMed Central

Huang, Fang; Hartwich, Tobias M. P.; Rivera-Molina, Felix E.; Lin, Yu; Duim, Whitney C.; Long, Jane J.; Uchil, Pradeep D.; Myers, Jordan R.; Baird, Michelle A.; Mothes, Walther; Davidson, Michael W.; Toomre, Derek; Bewersdorf, Joerg

2013-01-01

Newly developed scientific complementary metal–oxide–semiconductor (sCMOS) cameras have the potential to dramatically accelerate data acquisition in single-molecule switching nanoscopy (SMSN) while simultaneously increasing the effective quantum efficiency. However, sCMOS-intrinsic pixel-dependent readout noise substantially reduces the localization precision and introduces localization artifacts. Here we present algorithms that overcome these limitations and provide unbiased, precise localization of single molecules at the theoretical limit. In combination with a multi-emitter fitting algorithm, we demonstrate single-molecule localization super-resolution imaging at up to 32 reconstructed images/second (recorded at 1,600–3,200 camera frames/second) in both fixed and living cells. PMID:23708387

Asymmetric nanopore membranes: Single molecule detection and unique transport properties

NASA Astrophysics Data System (ADS)

Bishop, Gregory William

Biological systems rely on the transport properties of transmembrane channels. Such pores can display selective transport by allowing the passage of certain ions or molecules while rejecting others. Recent advances in nanoscale fabrication have allowed the production of synthetic analogs of such channels. Synthetic nanopores (pores with a limiting dimension of 1--100 nm) can be produced in a variety of materials by several different methods. In the Martin group, we have been exploring the track-etch method to produce asymmetric nanopores in thin films of polymeric or crystalline materials. Asymmetric nanopores are of particular interest due to their ability to serve as ion-current rectifiers. This means that when a membrane that contains such a pore or collection of pores is used to separate identical portions of electrolyte solution, the magnitude of the ionic current will depend not only on the magnitude of the applied potential (as expected) but also the polarity. Ion-current rectification is characterized by an asymmetric current--potential response. Here, the interesting transport properties of asymmetric nanopores (ion-current rectification and the related phenomenon of electroosmotic flow rectification) are explored. The effects of pore shape and pore density on these phenomena are investigated. Membranes that contain a single nanopore can serve as platforms for the single-molecule sensing technique known as resistive pulse sensing. The resistive-pulse sensing method is based on the Coulter principle. Thus, the selectivity of the technique is based largely upon size, making the analysis of mixtures by this method difficult in many cases. Here, the surface of a single nanopore membrane is modified with a molecular recognition agent in an attempt to obtain a more selective resistive-pulse sensor for a specific analyte.

Nanopore extended field-effect transistor for selective single-molecule biosensing.

PubMed

Ren, Ren; Zhang, Yanjun; Nadappuram, Binoy Paulose; Akpinar, Bernice; Klenerman, David; Ivanov, Aleksandar P; Edel, Joshua B; Korchev, Yuri

2017-09-19

There has been a significant drive to deliver nanotechnological solutions to biosensing, yet there remains an unmet need in the development of biosensors that are affordable, integrated, fast, capable of multiplexed detection, and offer high selectivity for trace analyte detection in biological fluids. Herein, some of these challenges are addressed by designing a new class of nanoscale sensors dubbed nanopore extended field-effect transistor (nexFET) that combine the advantages of nanopore single-molecule sensing, field-effect transistors, and recognition chemistry. We report on a polypyrrole functionalized nexFET, with controllable gate voltage that can be used to switch on/off, and slow down single-molecule DNA transport through a nanopore. This strategy enables higher molecular throughput, enhanced signal-to-noise, and even heightened selectivity via functionalization with an embedded receptor. This is shown for selective sensing of an anti-insulin antibody in the presence of its IgG isotype.Efficient detection of single molecules is vital to many biosensing technologies, which require analytical platforms with high selectivity and sensitivity. Ren et al. combine a nanopore sensor and a field-effect transistor, whereby gate voltage mediates DNA and protein transport through the nanopore.

Direct observation of λ-DNA molecule reversal movement within microfluidic channels under electric field with single molecule imaging technique

NASA Astrophysics Data System (ADS)

Fengyun, Yang; Kaige, Wang; Dan, Sun; Wei, Zhao; Hai-qing, Wang; Xin, He; Gui-ren, Wang; Jin-tao, Bai

2016-07-01

The electrodynamic characteristics of single DNA molecules moving within micro-/nano-fluidic channels are important in the design of biomedical chips and bimolecular sensors. In this study, the dynamic properties of λ-DNA molecules transferring along the microchannels driven by the external electrickinetic force were systemically investigated with the single molecule fluorescence imaging technique. The experimental results indicated that the velocity of DNA molecules was strictly dependent on the value of the applied electric field and the diameter of the channel. The larger the external electric field, the larger the velocity, and the more significant deformation of DNA molecules. More meaningfully, it was found that the moving directions of DNA molecules had two completely different directions: (i) along the direction of the external electric field, when the electric field intensity was smaller than a certain threshold value; (ii) opposite to the direction of the external electric field, when the electric field intensity was greater than the threshold electric field intensity. The reversal movement of DNA molecules was mainly determined by the competition between the electrophoresis force and the influence of electro-osmosis flow. These new findings will theoretically guide the practical application of fluidic channel sensors and lab-on-chips for precisely manipulating single DNA molecules. Project supported by the National Natural Science Foundation of China (Grant No. 61378083), the International Cooperation Foundation of the National Science and Technology Major Project of the Ministry of Science and Technology of China (Grant No. 2011DFA12220), the Major Research Plan of National Natural Science Foundation of China (Grant No. 91123030), and the Natural Science Foundation of Shaanxi Province of China (Grant Nos. 2010JS110 and 2013SZS03-Z01).

In Planta Single-Molecule Pull-Down Reveals Tetrameric Stoichiometry of HD-ZIPIII:LITTLE ZIPPER Complexes.

PubMed

Husbands, Aman Y; Aggarwal, Vasudha; Ha, Taekjip; Timmermans, Marja C P

2016-08-01

Deciphering complex biological processes markedly benefits from approaches that directly assess the underlying biomolecular interactions. Most commonly used approaches to monitor protein-protein interactions typically provide nonquantitative readouts that lack statistical power and do not yield information on the heterogeneity or stoichiometry of protein complexes. Single-molecule pull-down (SiMPull) uses single-molecule fluorescence detection to mitigate these disadvantages and can quantitatively interrogate interactions between proteins and other compounds, such as nucleic acids, small molecule ligands, and lipids. Here, we establish SiMPull in plants using the HOMEODOMAIN LEUCINE ZIPPER III (HD-ZIPIII) and LITTLE ZIPPER (ZPR) interaction as proof-of-principle. Colocalization analysis of fluorophore-tagged HD-ZIPIII and ZPR proteins provides strong statistical evidence of complex formation. In addition, we use SiMPull to directly quantify YFP and mCherry maturation probabilities, showing these differ substantially from values obtained in mammalian systems. Leveraging these probabilities, in conjunction with fluorophore photobleaching assays on over 2000 individual complexes, we determined HD-ZIPIII:ZPR stoichiometry. Intriguingly, these complexes appear as heterotetramers, comprising two HD-ZIPIII and two ZPR molecules, rather than heterodimers as described in the current model. This surprising result raises new questions about the regulation of these key developmental factors and is illustrative of the unique contribution SiMPull is poised to make to in planta protein interaction studies. © 2016 American Society of Plant Biologists. All rights reserved.

Hybrid error correction and de novo assembly of single-molecule sequencing reads

PubMed Central

Koren, Sergey; Schatz, Michael C.; Walenz, Brian P.; Martin, Jeffrey; Howard, Jason; Ganapathy, Ganeshkumar; Wang, Zhong; Rasko, David A.; McCombie, W. Richard; Jarvis, Erich D.; Phillippy, Adam M.

2012-01-01

Emerging single-molecule sequencing instruments can generate multi-kilobase sequences with the potential to dramatically improve genome and transcriptome assembly. However, the high error rate of single-molecule reads is challenging, and has limited their use to resequencing bacteria. To address this limitation, we introduce a novel correction algorithm and assembly strategy that utilizes shorter, high-identity sequences to correct the error in single-molecule sequences. We demonstrate the utility of this approach on Pacbio RS reads of phage, prokaryotic, and eukaryotic whole genomes, including the novel genome of the parrot Melopsittacus undulatus, as well as for RNA-seq reads of the corn (Zea mays) transcriptome. Our approach achieves over 99.9% read correction accuracy and produces substantially better assemblies than current sequencing strategies: in the best example, quintupling the median contig size relative to high-coverage, second-generation assemblies. Greater gains are predicted if read lengths continue to increase, including the prospect of single-contig bacterial chromosome assembly. PMID:22750884

Observation of Spectral Diffusion in Crystals Using Single Impurity Molecules

DTIC Science & Technology

1990-10-31

from 12pentacene photophysical parameters including intersystem crossing . Apparently (and not surprisingly), the local pentacene environment this... pentacene molecules inp-terphenyl, both stable as well as spectrally diffusing single molecules can be observed. 20 DISTRIBUTION/AVAILABILITY OF ABSTRACT 121...with ultrathin sublimed crystals have removed this obstacle. For the case of pentacene impurities in crystals of p-terphenyl, we observe two radically

Electrostatic melting in a single-molecule field-effect transistor with applications in genomic identification

PubMed Central

Vernick, Sefi; Trocchia, Scott M.; Warren, Steven B.; Young, Erik F.; Bouilly, Delphine; Gonzalez, Ruben L.; Nuckolls, Colin; Shepard, Kenneth L.

2017-01-01

The study of biomolecular interactions at the single-molecule level holds great potential for both basic science and biotechnology applications. Single-molecule studies often rely on fluorescence-based reporting, with signal levels limited by photon emission from single optical reporters. The point-functionalized carbon nanotube transistor, known as the single-molecule field-effect transistor, is a bioelectronics alternative based on intrinsic molecular charge that offers significantly higher signal levels for detection. Such devices are effective for characterizing DNA hybridization kinetics and thermodynamics and enabling emerging applications in genomic identification. In this work, we show that hybridization kinetics can be directly controlled by electrostatic bias applied between the device and the surrounding electrolyte. We perform the first single-molecule experiments demonstrating the use of electrostatics to control molecular binding. Using bias as a proxy for temperature, we demonstrate the feasibility of detecting various concentrations of 20-nt target sequences from the Ebolavirus nucleoprotein gene in a constant-temperature environment. PMID:28516911

Statistical inference in single molecule measurements of protein adsorption

NASA Astrophysics Data System (ADS)

Armstrong, Megan J.; Tsitkov, Stanislav; Hess, Henry

2018-02-01

Significant effort has been invested into understanding the dynamics of protein adsorption on surfaces, in particular to predict protein behavior at the specialized surfaces of biomedical technologies like hydrogels, nanoparticles, and biosensors. Recently, the application of fluorescent single molecule imaging to this field has permitted the tracking of individual proteins and their stochastic contribution to the aggregate dynamics of adsorption. However, the interpretation of these results is complicated by (1) the finite time available to observe effectively infinite adsorption timescales and (2) the contribution of photobleaching kinetics to adsorption kinetics. Here, we perform a protein adsorption simulation to introduce specific survival analysis methods that overcome the first complication. Additionally, we collect single molecule residence time data from the adsorption of fibrinogen to glass and use survival analysis to distinguish photobleaching kinetics from protein adsorption kinetics.

Resolving Single Molecule Lysozyme Dynamics with a Carbon Nanotube Electronic Circuit

NASA Astrophysics Data System (ADS)

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

2011-03-01

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

An all-electric single-molecule motor.

PubMed

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.

Single-Molecule Imaging of an in Vitro-Evolved RNA Aptamer Reveals Homogeneous Ligand Binding Kinetics

PubMed Central

2009-01-01

Many studies of RNA folding and catalysis have revealed conformational heterogeneity, metastable folding intermediates, and long-lived states with distinct catalytic activities. We have developed a single-molecule imaging approach for investigating the functional heterogeneity of in vitro-evolved RNA aptamers. Monitoring the association of fluorescently labeled ligands with individual RNA aptamer molecules has allowed us to record binding events over the course of multiple days, thus providing sufficient statistics to quantitatively define the kinetic properties at the single-molecule level. The ligand binding kinetics of the highly optimized RNA aptamer studied here displays a remarkable degree of uniformity and lack of memory. Such homogeneous behavior is quite different from the heterogeneity seen in previous single-molecule studies of naturally derived RNA and protein enzymes. The single-molecule methods we describe may be of use in analyzing the distribution of functional molecules in heterogeneous evolving populations or even in unselected samples of random sequences. PMID:19572753

A single molecule perspective on the functional diversity of in vitro evolved β-glucuronidase.

PubMed

Liebherr, Raphaela B; Renner, Max; Gorris, Hans H

2014-04-23

The mechanisms that drive the evolution of new enzyme activity have been investigated by comparing the kinetics of wild-type and in vitro evolved β-glucuronidase (GUS) at the single molecule level. Several hundred single GUS molecules were separated in large arrays of 62,500 ultrasmall reaction chambers etched into the surface of a fused silica slide to observe their individual substrate turnover rates in parallel by fluorescence microscopy. Individual GUS molecules feature long-lived but divergent activity states, and their mean activity is consistent with classic Michaelis-Menten kinetics. The large number of single molecule substrate turnover rates is representative of the activity distribution within an entire enzyme population. Partially evolved GUS displays a much broader activity distribution among individual enzyme molecules than wild-type GUS. The broader activity distribution indicates a functional division of work between individual molecules in a population of partially evolved enzymes that-as so-called generalists-are characterized by their promiscuous activity with many different substrates.

Video-rate confocal microscopy for single-molecule imaging in live cells and superresolution fluorescence imaging.

PubMed

Lee, Jinwoo; Miyanaga, Yukihiro; Ueda, Masahiro; Hohng, Sungchul

2012-10-17

There is no confocal microscope optimized for single-molecule imaging in live cells and superresolution fluorescence imaging. By combining the swiftness of the line-scanning method and the high sensitivity of wide-field detection, we have developed a, to our knowledge, novel confocal fluorescence microscope with a good optical-sectioning capability (1.0 μm), fast frame rates (<33 fps), and superior fluorescence detection efficiency. Full compatibility of the microscope with conventional cell-imaging techniques allowed us to do single-molecule imaging with a great ease at arbitrary depths of live cells. With the new microscope, we monitored diffusion motion of fluorescently labeled cAMP receptors of Dictyostelium discoideum at both the basal and apical surfaces and obtained superresolution fluorescence images of microtubules of COS-7 cells at depths in the range 0-85 μm from the surface of a coverglass. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Intrinsic property measurement of surfactant-templated mesoporous silica films using time-resolved single-molecule imaging

NASA Astrophysics Data System (ADS)

Kennard, Raymond; DeSisto, William J.; Giririjan, Thanu Praba; Mason, Michael D.

2008-04-01

,1-dioctadecyl-3,3,3,3-tetramethylindo-carbocyanine perchlorate, used to interrogate the available free volume in their vicinity, were loaded into the mesoporous silica membranes at subnanomolar concentrations. The mesoporous silica films were prepared using a nonionic ethylene oxide-propylene oxide-ethylene oxide triblock copolymer surfactant, Pluronic P123, on single crystal silicon substrates using dip coating of a silica sol. Membranes were prepared resulting in an average pore diameter of approximately 5nm as measured by helium, nitrogen permeance, and porosimetry. Fluorescent images and time transient experiments were recorded using a custom built single-molecule scanning confocal microscope at differing temperatures (10, 20, 30, 40, and 50°C). Time-dependent polarization anisotropy was used to obtain the enthalpy of adsorption and Henry's law constant of the probe molecule.

Intrinsic property measurement of surfactant-templated mesoporous silica films using time-resolved single-molecule imaging.

PubMed

Kennard, Raymond; DeSisto, William J; Giririjan, Thanu Praba; Mason, Michael D

2008-04-07

,1-dioctadecyl-3,3,3,3-tetramethylindo-carbocyanine perchlorate, used to interrogate the available free volume in their vicinity, were loaded into the mesoporous silica membranes at subnanomolar concentrations. The mesoporous silica films were prepared using a nonionic ethylene oxide-propylene oxide-ethylene oxide triblock copolymer surfactant, Pluronic P123, on single crystal silicon substrates using dip coating of a silica sol. Membranes were prepared resulting in an average pore diameter of approximately 5 nm as measured by helium, nitrogen permeance, and porosimetry. Fluorescent images and time transient experiments were recorded using a custom built single-molecule scanning confocal microscope at differing temperatures (10, 20, 30, 40, and 50 degrees C). Time-dependent polarization anisotropy was used to obtain the enthalpy of adsorption and Henry's law constant of the probe molecule.

Quantitative single-molecule imaging by confocal laser scanning microscopy.

PubMed

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

2008-11-25

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

A state space based approach to localizing single molecules from multi-emitter images.

PubMed

Vahid, Milad R; Chao, Jerry; Ward, E Sally; Ober, Raimund J

2017-01-28

Single molecule super-resolution microscopy is a powerful tool that enables imaging at sub-diffraction-limit resolution. In this technique, subsets of stochastically photoactivated fluorophores are imaged over a sequence of frames and accurately localized, and the estimated locations are used to construct a high-resolution image of the cellular structures labeled by the fluorophores. Available localization methods typically first determine the regions of the image that contain emitting fluorophores through a process referred to as detection. Then, the locations of the fluorophores are estimated accurately in an estimation step. We propose a novel localization method which combines the detection and estimation steps. The method models the given image as the frequency response of a multi-order system obtained with a balanced state space realization algorithm based on the singular value decomposition of a Hankel matrix, and determines the locations of intensity peaks in the image as the pole locations of the resulting system. The locations of the most significant peaks correspond to the locations of single molecules in the original image. Although the accuracy of the location estimates is reasonably good, we demonstrate that, by using the estimates as the initial conditions for a maximum likelihood estimator, refined estimates can be obtained that have a standard deviation close to the Cramér-Rao lower bound-based limit of accuracy. We validate our method using both simulated and experimental multi-emitter images.

Enzymatic production of single-molecule FISH and RNA capture probes.

PubMed

Gaspar, Imre; Wippich, Frank; Ephrussi, Anne

2017-10-01

Arrays of singly labeled short oligonucleotides that hybridize to a specific target revolutionized RNA biology, enabling quantitative, single-molecule microscopy analysis and high-efficiency RNA/RNP capture. Here, we describe a simple and efficient method that allows flexible functionalization of inexpensive DNA oligonucleotides by different fluorescent dyes or biotin using terminal deoxynucleotidyl transferase and custom-made functional group conjugated dideoxy-UTP. We show that (i) all steps of the oligonucleotide labeling-including conjugation, enzymatic synthesis, and product purification-can be performed in a standard biology laboratory, (ii) the process yields >90%, often >95% labeled product with minimal carryover of impurities, and (iii) the oligonucleotides can be labeled with different dyes or biotin, allowing single-molecule FISH, RNA affinity purification, and Northern blot analysis to be performed. © 2017 Gaspar et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Photon Antibunching in the Fluorescence of a Single Dye Molecule Trapped in a Solid

DTIC Science & Technology

1992-06-08

number) FIELD GROUP SUB-GROUP single-molecule spectroscopy in solids, photon antibunching, quantum-optics, nonclassical effects pentacene in p-terphenyl...emitted by an optically pumped single molecule of pentacene In a p-terphenyl host has been Investigated at short times. The correlation function...excitation tcclnique, certain individual pentacene impurity molecules in a p-terphenyl crystal 11 were observed to spectrally diffuse, i.e. their absorption

Single molecule fluorescence microscopy for ultra-sensitive RNA expression profiling

NASA Astrophysics Data System (ADS)

Hesse, Jan; Jacak, Jaroslaw; Regl, Gerhard; Eichberger, Thomas; Aberger, Fritz; Schlapak, Robert; Howorka, Stefan; Muresan, Leila; Frischauf, Anna-Maria; Schütz, Gerhard J.

2007-02-01

We developed a microarray analysis platform for ultra-sensitive RNA expression profiling of minute samples. It utilizes a novel scanning system for single molecule fluorescence detection on cm2 size samples in combination with specialized biochips, optimized for low autofluorescence and weak unspecific adsorption. 20 μg total RNA was extracted from 10 6 cells of a human keratinocyte cell line (HaCaT) and reversely transcribed in the presence of Alexa647-aha-dUTP. 1% of the resulting labeled cDNA was used for complex hybridization to a custom-made oligonucleotide microarray representing a set of 125 different genes. For low abundant genes, individual cDNA molecules hybridized to the microarray spots could be resolved. Single cDNA molecules hybridized to the chip surface appeared as diffraction limited features in the fluorescence images. The à trous wavelet method was utilized for localization and counting of the separated cDNA signals. Subsequently, the degree of labeling of the localized cDNA molecules was determined by brightness analysis for the different genes. Variations by factors up to 6 were found, which in conventional microarray analysis would result in a misrepresentation of the relative abundance of mRNAs.

Techniques for 3D tracking of single molecules with nanometer accuracy in living cells

NASA Astrophysics Data System (ADS)

Gardini, Lucia; Capitanio, Marco; Pavone, Francesco S.

2013-06-01

We describe a microscopy technique that, combining wide-field single molecule microscopy, bifocal imaging and Highly Inclined and Laminated Optical sheet (HILO) microscopy, allows a 3D tracking with nanometer accuracy of single fluorescent molecules in vitro and in living cells.

Single Molecule Spectroscopy of Amino Acids and Peptides by Recognition Tunneling

PubMed Central

Zhao, Yanan; Ashcroft, Brian; Zhang, Peiming; Liu, Hao; Sen, Suman; Song, Weisi; Im, JongOne; Gyarfas, Brett; Manna, Saikat; Biswas, Sovan; Borges, Chad; Lindsay, Stuart

2014-01-01

The human proteome has millions of protein variants due to alternative RNA splicing and post-translational modifications, and variants that are related to diseases are frequently present in minute concentrations. For DNA and RNA, low concentrations can be amplified using the polymerase chain reaction, but there is no such reaction for proteins. Therefore, the development of single molecule protein sequencing is a critical step in the search for protein biomarkers. Here we show that single amino acids can be identified by trapping the molecules between two electrodes that are coated with a layer of recognition molecules and measuring the electron tunneling current across the junction. A given molecule can bind in more than one way in the junction, and we therefore use a machine-learning algorithm to distinguish between the sets of electronic ‘fingerprints’ associated with each binding motif. With this recognition tunneling technique, we are able to identify D, L enantiomers, a methylated amino acid, isobaric isomers, and short peptides. The results suggest that direct electronic sequencing of single proteins could be possible by sequentially measuring the products of processive exopeptidase digestion, or by using a molecular motor to pull proteins through a tunnel junction integrated with a nanopore. PMID:24705512

Pulling monatomic gold wires with single molecules: an Ab initio simulation.

PubMed

Krüger, Daniel; Fuchs, Harald; Rousseau, Roger; Marx, Dominik; Parrinello, Michele

2002-10-28

Car-Parrinello molecular dynamics simulations demonstrate that pulling a single thiolate molecule anchored on a stepped gold surface does not preferentially break the sulfur-gold chemical bond. Instead, it is found that this process leads to the formation of a monoatomic gold nanowire, followed by breaking a gold-gold bond with a rupture force of about 1.2 nN. The simulations also indicate that previous single-molecule thiolate-gold and gold-gold rupture experiments both probe the same phenomenon, namely, the breaking of a gold-gold bond within a gold nanowire.

Combining nanofluidics and plasmonics for single molecule detection

NASA Astrophysics Data System (ADS)

West, Melanie M.

Single molecule detection is limited by the small scattering cross-section of molecules which leads to weak optical signals that can be obscured by background noise. The combination of plasmonics and nanofluidics in an integrated nano-device has the potential to provide the signal enhancement necessary for the detection of single molecules. The purpose of this investigation was to optimize the fabrication of an optofluidic device that integrates a nanochannel with a plasmonic bowtie antenna. The fluidic structure of the device was fabricated using UV-nanoimprint lithography, and the gold plasmonic antennas were fabricated using a shadow evaporation and lift-off process. The effect of electron beam lithography doses on the resolution of antenna-nanochannel configurations was studied to minimize antenna gap size while maintaining the integrity of the imprinted features. The smallest antenna gap size that was achieved was 46 nm. The antennas were characterized using dark field spectroscopy to find the resonance shift, which indicated the appropriate range for optical signal enhancement. The dark field scattering results showed antennas with a broad and well-defined resonance shift that ranged from 650--800 nm. The Raman scattering results showed the highest enhancement factor (EF = 2) for antennas with an "inverted configuration," which involved having the triangles of the antenna facing back-to-back rather than the more conventional tip-to-tip bowtie arrangement.

Polyproline and the “spectroscopic ruler” revisited with single-molecule fluorescence

PubMed Central

Schuler, Benjamin; Lipman, Everett A.; Steinbach, Peter J.; Kumke, Michael; Eaton, William A.

2005-01-01

To determine whether Förster resonance energy transfer (FRET) measurements can provide quantitative distance information in single-molecule fluorescence experiments on polypeptides, we measured FRET efficiency distributions for donor and acceptor dyes attached to the ends of freely diffusing polyproline molecules of various lengths. The observed mean FRET efficiencies agree with those determined from ensemble lifetime measurements but differ considerably from the values expected from Förster theory, with polyproline treated as a rigid rod. At donor–acceptor distances much less than the Förster radius R0, the observed efficiencies are lower than predicted, whereas at distances comparable to and greater than R0, they are much higher. Two possible contributions to the former are incomplete orientational averaging during the donor lifetime and, because of the large size of the dyes, breakdown of the point-dipole approximation assumed in Förster theory. End-to-end distance distributions and correlation times obtained from Langevin molecular dynamics simulations suggest that the differences for the longer polyproline peptides can be explained by chain bending, which considerably shortens the donor–acceptor distances. PMID:15699337

Visualizing single molecules interacting with nuclear pore complexes by narrow-field epifluorescence microscopy

PubMed Central

Yang, Weidong; Musser, Siegfried M.

2008-01-01

The utility of single molecule fluorescence (SMF) for understanding biological reactions has been amply demonstrated by a diverse series of studies over the last decade. In large part, the molecules of interest have been limited to those within a small focal volume or near a surface to achieve the high sensitivity required for detecting the inherently weak signals arising from individual molecules. Consequently, the investigation of molecular behavior with high time and spatial resolution deep within cells using SMF has remained challenging. Recently, we demonstrated that narrow-field epifluorescence microscopy allows visualization of nucleocytoplasmic transport at the single cargo level. We describe here the methodological approach that yields 2 ms and ∼15 nm resolution for a stationary particle. The spatial resolution for a mobile particle is inherently worse, and depends on how fast the particle is moving. The signal-to-noise ratio is sufficiently high to directly measure the time a single cargo molecule spends interacting with the nuclear pore complex. Particle tracking analysis revealed that cargo molecules randomly diffuse within the nuclear pore complex, exiting as a result of a single rate-limiting step. We expect that narrow-field epifluorescence microscopy will be useful for elucidating other binding and trafficking events within cells. PMID:16879979

Single Molecule Science for Personalized Nanomedicine: Atomic Force Microscopy of Biopolymer-Protein Interactions

NASA Astrophysics Data System (ADS)

Hsueh, Carlin

Nanotechnology has a unique and relatively untapped utility in the fields of medicine and dentistry at the level of single-biopolymer and -molecule diagnostics. In recent years atomic force microscopy (AFM) has garnered much interest due to its ability to obtain atomic-resolution of molecular structures and probe biophysical behaviors of biopolymers and proteins in a variety of biologically significant environments. The work presented in this thesis focuses on the nanoscale manipulation and observation of biopolymers to develop an innovative technology for personalized medicine while understanding complex biological systems. These studies described here primarily use AFM to observe biopolymer interactions with proteins and its surroundings with unprecedented resolution, providing a better understanding of these systems and interactions at the nanoscale. Transcriptional profiling, the measure of messenger RNA (mRNA) abundance in a single cell, is a powerful technique that detects "behavior" or "symptoms" at the tissue and cellular level. We have sought to develop an alternative approach, using our expertise in AFM and single molecule nanotechnology, to achieve a cost-effective high throughput method for sensitive detection and profiling of subtle changes in transcript abundance. The technique does not require amplification of the mRNA sample because the AFM provides three-dimensional views of molecules with unprecedented resolution, requires minimal sample preparation, and utilizes a simple tagging chemistry on cDNA molecules. AFM images showed collagen polymers in teeth and of Drebrin-A remodeling of filamentous actin structure and mechanics. AFM was used to image collagen on exposed dentine tubules and confirmed tubule occlusion with a desensitizing prophylaxis paste by Colgate-Palmolive. The AFM also superseded other microscopy tools in resolving F-actin helix remodeling and possible cooperative binding by a neuronal actin binding protein---Drebrin-A, an

Single-Molecule Fluorescence Imaging for Studying Organic, Organometallic, and Inorganic Reaction Mechanisms

DOE Office of Scientific and Technical Information (OSTI.GOV)

Blum, Suzanne A.

2016-05-24

The reactive behavior of individual molecules is seldom observed, because we usually measure the average properties of billions of molecules. What we miss is important: the catalytic activity of less than 1% of the molecules under observation can dominate the outcome of a chemical reaction seen at a macroscopic level. Currently available techniques to examine reaction mechanisms (such as nuclear magnetic resonance spectroscopy and mass spectrometry) study molecules as an averaged ensemble. These ensemble techniques are unable to detect minor components (under ~1%) in mixtures or determine which components in the mixture are responsible for reactivity and catalysis. In themore » field of mechanistic chemistry, there is a resulting heuristic device that if an intermediate is very reactive in catalysis, it often cannot be observed (termed “Halpern’s Rule” ). Ultimately, the development of single-molecule imaging technology could be a powerful tool to observe these “unobservable” intermediates and active catalysts. Single-molecule techniques have already transformed biology and the understanding of biochemical processes. The potential of single-molecule fluorescence microscopy to address diverse chemical questions, such as the chemical reactivity of organometallic or inorganic systems with discrete metal complexes, however, has not yet been realized. In this respect, its application to chemical systems lags significantly behind its application to biophysical systems. This transformative imaging technique has broad, multidisciplinary impact with the potential to change the way the chemistry community studies reaction mechanisms and reactivity distributions, especially in the core area of catalysis.« less

SINGLE MOLECULE APPROACHES TO BIOLOGY, 2010 GORDON RESEARCH CONFERENCE, JUNE 27-JULY 2, 2010, ITALY

DOE Office of Scientific and Technical Information (OSTI.GOV)

Professor William Moerner

2010-07-09

The 2010 Gordon Conference on Single-Molecule Approaches to Biology focuses on cutting-edge research in single-molecule science. Tremendous technical developments have made it possible to detect, identify, track, and manipulate single biomolecules in an ambient environment or even in a live cell. Single-molecule approaches have changed the way many biological problems are addressed, and new knowledge derived from these approaches continues to emerge. 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 biomolecular machines: what they do, how they work individually, how they work together,more » and finally, how they work inside live cells. The burgeoning use of single-molecule methods to elucidate biological problems is a highly multidisciplinary pursuit, involving both force- and fluorescence-based methods, the most up-to-date advances in microscopy, innovative biological and chemical approaches, and nanotechnology tools. This conference seeks to bring together top experts in molecular and cell biology with innovators in the measurement and manipulation of single molecules, and will provide opportunities for junior scientists and graduate students to present their work in poster format and to exchange ideas with leaders in the field. A number of excellent poster presenters will be selected for short oral talks. Topics as diverse as single-molecule sequencing, DNA/RNA/protein interactions, folding machines, cellular biophysics, synthetic biology and bioengineering, force spectroscopy, new method developments, superresolution imaging in cells, and novel probes for single-molecule imaging will be on the program. Additionally, the collegial atmosphere of this Conference, with programmed discussion sessions as well as opportunities for informal gatherings in the afternoons and evenings in the beauty of the Il Ciocco site

Inelastic electron tunneling spectroscopy of difurylethene-based photochromic single-molecule junctions

PubMed Central

Sysoiev, Dmytro; Huhn, Thomas; Pauly, Fabian

2017-01-01

Diarylethene-derived molecules alter their electronic structure upon transformation between the open and closed forms of the diarylethene core, when exposed to ultraviolet (UV) or visible light. This transformation results in a significant variation of electrical conductance and vibrational properties of corresponding molecular junctions. We report here a combined experimental and theoretical analysis of charge transport through diarylethene-derived single-molecule devices, which are created using the mechanically controlled break-junction technique. Inelastic electron tunneling (IET) spectroscopy measurements performed at 4.2 K are compared with first-principles calculations in the two distinct forms of diarylethenes connected to gold electrodes. The combined approach clearly demonstrates that the IET spectra of single-molecule junctions show specific vibrational features that can be used to identify different isomeric molecular states by transport experiments. PMID:29259875

Efficient use of single molecule time traces to resolve kinetic rates, models and uncertainties

NASA Astrophysics Data System (ADS)

Schmid, Sonja; Hugel, Thorsten

2018-03-01

Single molecule time traces reveal the time evolution of unsynchronized kinetic systems. Especially single molecule Förster resonance energy transfer (smFRET) provides access to enzymatically important time scales, combined with molecular distance resolution and minimal interference with the sample. Yet the kinetic analysis of smFRET time traces is complicated by experimental shortcomings—such as photo-bleaching and noise. Here we recapitulate the fundamental limits of single molecule fluorescence that render the classic, dwell-time based kinetic analysis unsuitable. In contrast, our Single Molecule Analysis of Complex Kinetic Sequences (SMACKS) considers every data point and combines the information of many short traces in one global kinetic rate model. We demonstrate the potential of SMACKS by resolving the small kinetic effects caused by different ionic strengths in the chaperone protein Hsp90. These results show an unexpected interrelation between conformational dynamics and ATPase activity in Hsp90.

Simulated single molecule microscopy with SMeagol.

PubMed

Lindén, Martin; Ćurić, Vladimir; Boucharin, Alexis; Fange, David; Elf, Johan

2016-08-01

SMeagol is a software tool to simulate highly realistic microscopy data based on spatial systems biology models, in order to facilitate development, validation and optimization of advanced analysis methods for live cell single molecule microscopy data. SMeagol runs on Matlab R2014 and later, and uses compiled binaries in C for reaction-diffusion simulations. Documentation, source code and binaries for Mac OS, Windows and Ubuntu Linux can be downloaded from http://smeagol.sourceforge.net johan.elf@icm.uu.se Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press.

Controlling Brownian motion of single protein molecules and single fluorophores in aqueous buffer.

PubMed

Cohen, Adam E; Moerner, W E

2008-05-12

We present an Anti-Brownian Electrokinetic trap (ABEL trap) capable of trapping individual fluorescently labeled protein molecules in aqueous buffer. The ABEL trap operates by tracking the Brownian motion of a single fluorescent particle in solution, and applying a time-dependent electric field designed to induce an electrokinetic drift that cancels the Brownian motion. The trapping strength of the ABEL trap is limited by the latency of the feedback loop. In previous versions of the trap, this latency was set by the finite frame rate of the camera used for video-tracking. In the present system, the motion of the particle is tracked entirely in hardware (without a camera or image-processing software) using a rapidly rotating laser focus and lock-in detection. The feedback latency is set by the finite rate of arrival of photons. We demonstrate trapping of individual molecules of the protein GroEL in buffer, and we show confinement of single fluorophores of the dye Cy3 in water.

Three-dimensional single-molecule localization with nanometer accuracy using Metal-Induced Energy Transfer (MIET) imaging

NASA Astrophysics Data System (ADS)

Karedla, Narain; Chizhik, Anna M.; Stein, Simon C.; Ruhlandt, Daja; Gregor, Ingo; Chizhik, Alexey I.; Enderlein, Jörg

2018-05-01

Our paper presents the first theoretical and experimental study using single-molecule Metal-Induced Energy Transfer (smMIET) for localizing single fluorescent molecules in three dimensions. Metal-Induced Energy Transfer describes the resonant energy transfer from the excited state of a fluorescent emitter to surface plasmons in a metal nanostructure. This energy transfer is strongly distance-dependent and can be used to localize an emitter along one dimension. We have used Metal-Induced Energy Transfer in the past for localizing fluorescent emitters with nanometer accuracy along the optical axis of a microscope. The combination of smMIET with single-molecule localization based super-resolution microscopy that provides nanometer lateral localization accuracy offers the prospect of achieving isotropic nanometer localization accuracy in all three spatial dimensions. We give a thorough theoretical explanation and analysis of smMIET, describe its experimental requirements, also in its combination with lateral single-molecule localization techniques, and present first proof-of-principle experiments using dye molecules immobilized on top of a silica spacer, and of dye molecules embedded in thin polymer films.

Correlating single-molecule and ensemble-average measurements of peptide adsorption onto different inorganic materials.

PubMed

Kim, Seong-Oh; Jackman, Joshua A; Mochizuki, Masahito; Yoon, Bo Kyeong; Hayashi, Tomohiro; Cho, Nam-Joon

2016-06-07

The coating of solid-binding peptides (SBPs) on inorganic material surfaces holds significant potential for improved surface functionalization at nano-bio interfaces. In most related studies, the goal has been to engineer peptides with selective and high binding affinity for a target material. The role of the material substrate itself in modulating the adsorption behavior of a peptide molecule remains less explored and there are few studies that compare the interaction of one peptide with different inorganic substrates. Herein, using a combination of two experimental techniques, we investigated the adsorption of a 16 amino acid-long random coil peptide to various inorganic substrates - gold, silicon oxide, titanium oxide and aluminum oxide. Quartz crystal microbalance-dissipation (QCM-D) experiments were performed in order to measure the peptide binding affinity for inorganic solid supports at the ensemble average level, and atomic force microscopy (AFM) experiments were conducted in order to determine the adhesion force of a single peptide molecule. A positive trend was observed between the total mass uptake of attached peptide and the single-molecule adhesion force on each substrate. Peptide affinity for gold was appreciably greater than for the oxide substrates. Collectively, the results obtained in this study offer insight into the ways in which inorganic materials can differentially influence and modulate the adhesion of SBPs.

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

PubMed Central

Zhang, Lei; Ren, Gang

2012-01-01

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

3D single-molecule super-resolution microscopy with a tilted light sheet.

PubMed

Gustavsson, Anna-Karin; Petrov, Petar N; Lee, Maurice Y; Shechtman, Yoav; Moerner, W E

2018-01-09

Tilted light sheet microscopy with 3D point spread functions (TILT3D) combines a novel, tilted light sheet illumination strategy with long axial range point spread functions (PSFs) for low-background, 3D super-localization of single molecules as well as 3D super-resolution imaging in thick cells. Because the axial positions of the single emitters are encoded in the shape of each single-molecule image rather than in the position or thickness of the light sheet, the light sheet need not be extremely thin. TILT3D is built upon a standard inverted microscope and has minimal custom parts. The result is simple and flexible 3D super-resolution imaging with tens of nm localization precision throughout thick mammalian cells. We validate TILT3D for 3D super-resolution imaging in mammalian cells by imaging mitochondria and the full nuclear lamina using the double-helix PSF for single-molecule detection and the recently developed tetrapod PSFs for fiducial bead tracking and live axial drift correction.

Optical Detection and Spectroscopy of Single Molecules in a Solid.

DTIC Science & Technology

1989-03-23

the optical absorption spectrum of single dopant molecules of pentacene in a p-terphenyl host crystal at liquid-helium temperatures. To achieve this...dopant molecules of pentacene in a p-terphenyl host crystal at liquid-helium temperatures. To achieve this, frequency-modulation spectroscopy was combined...solid would provide an important new tool for the study of local absorber-host interactions that would be uncomplicated by the normal averaging over

Single Molecule Fluorescence Measurements of Complex Systems

NASA Astrophysics Data System (ADS)

Sadegh, Sanaz

Single molecule methods are powerful tools for investigating the properties of complex systems that are generally concealed by ensemble measurements. Here we use single molecule fluorescent measurements to study two different complex systems: 1/ƒ noise in quantum dots and diffusion of the membrane proteins in live cells. The power spectrum of quantum dot (QD) fluorescence exhibits 1/ƒ beta noise, related to the intermittency of these nanosystems. As in other systems exhibiting 1/ƒ noise, this power spectrum is not integrable at low frequencies, which appears to imply infinite total power. We report measurements of individual QDs that address this long-standing paradox. We find that the level of 1/ƒbeta noise for QDs decays with the observation time. We show that the traditional description of the power spectrum with a single exponent is incomplete and three additional critical exponents characterize the dependence on experimental time. A broad range of membrane proteins display anomalous diffusion on the cell surface. Different methods provide evidence for obstructed subdiffusion and diffusion on a fractal space, but the underlying structure inducing anomalous diffusion has never been visualized due to experimental challenges. We addressed this problem by imaging the cortical actin at high resolution while simultaneously tracking individual membrane proteins in live mammalian cells. Our data show that actin introduces barriers leading to compartmentalization of the plasma membrane and that membrane proteins are transiently confined within actin fences. Furthermore, superresolution imaging shows that the cortical actin is organized into a self-similar fractal.

Single-molecule spectroscopy for plastic electronics: materials analysis from the bottom-up.

PubMed

Lupton, John M

2010-04-18

pi-conjugated polymers find a range of applications in electronic devices. These materials are generally highly disordered in terms of chain length and chain conformation, besides being influenced by a variety of chemical and physical defects. Although this characteristic can be of benefit in certain device applications, disorder severely complicates materials analysis. Accurate analytical techniques are, however, crucial to optimising synthetic procedures and assessing overall material purity. Fortunately, single-molecule spectroscopic techniques have emerged as an unlikely but uniquely powerful approach to unraveling intrinsic material properties from the bottom up. Building on the success of such techniques in the life sciences, single-molecule spectroscopy is finding increasing applicability in materials science, effectively enabling the dissection of the bulk down to the level of the individual molecular constituent. This article reviews recent progress in single molecule spectroscopy of conjugated polymers as used in organic electronics.

Single-Molecule Denaturation Mapping of Genomic DNA in Nanofluidic Channels

NASA Astrophysics Data System (ADS)

Reisner, Walter; Larsen, Niels; Kristensen, Anders; Tegenfeldt, Jonas O.; Flyvbjerg, Henrik

2009-03-01

We have developed a new DNA barcoding technique based on the partial denaturation of extended fluorescently labeled DNA molecules. We partially melt DNA extended in nanofluidic channels via a combination of local heating and added chemical denaturants. The melted molecules, imaged via a standard fluorescence videomicroscopy setup, exhibit a nonuniform fluorescence profile corresponding to a series of local dips and peaks in the intensity trace along the stretched molecule. We show that this barcode is consistent with the presence of locally melted regions and can be explained by calculations of sequence-dependent melting probability. We believe this melting mapping technology is the first optically based single molecule technique sensitive to genome wide sequence variation that does not require an additional enzymatic labeling or restriction scheme.

Vectorial nanoscale mapping of optical antenna fields by single molecule dipoles.

PubMed

Singh, Anshuman; Calbris, Gaëtan; van Hulst, Niek F

2014-08-13

Optical nanoantennas confine light on the nanoscale, enabling strong light-matter interactions and ultracompact optical devices. Such confined nanovolumes of light have nonzero field components in all directions (x, y, and z). Unfortunately mapping of the actual nanoscale field vectors has so far remained elusive, though antenna hotspots have been explored by several techniques. In this paper, we present a novel method to probe all three components of the local antenna field. To this end a resonant nanoantenna is fabricated at the vertex of a scanning tip. Next, the nanoantenna is deterministically scanned in close proximity to single fluorescent molecules, whose fixed excitation dipole moment reads out the local field vector. With nanometer molecular resolution, we distinctly map x-, y-, and z-field components of the dipole antenna, i.e. a full vectorial mode map, and show good agreement with full 3D FDTD simulations. Moreover, the fluorescence polarization maps the localized coupling, with emission through the longitudinal antenna mode. Finally, the resonant antenna probe is used for single molecule imaging with 40 nm fwhm response function. The total fluorescence enhancement is 7.6 times, while out-of-plane molecules, almost undetectable in far-field, are made visible by the strong antenna z-field with a fluorescence enhancement up to 100 times. Interestingly, the apparent position of molecules shifts up to 20 nm depending on their orientation. The capability to resolve orientational information on the single molecule level makes the scanning resonant antenna an ideal tool for extreme resolution bioimaging.

Winding single-molecule double-stranded DNA on a nanometer-sized reel

PubMed Central

You, Huijuan; Iino, Ryota; Watanabe, Rikiya; Noji, Hiroyuki

2012-01-01

A molecular system of a nanometer-sized reel was developed from F1–ATPase, a rotary motor protein. By combination with magnetic tweezers and optical tweezers, single-molecule double-stranded DNA (dsDNA) was wound around the molecular reel. The bending stiffness of dsDNA was determined from the winding tension (0.9–6.0 pN) and the diameter of the wound loop (21.4–8.5 nm). Our results were in good agreement with the conventional worm-like chain model and a persistence length of 54 ± 9 nm was estimated. This molecular reel system offers a new platform for single-molecule study of micromechanics of sharply bent DNA molecules and is expected to be applicable to the elucidation of the molecular mechanism of DNA-associating proteins on sharply bent DNA strands. PMID:22772992

On the uncertainty in single molecule fluorescent lifetime and energy emission measurements

NASA Technical Reports Server (NTRS)

Brown, Emery N.; Zhang, Zhenhua; Mccollom, Alex D.

1995-01-01

Time-correlated single photon counting has recently been combined with mode-locked picosecond pulsed excitation to measure the fluorescent lifetimes and energy emissions of single molecules in a flow stream. Maximum likelihood (ML) and least square methods agree and are optimal when the number of detected photons is large however, in single molecule fluorescence experiments the number of detected photons can be less than 20, 67% of those can be noise and the detection time is restricted to 10 nanoseconds. Under the assumption that the photon signal and background noise are two independent inhomogeneous poisson processes, we derive the exact joint arrival time probably density of the photons collected in a single counting experiment performed in the presence of background noise. The model obviates the need to bin experimental data for analysis, and makes it possible to analyze formally the effect of background noise on the photon detection experiment using both ML or Bayesian methods. For both methods we derive the joint and marginal probability densities of the fluorescent lifetime and fluorescent emission. the ML and Bayesian methods are compared in an analysis of simulated single molecule fluorescence experiments of Rhodamine 110 using different combinations of expected background nose and expected fluorescence emission. While both the ML or Bayesian procedures perform well for analyzing fluorescence emissions, the Bayesian methods provide more realistic measures of uncertainty in the fluorescent lifetimes. The Bayesian methods would be especially useful for measuring uncertainty in fluorescent lifetime estimates in current single molecule flow stream experiments where the expected fluorescence emission is low. Both the ML and Bayesian algorithms can be automated for applications in molecular biology.

On the Uncertainty in Single Molecule Fluorescent Lifetime and Energy Emission Measurements

NASA Technical Reports Server (NTRS)

Brown, Emery N.; Zhang, Zhenhua; McCollom, Alex D.

1996-01-01

Time-correlated single photon counting has recently been combined with mode-locked picosecond pulsed excitation to measure the fluorescent lifetimes and energy emissions of single molecules in a flow stream. Maximum likelihood (ML) and least squares methods agree and are optimal when the number of detected photons is large, however, in single molecule fluorescence experiments the number of detected photons can be less than 20, 67 percent of those can be noise, and the detection time is restricted to 10 nanoseconds. Under the assumption that the photon signal and background noise are two independent inhomogeneous Poisson processes, we derive the exact joint arrival time probability density of the photons collected in a single counting experiment performed in the presence of background noise. The model obviates the need to bin experimental data for analysis, and makes it possible to analyze formally the effect of background noise on the photon detection experiment using both ML or Bayesian methods. For both methods we derive the joint and marginal probability densities of the fluorescent lifetime and fluorescent emission. The ML and Bayesian methods are compared in an analysis of simulated single molecule fluorescence experiments of Rhodamine 110 using different combinations of expected background noise and expected fluorescence emission. While both the ML or Bayesian procedures perform well for analyzing fluorescence emissions, the Bayesian methods provide more realistic measures of uncertainty in the fluorescent lifetimes. The Bayesian methods would be especially useful for measuring uncertainty in fluorescent lifetime estimates in current single molecule flow stream experiments where the expected fluorescence emission is low. Both the ML and Bayesian algorithms can be automated for applications in molecular biology.

Theoretical electrical conductivity of hydrogen-bonded benzamide-derived molecules and single DNA bases.

PubMed

Chen, Xiang

2013-09-01

A benzamide molecule is used as a "reader" molecule to form hydrogen bonds with five single DNA bases, i.e., four normal single DNA bases A,T,C,G and one for 5methylC. The whole molecule is then attached to the gold surface so that a meta-molecule junction is formed. We calculate the transmission function and conductance for the five metal-molecule systems, with the implementation of density functional theory-based non-equilibrium Green function method. Our results show that each DNA base exhibits a unique conductance and most of them are on the pS level. The distinguishable conductance of each DNA base provides a way for the fast sequencing of DNA. We also investigate the dependence of conductivity of such a metal-molecule system on the hydrogen bond length between the "reader" molecule and DNA base, which shows that conductance follows an exponential decay as the hydrogen bond length increases, i.e., the conductivity is highly sensitive to the change in hydrogen bond length.

Single Molecule Study of Cellulase Hydrolysis of Crystalline Cellulose

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Y.-S.; Luo, Y.; Baker, J. O.

This report seeks to elucidate the role of cellobiohydrolase-I (CBH I) in the hydrolysis of crystalline cellulose. A single-molecule approach uses various imaging techniques to investigate the surface structure of crystalline cellulose and changes made in the structure by CBH I.

Atomic force microscope observation of branching in single transcript molecules derived from human cardiac muscle

NASA Astrophysics Data System (ADS)

Reed, Jason; Hsueh, Carlin; Mishra, Bud; Gimzewski, James K.

2008-09-01

We have used an atomic force microscope to examine a clinically derived sample of single-molecule gene transcripts, in the form of double-stranded cDNA, (c: complementary) obtained from human cardiac muscle without the use of polymerase chain reaction (PCR) amplification. We observed a log-normal distribution of transcript sizes, with most molecules being in the range of 0.4-7.0 kilobase pairs (kb) or 130-2300 nm in contour length, in accordance with the expected distribution of mRNA (m: messenger) sizes in mammalian cells. We observed novel branching structures not previously known to exist in cDNA, and which could have profound negative effects on traditional analysis of cDNA samples through cloning, PCR and DNA sequencing.

Visualizing long-term single-molecule dynamics in vivo by stochastic protein labeling.

PubMed

Liu, Hui; Dong, Peng; Ioannou, Maria S; Li, Li; Shea, Jamien; Pasolli, H Amalia; Grimm, Jonathan B; Rivlin, Patricia K; Lavis, Luke D; Koyama, Minoru; Liu, Zhe

2018-01-09

Our ability to unambiguously image and track individual molecules in live cells is limited by packing of multiple copies of labeled molecules within the resolution limit. Here we devise a universal genetic strategy to precisely control copy number of fluorescently labeled molecules in a cell. This system has a dynamic range of ∼10,000-fold, enabling sparse labeling of proteins expressed at different abundance levels. Combined with photostable labels, this system extends the duration of automated single-molecule tracking by two orders of magnitude. We demonstrate long-term imaging of synaptic vesicle dynamics in cultured neurons as well as in intact zebrafish. We found axon initial segment utilizes a "waterfall" mechanism gating synaptic vesicle transport polarity by promoting anterograde transport processivity. Long-time observation also reveals that transcription factor hops between clustered binding sites in spatially restricted subnuclear regions, suggesting that topological structures in the nucleus shape local gene activities by a sequestering mechanism. This strategy thus greatly expands the spatiotemporal length scales of live-cell single-molecule measurements, enabling new experiments to quantitatively understand complex control of molecular dynamics in vivo.

Elastin-like Polypeptide Linkers for Single-Molecule Force Spectroscopy.

PubMed

Ott, Wolfgang; Jobst, Markus A; Bauer, Magnus S; Durner, Ellis; Milles, Lukas F; Nash, Michael A; Gaub, Hermann E

2017-06-27

Single-molecule force spectroscopy (SMFS) is by now well established as a standard technique in biophysics and mechanobiology. In recent years, the technique has benefitted greatly from new approaches to bioconjugation of proteins to surfaces. Indeed, optimized immobilization strategies for biomolecules and refined purification schemes are being steadily adapted and improved, which in turn has enhanced data quality. In many previously reported SMFS studies, poly(ethylene glycol) (PEG) was used to anchor molecules of interest to surfaces and/or cantilever tips. The limitation, however, is that PEG exhibits a well-known trans-trans-gauche to all-trans transition, which results in marked deviation from standard polymer elasticity models such as the worm-like chain, particularly at elevated forces. As a result, the assignment of unfolding events to protein domains based on their corresponding amino acid chain lengths is significantly obscured. Here, we provide a solution to this problem by implementing unstructured elastin-like polypeptides as linkers to replace PEG. We investigate the suitability of tailored elastin-like polypeptides linkers and perform direct comparisons to PEG, focusing on attributes that are critical for single-molecule force experiments such as linker length, monodispersity, and bioorthogonal conjugation tags. Our results demonstrate that by avoiding the ambiguous elastic response of mixed PEG/peptide systems and instead building the molecular mechanical systems with only a single bond type with uniform elastic properties, we improve data quality and facilitate data analysis and interpretation in force spectroscopy experiments. The use of all-peptide linkers allows alternative approaches for precisely defining elastic properties of proteins linked to surfaces.

Single-Molecule Fluorescence In Situ Hybridization (FISH) of Circular RNA CDR1as.

PubMed

Kocks, Christine; Boltengagen, Anastasiya; Piwecka, Monika; Rybak-Wolf, Agnieszka; Rajewsky, Nikolaus

2018-01-01

Individual mRNA molecules can be imaged in fixed cells by hybridization with multiple, singly labeled oligonucleotide probes, followed by computational identification of fluorescent signals. This approach, called single-molecule RNA fluorescence in situ hybridization (smRNA FISH), allows subcellular localization and absolute quantification of RNA molecules in individual cells. Here, we describe a simple smRNA FISH protocol for two-color imaging of a circular RNA, CDR1as, simultaneously with an unrelated messenger RNA. The protocol can be adapted to circRNAs that coexist with overlapping, noncircular mRNA isoforms produced from the same genetic locus.

Single-molecule live-cell imaging of bacterial DNA repair and damage tolerance.

PubMed

Ghodke, Harshad; Ho, Han; van Oijen, Antoine M

2018-02-19

Genomic DNA is constantly under threat from intracellular and environmental factors that damage its chemical structure. Uncorrected DNA damage may impede cellular propagation or even result in cell death, making it critical to restore genomic integrity. Decades of research have revealed a wide range of mechanisms through which repair factors recognize damage and co-ordinate repair processes. In recent years, single-molecule live-cell imaging methods have further enriched our understanding of how repair factors operate in the crowded intracellular environment. The ability to follow individual biochemical events, as they occur in live cells, makes single-molecule techniques tremendously powerful to uncover the spatial organization and temporal regulation of repair factors during DNA-repair reactions. In this review, we will cover practical aspects of single-molecule live-cell imaging and highlight recent advances accomplished by the application of these experimental approaches to the study of DNA-repair processes in prokaryotes. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

[The principle and application of the single-molecule real-time sequencing technology].

PubMed

Yanhu, Liu; Lu, Wang; Li, Yu

2015-03-01

Last decade witnessed the explosive development of the third-generation sequencing strategy, including single-molecule real-time sequencing (SMRT), true single-molecule sequencing (tSMSTM) and the single-molecule nanopore DNA sequencing. In this review, we summarize the principle, performance and application of the SMRT sequencing technology. Compared with the traditional Sanger method and the next-generation sequencing (NGS) technologies, the SMRT approach has several advantages, including long read length, high speed, PCR-free and the capability of direct detection of epigenetic modifications. However, the disadvantage of its low accuracy, most of which resulted from insertions and deletions, is also notable. So, the raw sequence data need to be corrected before assembly. Up to now, the SMRT is a good fit for applications in the de novo genomic sequencing and the high-quality assemblies of small genomes. In the future, it is expected to play an important role in epigenetics, transcriptomic sequencing, and assemblies of large genomes.

Single-molecule detection of dihydroazulene photo-thermal reaction using break junction technique

NASA Astrophysics Data System (ADS)

Huang, Cancan; Jevric, Martyn; Borges, Anders; Olsen, Stine T.; Hamill, Joseph M.; Zheng, Jue-Ting; Yang, Yang; Rudnev, Alexander; Baghernejad, Masoud; Broekmann, Peter; Petersen, Anne Ugleholdt; Wandlowski, Thomas; Mikkelsen, Kurt V.; Solomon, Gemma C.; Brøndsted Nielsen, Mogens; Hong, Wenjing

2017-05-01

Charge transport by tunnelling is one of the most ubiquitous elementary processes in nature. Small structural changes in a molecular junction can lead to significant difference in the single-molecule electronic properties, offering a tremendous opportunity to examine a reaction on the single-molecule scale by monitoring the conductance changes. Here, we explore the potential of the single-molecule break junction technique in the detection of photo-thermal reaction processes of a photochromic dihydroazulene/vinylheptafulvene system. Statistical analysis of the break junction experiments provides a quantitative approach for probing the reaction kinetics and reversibility, including the occurrence of isomerization during the reaction. The product ratios observed when switching the system in the junction does not follow those observed in solution studies (both experiment and theory), suggesting that the junction environment was perturbing the process significantly. This study opens the possibility of using nano-structured environments like molecular junctions to tailor product ratios in chemical reactions.

Probing Biomolecular Structures and Dynamics of Single Molecules Using In-Gel Alternating-Laser Excitation

PubMed Central

Santoso, Yusdi; Kapanidis, Achillefs N.

2009-01-01

Gel electrophoresis is a standard biochemical technique used for separating biomolecules on the basis of size and charge. Despite the use of gels in early single-molecule experiments, gel electrophoresis has not been widely adopted for single-molecule fluorescence spectroscopy. We present a novel method that combines gel electrophoresis and single-molecule fluorescence spectroscopy to simultaneously purify and analyze biomolecules in a gel matrix. Our method, in-gel ALEX, uses non-denaturing gels to purify biomolecular complexes of interest from free components, aggregates, and non-specific complexes. The gel matrix also slows down translational diffusion of molecules, giving rise to long, high-resolution time traces without surface immobilization, which allow extended observations of conformational dynamics in a biologically friendly environment. We demonstrated the compatibility of this method with different types of single molecule spectroscopy techniques, including confocal detection and fluorescence-correlation spectroscopy. We demonstrated that in-gel ALEX can be used to study conformational dynamics at the millisecond timescale; by studying a DNA hairpin in gels, we directly observed fluorescence fluctuations due to conformational interconversion between folded and unfolded states. Our method is amenable to the addition of small molecules that can alter the equilibrium and dynamic properties of the system. In-gel ALEX will be a versatile tool for studying structures and dynamics of complex biomolecules and their assemblies. PMID:19863108

Single molecule dynamics at a mechanically controllable break junction in solution at room temperature.

PubMed

Konishi, Tatsuya; Kiguchi, Manabu; Takase, Mai; Nagasawa, Fumika; Nabika, Hideki; Ikeda, Katsuyoshi; Uosaki, Kohei; Ueno, Kosei; Misawa, Hiroaki; Murakoshi, Kei

2013-01-23

The in situ observation of geometrical and electronic structural dynamics of a single molecule junction is critically important in order to further progress in molecular electronics. Observations of single molecular junctions are difficult, however, because of sensitivity limits. Here, we report surface-enhanced Raman scattering (SERS) of a single 4,4'-bipyridine molecule under conditions of in situ current flow in a nanogap, by using nano-fabricated, mechanically controllable break junction (MCBJ) electrodes. When adsorbed at room temperature on metal nanoelectrodes in solution to form a single molecule junction, statistical analysis showed that nontotally symmetric b(1) and b(2) modes of 4,4'-bipyridine were strongly enhanced relative to observations of the same modes in solid or aqueous solutions. Significant changes in SERS intensity, energy (wavenumber), and selectivity of Raman vibrational bands that are coincident with current fluctuations provide information on distinct states of electronic and geometrical structure of the single molecule junction, even under large thermal fluctuations occurring at room temperature. We observed the dynamics of 4,4'-bipyridine motion between vertical and tilting configurations in the Au nanogap via b(1) and b(2) mode switching. A slight increase in the tilting angle of the molecule was also observed by noting the increase in the energies of Raman modes and the decrease in conductance of the molecular junction.

Meeting report: SMART timing--principles of single molecule techniques course at the University of Michigan 2014.

PubMed

Bartke, Rebecca M; Cameron, Elizabeth L; Cristie-David, Ajitha S; Custer, Thomas C; Denies, Maxwell S; Daher, May; Dhakal, Soma; Ghosh, Soumi; Heinicke, Laurie A; Hoff, J Damon; Hou, Qian; Kahlscheuer, Matthew L; Karslake, Joshua; Krieger, Adam G; Li, Jieming; Li, Xiang; Lund, Paul E; Vo, Nguyen N; Park, Jun; Pitchiaya, Sethuramasundaram; Rai, Victoria; Smith, David J; Suddala, Krishna C; Wang, Jiarui; Widom, Julia R; Walter, Nils G

2015-05-01

Four days after the announcement of the 2014 Nobel Prize in Chemistry for "the development of super-resolved fluorescence microscopy" based on single molecule detection, the Single Molecule Analysis in Real-Time (SMART) Center at the University of Michigan hosted a "Principles of Single Molecule Techniques 2014" course. Through a combination of plenary lectures and an Open House at the SMART Center, the course took a snapshot of a technology with an especially broad and rapidly expanding range of applications in the biomedical and materials sciences. Highlighting the continued rapid emergence of technical and scientific advances, the course underscored just how brightly the future of the single molecule field shines. © 2014 Wiley Periodicals, Inc.

Single molecule characterization of DNA binding and strand displacement reactions on lithographic DNA origami microarrays.

PubMed

Scheible, Max B; Pardatscher, Günther; Kuzyk, Anton; Simmel, Friedrich C

2014-03-12

The combination of molecular self-assembly based on the DNA origami technique with lithographic patterning enables the creation of hierarchically ordered nanosystems, in which single molecules are positioned at precise locations on multiple length scales. Based on a hybrid assembly protocol utilizing DNA self-assembly and electron-beam lithography on transparent glass substrates, we here demonstrate a DNA origami microarray, which is compatible with the requirements of single molecule fluorescence and super-resolution microscopy. The spatial arrangement allows for a simple and reliable identification of single molecule events and facilitates automated read-out and data analysis. As a specific application, we utilize the microarray to characterize the performance of DNA strand displacement reactions localized on the DNA origami structures. We find considerable variability within the array, which results both from structural variations and stochastic reaction dynamics prevalent at the single molecule level.

Binding and Translocation of Termination Factor Rho Studied at the Single-Molecule Level

PubMed Central

Koslover, Daniel J.; Fazal, Furqan M.; Mooney, Rachel A.; Landick, Robert; Block, Steven M.

2012-01-01

Rho termination factor is an essential hexameric helicase responsible for terminating 20–50% of all mRNA synthesis in E. coli. We used single- molecule force spectroscopy to investigate Rho-RNA binding interactions at the Rho- utilization (rut) site of the ? tR1 terminator. Our results are consistent with Rho complexes adopting two states, one that binds 57 ±2 nucleotides of RNA across all six of the Rho primary binding sites, and another that binds 85 ±2 nucleotides at the six primary sites plus a single secondary site situated at the center of the hexamer. The single-molecule data serve to establish that Rho translocates 5′-to-3′ towards RNA polymerase (RNAP) by a tethered-tracking mechanism, looping out the intervening RNA between the rut site and RNAP. These findings lead to a general model for Rho binding and translocation, and establish a novel experimental approach that should facilitate additional single- molecule studies of RNA-binding proteins. PMID:22885804

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

NASA Astrophysics Data System (ADS)

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.

PubMed

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.

Effect of impurity molecules on the low-temperature vibrational dynamics of polyisobutylene: Investigation by single-molecule spectroscopy

NASA Astrophysics Data System (ADS)

Eremchev, I. Yu.; Naumov, A. V.; Vainer, Yu. G.; Kador, L.

2009-05-01

The influence of impurity chromophore molecules—tetra-tert-butylterrylene (TBT) and dibenzo-anthanthrene (DBATT)—on the vibrational dynamics of the amorphous polymer polyisobutylene (PIB) has been studied via single-molecule spectroscopy. The measurements were performed in the temperature region of 7-30 K, where the interaction of the chromophores with quasilocalized low-frequency vibrational modes (LFMs) determines the observed spectral line broadening. The analysis of the individual temperature dependences of the linewidths for a large number of single probe molecules yielded effective frequency values of those LFMs which are located near the respective chromophores. In this way the distributions of the LFM frequencies were measured for the two systems, and they were found to be similar. Moreover, they are in good agreement with the vibrational density of states as measured in pure PIB by inelastic neutron scattering. This allows us to conclude that, at least in the case of PIB, doping with low concentrations of the nonpolar and neutral molecules TBT and DBATT does not affect the vibrational dynamics of the matrix markedly.

Modeling single molecule junction mechanics as a probe of interface bonding

NASA Astrophysics Data System (ADS)

Hybertsen, Mark S.

2017-03-01

Using the atomic force microscope based break junction approach, applicable to metal point contacts and single molecule junctions, measurements can be repeated thousands of times resulting in rich data sets characterizing the properties of an ensemble of nanoscale junction structures. This paper focuses on the relationship between the measured force extension characteristics including bond rupture and the properties of the interface bonds in the junction. A set of exemplary model junction structures has been analyzed using density functional theory based calculations to simulate the adiabatic potential surface that governs the junction elongation. The junction structures include representative molecules that bond to the electrodes through amine, methylsulfide, and pyridine links. The force extension characteristics are shown to be most effectively analyzed in a scaled form with maximum sustainable force and the distance between the force zero and force maximum as scale factors. Widely used, two parameter models for chemical bond potential energy versus bond length are found to be nearly identical in scaled form. Furthermore, they fit well to the present calculations of N-Au and S-Au donor-acceptor bonds, provided no other degrees of freedom are allowed to relax. Examination of the reduced problem of a single interface, but including relaxation of atoms proximal to the interface bond, shows that a single-bond potential form renormalized by an effective harmonic potential in series fits well to the calculated results. This allows relatively accurate extraction of the interface bond energy. Analysis of full junction models shows cooperative effects that go beyond the mechanical series inclusion of the second bond in the junction, the spectator bond that does not rupture. Calculations for a series of diaminoalkanes as a function of molecule length indicate that the most important cooperative effect is due to the interactions between the dipoles induced by the donor

Modeling single molecule junction mechanics as a probe of interface bonding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hybertsen, Mark S.

Using the atomic force microscope based break junction approach, applicable to metal point contacts and single molecule junctions, measurements can be repeated thousands of times resulting in rich data sets characterizing the properties of an ensemble of nanoscale junction structures. This paper focuses on the relationship between the measured force extension characteristics including bond rupture and the properties of the interface bonds in the junction. We analyzed a set of exemplary model junction structures using density functional theory based calculations to simulate the adiabatic potential surface that governs the junction elongation. The junction structures include representative molecules that bond tomore » the electrodes through amine, methylsulfide, and pyridine links. The force extension characteristics are shown to be most effectively analyzed in a scaled form with maximum sustainable force and the distance between the force zero and force maximum as scale factors. Widely used, two parameter models for chemical bond potential energy versus bond length are found to be nearly identical in scaled form. Furthermore, they fit well to the present calculations of N–Au and S–Au donor-acceptor bonds, provided no other degrees of freedom are allowed to relax. Examination of the reduced problem of a single interface, but including relaxation of atoms proximal to the interface bond, shows that a single-bond potential form renormalized by an effective harmonic potential in series fits well to the calculated results. This, then, allows relatively accurate extraction of the interface bond energy. Analysis of full junction models shows cooperative effects that go beyond the mechanical series inclusion of the second bond in the junction, the spectator bond that does not rupture. Calculations for a series of diaminoalkanes as a function of molecule length indicate that the most important cooperative effect is due to the interactions between the dipoles induced by

Modeling single molecule junction mechanics as a probe of interface bonding

DOE PAGES

Hybertsen, Mark S.

2017-03-07

Using the atomic force microscope based break junction approach, applicable to metal point contacts and single molecule junctions, measurements can be repeated thousands of times resulting in rich data sets characterizing the properties of an ensemble of nanoscale junction structures. This paper focuses on the relationship between the measured force extension characteristics including bond rupture and the properties of the interface bonds in the junction. We analyzed a set of exemplary model junction structures using density functional theory based calculations to simulate the adiabatic potential surface that governs the junction elongation. The junction structures include representative molecules that bond tomore » the electrodes through amine, methylsulfide, and pyridine links. The force extension characteristics are shown to be most effectively analyzed in a scaled form with maximum sustainable force and the distance between the force zero and force maximum as scale factors. Widely used, two parameter models for chemical bond potential energy versus bond length are found to be nearly identical in scaled form. Furthermore, they fit well to the present calculations of N–Au and S–Au donor-acceptor bonds, provided no other degrees of freedom are allowed to relax. Examination of the reduced problem of a single interface, but including relaxation of atoms proximal to the interface bond, shows that a single-bond potential form renormalized by an effective harmonic potential in series fits well to the calculated results. This, then, allows relatively accurate extraction of the interface bond energy. Analysis of full junction models shows cooperative effects that go beyond the mechanical series inclusion of the second bond in the junction, the spectator bond that does not rupture. Calculations for a series of diaminoalkanes as a function of molecule length indicate that the most important cooperative effect is due to the interactions between the dipoles induced by

Quantitative gene expression analysis in Caenorhabditis elegans using single molecule RNA FISH.

PubMed

Bolková, Jitka; Lanctôt, Christian

2016-04-01

Advances in fluorescent probe design and synthesis have allowed the uniform in situ labeling of individual RNA molecules. In a technique referred to as single molecule RNA FISH (smRNA FISH), the labeled RNA molecules can be imaged as diffraction-limited spots and counted using image analysis algorithms. Single RNA counting has provided valuable insights into the process of gene regulation. This microscopy-based method has often revealed a high cell-to-cell variability in expression levels, which has in turn led to a growing interest in investigating the biological significance of gene expression noise. Here we describe the application of the smRNA FISH technique to samples of Caenorhabditis elegans, a well-characterized model organism. Copyright © 2015 Elsevier Inc. All rights reserved.

Nanopipette Delivery of Individual Molecules to Cellular Compartments for Single-Molecule Fluorescence Tracking

PubMed Central

Bruckbauer, Andreas; James, Peter; Zhou, Dejian; Yoon, Ji Won; Excell, David; Korchev, Yuri; Jones, Roy; Klenerman, David

2007-01-01

We have developed a new method, using a nanopipette, for controlled voltage-driven delivery of individual fluorescently labeled probe molecules to the plasma membrane which we used for single-molecule fluorescence tracking (SMT). The advantages of the method are 1), application of the probe to predefined regions on the membrane; 2), release of only one or a few molecules onto the cell surface; 3), when combined with total internal reflection fluorescence microscopy, very low background due to unbound molecules; and 4), the ability to first optimize the experiment and then repeat it on the same cell. We validated the method by performing an SMT study of the diffusion of individual membrane glycoproteins labeled with Atto 647-wheat germ agglutin in different surface domains of boar spermatozoa. We found little deviation from Brownian diffusion with a mean diffusion coefficient of 0.79 ± 0.04 μm2/s in the acrosomal region and 0.10 ± 0.02 μm2/s in the postacrosomal region; this difference probably reflects different membrane structures. We also showed that we can analyze diffusional properties of different subregions of the cell membrane and probe for the presence of diffusion barriers. It should be straightforward to extend this new method to other probes and cells, and it can be used as a new tool to investigate the cell membrane. PMID:17631532

Smart SERS Hot Spots: Single Molecules Can Be Positioned in a Plasmonic Nanojunction Using Host-Guest Chemistry.

PubMed

Kim, Nam Hoon; Hwang, Wooseup; Baek, Kangkyun; Rohman, Md Rumum; Kim, Jeehong; Kim, Hyun Woo; Mun, Jungho; Lee, So Young; Yun, Gyeongwon; Murray, James; Ha, Ji Won; Rho, Junsuk; Moskovits, Martin; Kim, Kimoon

2018-04-04

Single-molecule surface-enhanced Raman spectroscopy (SERS) offers new opportunities for exploring the complex chemical and biological processes that cannot be easily probed using ensemble techniques. However, the ability to place the single molecule of interest reliably within a hot spot, to enable its analysis at the single-molecule level, remains challenging. Here we describe a novel strategy for locating and securing a single target analyte in a SERS hot spot at a plasmonic nanojunction. The "smart" hot spot was generated by employing a thiol-functionalized cucurbit[6]uril (CB[6]) as a molecular spacer linking a silver nanoparticle to a metal substrate. This approach also permits one to study molecules chemically reluctant to enter the hot spot, by conjugating them to a moiety, such as spermine, that has a high affinity for CB[6]. The hot spot can accommodate at most a few, and often only a single, analyte molecule. Bianalyte experiments revealed that one can reproducibly treat the SERS substrate such that 96% of the hot spots contain a single analyte molecule. Furthermore, by utilizing a series of molecules each consisting of spermine bound to perylene bisimide, a bright SERS molecule, with polymethylene linkers of varying lengths, the SERS intensity as a function of distance from the center of the hot spot could be measured. The SERS enhancement was found to decrease as 1 over the square of the distance from the center of the hot spot, and the single-molecule SERS cross sections were found to increase with AgNP diameter.

Scanning confocal fluorescence microscopy for single molecule analysis of nucleotide excision repair complexes.

PubMed

Segers-Nolten, G M J; Wyman, C; Wijgers, N; Vermeulen, W; Lenferink, A T M; Hoeijmakers, J H J; Greve, J; Otto, C

2002-11-01

We used scanning confocal fluorescence microscopy to observe and analyze individual DNA- protein complexes formed between human nucleotide excision repair (NER) proteins and model DNA substrates. For this purpose human XPA protein was fused to EGFP, purified and shown to be functional. Binding of EGFP-labeled XPA protein to a Cy3.5-labeled DNA substrate, in the presence and absence of RPA, was assessed quantitatively by simultaneous excitation and emission detection of both fluorophores. Co-localization of Cy3.5 and EGFP signals within one diffraction limited spot indicated complexes of XPA with DNA. Measurements were performed on samples in a 1% agarose matrix in conditions that are compatible with protein activity and where reactions can be studied under equilibrium conditions. In these samples DNA alone was freely diffusing and protein-bound DNA was immobile, whereby they could be discriminated resulting in quantitative data on DNA binding. On the single molecule level approximately 10% of XPA co-localized with DNA; this increased to 32% in the presence of RPA. These results, especially the enhanced binding of XPA in the presence of RPA, are similar to those obtained in bulk experiments, validating the utility of scanning confocal fluorescence microscopy for investigating functional interactions at the single molecule level.

Single-Molecule Microscopy and Force Spectroscopy of Membrane Proteins

NASA Astrophysics Data System (ADS)

Engel, Andreas; Janovjak, Harald; Fotiadis, Dimtrios; Kedrov, Alexej; Cisneros, David; Müller, Daniel J.

Single-molecule atomic force microscopy (AFM) provides novel ways to characterize the structure-function relationship of native membrane proteins. High-resolution AFM topographs allow observing the structure of single proteins at sub-nanometer resolution as well as their conformational changes, oligomeric state, molecular dynamics and assembly. We will review these feasibilities illustrating examples of membrane proteins in native and reconstituted membranes. Classification of individual topographs of single proteins allows understanding the principles of motions of their extrinsic domains, to learn about their local structural flexibilities and to find the entropy minima of certain conformations. Combined with the visualization of functionally related conformational changes these insights allow understanding why certain flexibilities are required for the protein to function and how structurally flexible regions allow certain conformational changes. Complementary to AFM imaging, single-molecule force spectroscopy (SMFS) experiments detect molecular interactions established within and between membrane proteins. The sensitivity of this method makes it possible to measure interactions that stabilize secondary structures such as transmembrane α-helices, polypeptide loops and segments within. Changes in temperature or protein-protein assembly do not change the locations of stable structural segments, but influence their stability established by collective molecular interactions. Such changes alter the probability of proteins to choose a certain unfolding pathway. Recent examples have elucidated unfolding and refolding pathways of membrane proteins as well as their energy landscapes.

Fluorescence quenching by TEMPO: a sub-30 A single-molecule ruler.

PubMed

Zhu, Peizhi; Clamme, Jean-Pierre; Deniz, Ashok A

2005-11-01

A series of DNA molecules labeled with 5-carboxytetramethylrhodamine (5-TAMRA) and the small nitroxide radical TEMPO were synthesized and tested to investigate whether the intramolecular quenching efficiency can be used to measure short intramolecular distances in small ensemble and single-molecule experiments. In combination with distance calculations using molecular mechanics modeling, the experimental results from steady-state ensemble fluorescence and fluorescence correlation spectroscopy measurements both show an exponential decrease in the quenching rate constant with the dye-quencher distance in the 10-30 A range. The results demonstrate that TEMPO-5-TAMRA fluorescence quenching is a promising method to measure short distance changes within single biomolecules.

Fluorescence Quenching by TEMPO: A Sub-30 Å Single-Molecule Ruler

PubMed Central

Zhu, Peizhi; Clamme, Jean-Pierre; Deniz, Ashok A.

2005-01-01

A series of DNA molecules labeled with 5-carboxytetramethylrhodamine (5-TAMRA) and the small nitroxide radical TEMPO were synthesized and tested to investigate whether the intramolecular quenching efficiency can be used to measure short intramolecular distances in small ensemble and single-molecule experiments. In combination with distance calculations using molecular mechanics modeling, the experimental results from steady-state ensemble fluorescence and fluorescence correlation spectroscopy measurements both show an exponential decrease in the quenching rate constant with the dye-quencher distance in the 10–30 Å range. The results demonstrate that TEMPO-5-TAMRA fluorescence quenching is a promising method to measure short distance changes within single biomolecules. PMID:16199509

Fluorescence lifetime imaging system with nm-resolution and single-molecule sensitivity

NASA Astrophysics Data System (ADS)

Wahl, Michael; Rahn, Hans-Juergen; Ortmann, Uwe; Erdmann, Rainer; Boehmer, Martin; Enderlein, Joerg

2002-03-01

Fluorescence lifetime measurement of organic fluorophores is a powerful tool for distinguishing molecules of interest from background or other species. This is of interest in sensitive analysis and Single Molecule Detection (SMD). A demand in many applications is to provide 2-D imaging together with lifetime information. The method of choice is then Time-Correlated Single Photon Counting (TCSPC). We have devloped a compact system on a single PC board that can perform TCSPC at high throughput, while synchronously driving a piezo scanner holding the immobilized sample. The system allows count rates up to 3 MHz and a resolution down to 30 ps. An overall Instrument Response Function down to 300ps is achieved with inexpensive detectors and diode lasers. The board is designed for the PCI bus, permitting high throughput without loss of counts. It is reconfigurable to operate in different modes. The Time-Tagged Time-Resolved (TTTR) mode permits the recording of all photon events with a real-time tag allowing data analysis with unlimited flexibility. We use the Time-Tag clock for an external piezo scanner that moves the sample. As the clock source is common for scanning and tagging, the individual photons can be matched to pixels. Demonstrating the capablities of the system we studied single molecule solutions. Lifetime imaging can be performed at high resolution with as few as 100 photons per pixel.

Tracking the ultrafast motion of a single molecule by femtosecond orbital imaging

PubMed Central