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Single molecule imaging

Satoshi Habuchi, Takuya Yamamoto, Yasuyuki Tezuka
We demonstrate a method for the synthesis of cyclic polymers and a protocol for characterizing their diffusive motion in a melt state at the single molecule level. An electrostatic self-assembly and covalent fixation (ESA-CF) process is used for the synthesis of the cyclic poly(tetrahydrofuran) (poly(THF)). The diffusive motion of individual cyclic polymer chains in a melt state is visualized using single molecule fluorescence imaging by incorporating a fluorophore unit in the cyclic chains. The diffusive motion of the chains is quantitatively characterized by means of a combination of mean-squared displacement (MSD) analysis and a cumulative distribution function (CDF) analysis...
September 26, 2016: Journal of Visualized Experiments: JoVE
Gil Henkin, Daniel Berard, Francis Stabile, Marjan Shayegan, Jason S Leith, Sabrina R Leslie
We present a dynamically adjustable nanofluidic platform for formatting the conformations of and visualizing the interaction kinetics between biomolecules in solution, offering new time resolution and control of the reaction processes. This platform extends Convex Lens-induced Confinement (CLiC), a technique for imaging molecules under confinement, by introducing a system for in-situ modification of the chemical environment; this system uses a deep microchannel to diffusively exchange reagents within the nanoscale imaging region, whose height is fixed by a nanopost array...
October 21, 2016: Analytical Chemistry
Jinglin Fu, Yuhe Renee Yang, Soma Dhakal, Zhao Zhao, Minghui Liu, Ting Zhang, Nils G Walter, Hao Yan
In nature, the catalytic efficiency of multienzyme complexes highly depends on their spatial organization. The positions and orientations of the composite enzymes are often precisely controlled to facilitate substrate transport between them. Self-assembled DNA nanostructures hold great promise for organizing biomolecules at the nanoscale. Here, we present detailed protocols for exploiting DNA nanostructures as assembly scaffolds that organize the spatial arrangements of multienzyme cascades with control over their relative distance, compartmentalization and substrate diffusion paths...
November 2016: Nature Protocols
Zheng Liu, Cristina Gutierrez-Vargas, Jia Wei, Robert A Grassucci, Noel Espina, Susan Madison-Antenucci, Liang Tong, Joachim Frank
With the advance of new instruments and algorithms, and the accumulation of experience over decades, single-particle cryo-EM has become a pivotal part of structural biology. Recently, we determined the structure of a eukaryotic ribosome at 2.5 Å for the large subunit. The ribosome was derived from Trypanosoma cruzi, the protozoan pathogen of Chagas disease. The high-resolution density map allowed us to discern a large number of unprecedented details including rRNA modifications, water molecules, and ions such as Mg(2+) and Zn(2+) ...
October 17, 2016: Protein Science: a Publication of the Protein Society
Guohua Tao
Accurately describing nuclear motion is crucial in electronically nonadiabatic dynamics simulations. In this work, a coherence-controlled (CC) approach is proposed based on the mapping between the classical state space and the full electronic matrix and that between the decomposed state space and different nuclear dynamics that allows nuclear motion to properly follow either Ehrenfest dynamics in the coherence domain or Born-Oppenheimer-like dynamics in the single-state domain in a consistent manner. This new method is applied to several benchmark models involving nonadiabatic transitions in two-state or three-state systems, and the obtained results are in excellent agreement with exact quantum calculations...
October 17, 2016: Journal of Physical Chemistry Letters
Carmine Di Rienzo, Paolo Annibale
The determination of the mode and rapidity of motion of individual molecules within a biological sample is becoming a more and more common analysis in biophysical investigations. Single molecule tracking (SMT) techniques allow reconstructing the trajectories of individual molecules within a movie, provided that the position from one frame to the other can be correctly linked. The outcomes, however, appear to depend on the specific method used, and most techniques display a limitation to capture fast modes of motion in a crowded environment...
October 1, 2016: Optics Letters
Adam J M Wollman, Helen Miller, Simon Foster, Mark C Leake
Staphylococcus aureus is an important pathogen, giving rise to antimicrobial resistance in cell strains such as Methicillin Resistant S. aureus (MRSA). Here we report an image analysis framework for automated detection and image segmentation of cells in S. aureus cell clusters, and explicit identification of their cell division planes. We use a new combination of several existing analytical tools of image analysis to detect cellular and subcellular morphological features relevant to cell division from millisecond time scale sampled images of live pathogens at a detection precision of single molecules...
October 17, 2016: Physical Biology
Ximiao Hou, Michael C DeSantis, Chunjuan Tian, Wei Cheng
Although Ashkin and Dziedzic first demonstrated optical trapping of individual tobacco mosaic viruses in suspension as early as 1987, this pioneering work has not been followed up only until recently. Using human immunodeficiency virus type 1 (HIV-1) as a model virus, we have recently demonstrated that a single HIV-1 virion can be stabled trapped, manipulated and measured in physiological media with high precision. The capability to optically trap a single virion in suspension not only allows us to determine, for the first time, the refractive index of a single virus with high precision, but also quantitate the heterogeneity among individual virions with single-molecule resolution, the results of which shed light on the molecular mechanisms of virion infectivity...
August 2016: Proceedings of SPIE
Wen-Liang Gong, Jie Yan, Ling-Xi Zhao, Chong Li, Zhen-Li Huang, Ben Zhong Tang, Ming-Qiang Zhu
Photoswitchable fluorophores are promising in single-molecule optical devices and super-resolution fluorescence imaging, especially in single-molecule photo-activated localization microscopy (PALM) or stochastic optical reconstruction microscopy (STORM). However, the scarcity of current photoswitchable fluorophores stimulates researchers to develop complicated optical systems and processing software, in accordance with the limited photoswitchable fluorescent proteins and organic fluorophores. Previous efforts to develop synthetic photoswitchable fluorophores have exhibited their promising potential in super-resolution fluorescence imaging...
October 14, 2016: Photochemical & Photobiological Sciences
Younghyoun Yoon, Aaron M Mohs, Michael C Mancini, Shuming Nie, Hyunsuk Shim
Despite major advances in targeted drug therapy and radiation therapy, surgery remains the most effective treatment for most solid tumors. The single most important predictor of patient survival is a complete surgical resection of the primary tumor, draining lymph nodes, and metastatic lesions. Presently, however, 20%-30% of patients with head and neck cancer who undergo surgery still leave the operating room without complete resection because of missed lesions. Thus, major opportunities exist to develop advanced imaging tracers and intraoperative instrumentation that would allow surgeons to visualize microscopic tumors during surgery...
September 2016: Tomography: a Journal for Imaging Research
Bert Nitzsche, Elzbieta Dudek, Lukasz Hajdo, Andrzej A Kasprzak, Andrej Vilfan, Stefan Diez
Single-molecule experiments have been used with great success to explore the mechanochemical cycles of processive motor proteins such as kinesin-1, but it has proven difficult to apply these approaches to nonprocessive motors. Therefore, the mechanochemical cycle of kinesin-14 (ncd) is still under debate. Here, we use the readout from the collective activity of multiple motors to derive information about the mechanochemical cycle of individual ncd motors. In gliding motility assays we performed 3D imaging based on fluorescence interference contrast microscopy combined with nanometer tracking to simultaneously study the translation and rotation of microtubules...
October 11, 2016: Proceedings of the National Academy of Sciences of the United States of America
Birol Cabukusta, Jan A Köhlen, Christian P Richter, Changjiang You, Joost C M Holthuis
Single-molecule photobleaching has emerged as a powerful non-invasive approach to extract the stoichiometry of multimeric membrane proteins in their native cellular environment. However, this method has mainly been used to determine the subunit composition of ion channels and receptors at the plasma membrane. In here we applied single-molecule photobleaching to analyse the oligomeric state of an ER-resident candidate ceramide sensor protein, SMSr/SAMD8. Co-immunoprecipitation and chemical crosslinking studies previously revealed that the N-terminal sterile alpha motif or SAM domain of SMSr drives self-assembly of the protein into oligomers and that SMSr oligomerization is promoted by curcumin, a drug known to perturb ER ceramide and calcium homeostasis...
October 10, 2016: Journal of Biological Chemistry
Erin Gemperline, Caitlin Keller, Dhileepkumar Jayaraman, Junko Maeda, Michael R Sussman, Jean-Michel Ané, Lingjun Li
Plant science is an important, rapidly developing area of study. Within plant science, one area of study that has grown tremendously with recent technological advances, such as mass spectrometry, is the field of plant-omics; however, plant peptidomics is relatively underdeveloped in comparison with proteomics and metabolomics. Endogenous plant peptides can act as signaling molecules and have been shown to affect cell division, development, nodulation, reproduction, symbiotic associations, and defense reactions...
October 11, 2016: Journal of Proteome Research
V S Perunicic, C D Hill, L T Hall, L C L Hollenberg
Imaging the atomic structure of a single biomolecule is an important challenge in the physical biosciences. Whilst existing techniques all rely on averaging over large ensembles of molecules, the single-molecule realm remains unsolved. Here we present a protocol for 3D magnetic resonance imaging of a single molecule using a quantum spin probe acting simultaneously as the magnetic resonance sensor and source of magnetic field gradient. Signals corresponding to specific regions of the molecule's nuclear spin density are encoded on the quantum state of the probe, which is used to produce a 3D image of the molecular structure...
October 11, 2016: Nature Communications
Carlos J Villagómez, Fabien Castanié, Cristina Momblona, Sébastien Gauthier, Tomaso Zambelli, Xavier Bouju
Single 1,8-octanedithiol (ODT) molecules adsorbed onto the Cu(100) surface have been characterized by using scanning tunneling microscopy (STM) and studied by semi-empirical calculations. STM images have revealed two types of chiral molecules on the surface upon adsorption and both types of molecules showed two bright spots at the extremities of a small rod due to the enhanced electronic density contrast of the chemisorbed sulfur atoms. In sub-monolayer regime deposition, ODT molecules exhibit preferential adsorption directions and the relaxation mechanism is driven by the chemisorption of the two sulfur atoms in a hollow site of the surface...
October 5, 2016: Physical Chemistry Chemical Physics: PCCP
Adam S Backer, Maurice Y Lee, W E Moerner
Single-molecule orientation measurements provide unparalleled insight into a multitude of biological and polymeric systems. We report a simple, high-throughput technique for measuring the azimuthal orientation and rotational dynamics of single fluorescent molecules, which is compatible with localization microscopy. Our method involves modulating the polarization of an excitation laser, and analyzing the corresponding intensities emitted by single dye molecules and their modulation amplitudes. To demonstrate our approach, we use intercalating and groove-binding dyes to obtain super-resolved images of stretched DNA strands through binding-induced turn-on of fluorescence...
2016: Optica
Muwen Kong, Lili Liu, Xuejing Chen, Katherine I Driscoll, Peng Mao, Stefanie Böhm, Neil M Kad, Simon C Watkins, Kara A Bernstein, John J Wyrick, Jung-Hyun Min, Bennett Van Houten
Nucleotide excision repair (NER) is an evolutionarily conserved mechanism that processes helix-destabilizing and/or -distorting DNA lesions, such as UV-induced photoproducts. Here, we investigate the dynamic protein-DNA interactions during the damage recognition step using single-molecule fluorescence microscopy. Quantum dot-labeled Rad4-Rad23 (yeast XPC-RAD23B ortholog) forms non-motile complexes or conducts a one-dimensional search via either random diffusion or constrained motion. Atomic force microcopy analysis of Rad4 with the β-hairpin domain 3 (BHD3) deleted reveals that this motif is non-essential for damage-specific binding and DNA bending...
October 20, 2016: Molecular Cell
E F Silva, R F Bazoni, E B Ramos, M S Rocha
We have investigated the interaction of the DNA molecule with the anticancer drug doxorubicin (doxo) by using three different experimental techniques: single molecule stretching, single molecule imaging and dynamic light scattering. Such techniques allowed us to get new insights on the mechanical behavior of the DNA-doxo complexes as well as on the physical chemistry of the interaction. Firstly, the contour length data obtained from single molecule stretching were used to extract the physicochemical parameters of the DNA-doxo interaction under different buffer conditions...
October 7, 2016: Biopolymers
Feriel Melaine, Clothilde Coilhac, Yoann Roupioz, Arnaud Buhot
Small molecules (MW < 1000 Da) represent a large class of biomarkers of interest. Recently, a new class of biosensors has been emerging thanks to the recognition properties of aptamers, short DNA or RNA single strands, selected against such small molecular targets. Among them, an adenosine-specific aptamer has been largely described and used due to its remarkable affinity to this small target (KD = 6 μM). In this paper, we achieved the proof-of-principle of an aptasensor based on the thermodynamic follow-up of adenosine binding with engineered split-aptamer sequences...
September 29, 2016: Nanoscale
Christos Boutopoulos, Adrien Dagallier, Maria Sansone, Andre-Pierre Blanchard-Dionne, Évelyne Lecavalier-Hurtubise, Étienne Boulais, Michel Meunier
Precise spatial and temporal control of pressure stimulation at the nanometer scale is essential for the fabrication and manipulation of nano-objects, and for exploring single-molecule behaviour of matter under extreme conditions. However, state-of-the-art nano-mechanical transducers require sophisticated driving hardware and are currently limited to moderate pressure regimes. Here we report a gold plasmonic bowtie (AuBT) nano-antennas array that can generate extreme pressure stimulus of ∼100 GPa in the ps (10(-12) s) time scale with sub-wavelength resolution upon irradiation with ultra-short laser pulses...
October 6, 2016: Nanoscale
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