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Single molecule microscopy

Bernardo A Nogueira, Gulce O Ildiz, João Canotilho, M Ermelinda S Eusébio, Marta Sofia C Henriques, José A Paixão, Rui Fausto
The molecular structure, vibrational spectra and photochemistry of 5-methylhydantoin (C4H6N2O2; 5-MH) were studied by matrix isolation infrared spectroscopy and theoretical calculations at the DFT(B3LYP)/6-311++G(d,p) theory level. The Natural Bond Orbital (NBO) analysis approach was used to study in detail the electronic structure of the minimum energy structure of 5-MH, namely the specific characteristics of the σ and π electronic systems of the molecule, and the stabilizing orbital interactions. UV irradiation of 5-MH isolated in argon matrix resulted in its photofragmentation through a single photochemical pathway, yielding isocyanic acid, ethanimine and carbon monoxide, thus following a pattern already observed before for the parent hydantoin and 1-methylhydantoin molecules...
June 22, 2017: Journal of Physical Chemistry. A
Hai Pan, Stephanie M Bilinovich, Parminder Kaur, Robert Riehn, Hong Wang, David C Williams
The methylcytosine-binding domain 2 (MBD2) protein recruits the nucleosome remodeling and deacetylase complex (NuRD) to methylated DNA to modify chromatin and regulate transcription. Importantly, MBD2 functions within CpG islands that contain 100s to 1000s of potential binding sites. Since NuRD physically rearranges nucleosomes, the dynamic mobility of this complex is directly related to function. In these studies, we use NMR and single-molecule atomic force microscopy and fluorescence imaging to study DNA binding dynamics of MBD2...
June 20, 2017: Nucleic Acids Research
Keith J Mickolajczyk, William O Hancock
Kinesin processivity, defined as the average number of steps that occur per interaction with a microtubule, is an important biophysical determinant of the motor's intracellular capabilities. Despite its fundamental importance to the diversity of tasks that kinesins carry out in cells, no existing quantitative model fully explains how structural differences between kinesins alter kinetic rates in the ATPase cycle to produce functional changes in processivity. Here we use high-resolution single-molecule microscopy to directly observe the stepping behavior of kinesin-1 and -2 family motors with different length neck-linker domains...
June 20, 2017: Biophysical Journal
Shenghang Jiang, Seongjin Park, Sai Divya Challapalli, Jingyi Fei, Yong Wang
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 ([Formula: see text]) depends exclusively on the density of clustering molecules (ρc)...
2017: PloS One
Ville Paakinaho, Diego M Presman, David A Ball, Thomas A Johnson, R Louis Schiltz, Peter Levitt, Davide Mazza, Tatsuya Morisaki, Tatiana S Karpova, Gordon L Hager
Population-based assays have been employed extensively to investigate the interactions of transcription factors (TFs) with chromatin and are often interpreted in terms of static and sequential binding. However, fluorescence microscopy techniques reveal a more dynamic binding behaviour of TFs in live cells. Here we analyse the strengths and limitations of in vivo single-molecule tracking and performed a comprehensive analysis on the intranuclear dwell times of four steroid receptors and a number of known cofactors...
June 21, 2017: Nature Communications
Daniel Halwidl, Wernfried Mayr-Schmölzer, David Fobes, Jin Peng, Zhiqiang Mao, Michael Schmid, Florian Mittendorfer, Josef Redinger, Ulrike Diebold
As complex ternary perovskite-type oxides are increasingly used in solid oxide fuel cells, electrolysis and catalysis, it is desirable to obtain a better understanding of their surface chemical properties. Here we report a pronounced ordering of hydroxyls on the cleaved (001) surface of the Ruddlesden-Popper perovskite Ca3Ru2O7 upon water adsorption at 105 K and subsequent annealing to room temperature. Density functional theory calculations predict the dissociative adsorption of a single water molecule (E ads = 1...
June 20, 2017: Nature Communications
E L S Souza, J C Sczancoski, I C Nogueira, M A P Almeida, M O Orlandi, M S Li, R A S Luz, M G R Filho, E Longo, L S Cavalcante
Copper tungstate (CuWO4) crystals were synthesized by the sonochemistry (SC) method, and then, heat treated in a conventional furnace at different temperatures for 1h. The structural evolution, growth mechanism and photoluminescence (PL) properties of these crystals were thoroughly investigated. X-ray diffraction patterns, micro-Raman spectra and Fourier transformed infrared spectra indicated that crystals heat treated and 100°C and 200°C have water molecules in their lattice (copper tungstate dihydrate (CuWO4·2H2O) with monoclinic structure), when the crystals are calcinated at 300°C have the presence of two phase (CuWO4·2H2O and CuWO4), while the others heat treated at 400°C and 500°C have a single CuWO4 triclinic structure...
September 2017: Ultrasonics Sonochemistry
Alexander V Rudnev, Veerabhadrarao Kaliginedi, Andrea Droghetti, Hiroaki Ozawa, Akiyoshi Kuzume, Masa-Aki Haga, Peter Broekmann, Ivan Rungger
An open challenge for single-molecule electronics is to find stable contacts at room temperature with a well-defined conductance. Common coinage metal electrodes pose fabrication and operational problems due to the high mobility of the surface atoms. We demonstrate how molecules covalently grafted onto mechanically robust graphite/graphene substrates overcome these limitations. To this aim, we explore the effect of the anchoring group chemistry on the charge transport properties of graphite-molecule contacts by means of the scanning tunneling microscopy break-junction technique and ab initio simulations...
June 2017: Science Advances
Bas M C Cloin, Elke De Zitter, Desiree Salas, Vincent Gielen, Gert E Folkers, Marina Mikhaylova, Maike Bergeler, Bartosz Krajnik, Jeremy Harvey, Casper C Hoogenraad, Luc Van Meervelt, Peter Dedecker, Lukas C Kapitein
Fluorophores with dynamic or controllable fluorescence emission have become essential tools for advanced imaging, such as superresolution imaging. These applications have driven the continuing development of photoactivatable or photoconvertible labels, including genetically encoded fluorescent proteins. These new probes work well but require the introduction of new labels that may interfere with the proper functioning of existing constructs and therefore require extensive functional characterization. In this work we show that the widely used red fluorescent protein mCherry can be brought to a purely chemically induced blue-fluorescent state by incubation with β-mercaptoethanol (βME)...
June 19, 2017: Proceedings of the National Academy of Sciences of the United States of America
Joachim Frank
Time-resolved cryo-electron microscopy (cryo-EM) combines the known advantages of single-particle cryo-EM in visualizing molecular structure with the ability to dissect the time progress of a reaction between molecules in vitro. Here some of the recent progress of this methodology and its first biological applications are outlined.
June 15, 2017: Journal of Structural Biology
Seung-Ryoung Jung, Bryant S Fujimoto, Daniel T Chiu
Quantitative microscopy is needed to understand reactions or phenomena carried out by biological molecules such as enzymes, receptors, and membrane-localized proteins. Counting the biomolecules of interest in single organelles or cellular compartments is critical in these approaches. In this brief perspective, we focus on the development of quantitative fluorescence microscopies that measure the precise copy numbers of proteins in cellular organelles or purified samples. We introduce recent improvements in quantitative microscopies to overcome undercounting or overcounting errors in certain conditions...
June 14, 2017: Current Opinion in Chemical Biology
Flynn R Hill, Enrico Monachino, Antoine M van Oijen
The single-molecule approach seeks to understand molecular mechanisms by observing biomolecular processes at the level of individual molecules. These methods have led to a developing understanding that for many processes, a diversity of behaviours will be observed, representing a multitude of pathways. This realisation necessitates that an adequate number of observations are recorded to fully characterise this diversity. The requirement for large numbers of observations to adequately sample distributions, subpopulations, and rare events presents a significant challenge for single-molecule techniques, which by their nature do not typically provide very high throughput...
June 15, 2017: Biochemical Society Transactions
Geelsu Hwang, Yuan Liu, Dongyeop Kim, Yong Li, Damian J Krysan, Hyun Koo
Candida albicans is frequently detected with heavy infection by Streptococcus mutans in plaque-biofilms from children with early-childhood caries (ECC). This cross-kingdom biofilm contains an extensive matrix of extracellular α-glucans that is produced by an exoenzyme (GtfB) secreted by S. mutans. Here, we report that mannans located on the outer surface of C. albicans cell-wall mediates GtfB binding, enhancing glucan-matrix production and modulating bacterial-fungal association within biofilms formed in vivo...
June 2017: PLoS Pathogens
Antoine Hinaut, Baran Eren, Roland Steiner, Sara Freund, Res Jöhr, Thilo Glatzel, Laurent Marot, Ernst Meyer, Shigeki Kawai
Templating insulating surfaces at the nanoscale is an interesting prospect for applications that involve the adsorption of molecules or nanoparticles where electronic decoupling of the adsorbed species from the substrate is needed. In this study, we present a method to structure alkali halide surfaces at the nanoscale using a combination of low temperature plasma exposure and annealing, and characterize the surfaces by atomic force microscopy. We find that nanostructurating can be controlled by the duration of the exposure, the atomic mass of the plasma gas and the subsequent step-by-step annealing process...
June 21, 2017: Physical Chemistry Chemical Physics: PCCP
Tatjana Trcek, Timothée Lionnet, Hari Shroff, Ruth Lehmann
Spatial information is critical to the interrogation of developmental and tissue-level regulation of gene expression. However, this information is usually lost when global mRNA levels from tissues are measured using reverse transcriptase PCR, microarray analysis or high-throughput sequencing. By contrast, single-molecule fluorescence in situ hybridization (smFISH) preserves the spatial information of the cellular mRNA content with subcellular resolution within tissues. Here we describe an smFISH protocol that allows for the quantification of single mRNAs in Drosophila embryos, using commercially available smFISH probes (e...
July 2017: Nature Protocols
Ju Lu, Shenfei Zong, Zhuyuan Wang, Chen Chen, Yizhi Zhang, Yiping Cui
A new yolk-shell type nanoprobe for super-resolution imaging is demonstrated. Using the proposed nanoprobe and single molecule localization based super resolution imaging strategy, intracellular nanoparticle tracking and super-resolution imaging are realized. The localization precision is about 50 nm and single-molecule localization microscopy using the proposed nanoprobe requires only one single excitation laser and no specific imaging buffer.
June 8, 2017: Nanotechnology
Jamie L Gilmore, Aiko Yoshida, James A Hejna, Kunio Takeyasu
We demonstrate an application of atomic force microscopy (AFM) for the structural analysis of long single-stranded RNA (>1 kb), focusing on 28S ribosomal RNA (rRNA). Generally, optimization of the conditions required to obtain three-dimensional (3D) structures of long RNA molecules is a challenging or nearly impossible process. In this study, we overcome these limitations by developing a method using AFM imaging combined with automated, MATLAB-based image analysis algorithms for extracting information about the domain organization of single RNA molecules...
June 7, 2017: Nucleic Acids Research
Meng Li, Lijun Wang, Christine V Putnis
Calcium oxalate kidney stones form attached to Randall's plaques (RP), calcium phosphate (Ca-P) deposits on the renal papillary surface. Osteopontin (OPN) suppresses crystal growth in the complex process of urinary stone formation, but the inhibitory role of active domains of OPN involved in the initial formation of the RPs attached to epithelial cells has yet to be clarified. Here we demonstrate the thermodynamic basis for how OPN sequences regulate the onset of Ca-P mineral formation on lipid rafts as a model membrane...
June 12, 2017: Journal of Physical Chemistry. B
Yifan Yao, Huanli Dong, Feng Liu, Thomas P Russell, Wenping Hu
Charge transport of small molecules is measured well with scanning tunneling microscopy, conducting atomic force microscopy, break junction, nanopore, and covalently bridging gaps. However, the manipulation and measurement of polymer chains remain a long-standing fundamental issue in conjugated polymers and full of challenge since conjugated polymers are naturally disordered materials. Here, a fundamental breakthrough in generating high-quality conjugated-polymer nanocrystals with extended conjugation and exceptionally high degrees of order using a surface-supported topochemical polymerization method is demonstrated...
June 6, 2017: Advanced Materials
Lars Müller, Seon-Young Rhim, Vipilan Sivanesan, Dongxiang Wang, Sebastian Hietzschold, Patrick Reiser, Eric Mankel, Sebastian Beck, Stephen Barlow, Seth R Marder, Annemarie Pucci, Wolfgang Kowalsky, Robert Lovrincic
Stable electrical doping of organic semiconductors is fundamental for the functionality of high performance devices. It is known that dopants can be subjected to strong diffusion in certain organic semiconductors. This work studies the impact of operating conditions on thin films of the polymer poly(3-hexylthiophene) (P3HT) and the small molecule Spiro-MeOTAD, doped with two differently sized p-type dopants. The negatively charged dopants can drift upon application of an electric field in thin films of doped P3HT over surprisingly large distances...
June 6, 2017: Advanced Materials
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