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Single molecule microscopy

Takashi Kumagai, Janina N Ladenthin, Yair Litman, Mariana Rossi, Leonhard Grill, Sylwester Gawinkowski, Jacek Waluk, Mats Persson
Tautomerization in single porphycene molecules is investigated on Cu(111), Ag(111), and Au(111) surfaces by a combination of low-temperature scanning tunneling microscopy (STM) experiments and density functional theory (DFT) calculations. It is revealed that the trans configuration is the thermodynamically stable form of porphycene on Cu(111) and Ag(111), whereas the cis configuration occurs as a meta-stable form. The trans → cis or cis → trans conversion on Cu(111) can be induced in an unidirectional fashion by injecting tunneling electrons from the STM tip or heating the surface, respectively...
March 14, 2018: Journal of Chemical Physics
Qing Li, Biao Yang, Jonas Björk, Qigang Zhong, Huanxin Ju, Junjie Zhang, Nan Cao, Ziliang Shi, Haiming Zhang, Daniel Ebeling, Andre Schirmeisen, Junfa Zhu, Lifeng Chi
Hierarchical control of chemical reactions is being considered as one of the most ambitious and challenging topics in modern organic chemistry. In this study, we have realized the one-by-one scission of the X-H bonds (X=N and C) of aromatic amines in a controlled fashion on the Cu(111) surface. Each dehydrogenation reaction leads to certain metal-organic supramolecular structures, which were monitored in single-bond resolution via scanning tunneling microscopy and non-contact atomic force microscopy. Moreover, the reaction pathways were elucidated from X-ray photoelectron spectroscopy measurements and density functional theory calculations...
March 15, 2018: Journal of the American Chemical Society
David R Glenn, Dominik B Bucher, Junghyun Lee, Mikhail D Lukin, Hongkun Park, Ronald L Walsworth
Quantum systems that consist of solid-state electronic spins can be sensitive detectors of nuclear magnetic resonance (NMR) signals, particularly from very small samples. For example, nitrogen-vacancy centres in diamond have been used to record NMR signals from nanometre-scale samples, with sensitivity sufficient to detect the magnetic field produced by a single protein. However, the best reported spectral resolution for NMR of molecules using nitrogen-vacancy centres is about 100 hertz. This is insufficient to resolve the key spectral identifiers of molecular structure that are critical to NMR applications in chemistry, structural biology and materials research, such as scalar couplings (which require a resolution of less than ten hertz) and small chemical shifts (which require a resolution of around one part per million of the nuclear Larmor frequency)...
March 14, 2018: Nature
Shawulienu Kezilebieke, Marc Dvorak, Teemu Ojanen, Peter Liljeroth
Magnetic impurities have a dramatic effect on superconductivity by breaking the time-reversal symmetry and inducing so-called Yu-Shiba-Rusinov (YSR) low energy bound states within the superconducting gap. The spatial extent of YSR states is greatly enhanced in 2D systems, which should facilitate the formation of coupled states. Here, we observe YSR states on single cobalt phthalocyanine (CoPC) molecules on a 2D superconductor NbSe2 using low-temperature scanning tunneling microscopy (STM) and spectroscopy (STS)...
March 13, 2018: Nano Letters
Joerg Schnitzbauer, Yina Wang, Shijie Zhao, Matthew Bakalar, Tulip Nuwal, Baohui Chen, Bo Huang
Superresolution images reconstructed from single-molecule localizations can reveal cellular structures close to the macromolecular scale and are now being used routinely in many biomedical research applications. However, because of their coordinate-based representation, a widely applicable and unified analysis platform that can extract a quantitative description and biophysical parameters from these images is yet to be established. Here, we propose a conceptual framework for correlation analysis of coordinate-based superresolution images using distance histograms...
March 12, 2018: Proceedings of the National Academy of Sciences of the United States of America
Weixiang Jin, M Fethullah Simsek, Arnd Pralle
It has been long recognized that the cell membrane is heterogeneous on scales ranging from a couple of molecules to micrometers in size and hence diffusiong of receptors is length scale dependent. This heterogeneity modulates many cell-membrane-associated processes requiring transient spatiotemporal separation of components. The transient increase in local concentration of interacting signal components enables robust signaling in an otherwise thermally noisy system. Understanding how lipids and proteins self-organize and interact with the cell cortex requires quantifying the motion of the components...
March 9, 2018: Methods: a Companion to Methods in Enzymology
Yuejin Li, Xili Shang, Changhai Li, Xiaoming Huang, Jingjing Zheng
Novel visible-light-induced UiO-66/BiOI photocatalysts with a p-n junction structure have been prepared for the first time through a facile hydrothermal method. The prepared photocatalysts were characterized using the powder X-ray diffraction, high resolution transmission electron microscopy, scanning electron microscopy, UV-visible diffuse reflectance spectra, and N2 adsorption-desorption (Brunauer-Emmett-Teller) techniques respectively. The photodegradation performances of UiO-66/BiOI photocatalysts were evaluated by photodegrading salicylic acid under visible-light irradiation...
March 2018: Water Science and Technology: a Journal of the International Association on Water Pollution Research
Margarete Poppelreuther, Simone Sander, Fadil Minden, Marina S Dietz, Tarik Exner, Chen Du, Ingrid Zhang, Friedrich Ehehalt, Laura Knüppel, Susanne Domschke, Anna Badenhop, Sarah Staudacher, Robert Ehehalt, Wolfgang Stremmel, Christoph Thiele, Mike Heilemann, Joachim Füllekrug
ACSL3 is the only long chain fatty acyl-CoA synthetase consistently found on growing and mature lipid droplets (LDs), suggesting that this specific localization has biological relevance. Current models for LD growth propose that triglycerides are synthesized by enzymes at the LD surface, with activated fatty acids provided by LD localized ACSL3, thus allowing growth independent of the ER. Here, we tested this hypothesis by quantifying ACSL3 on LDs from human A431 cells. RNAi of ACSL3 reduced the oleoyl-CoA synthetase activity by 83%, suggesting that ACSL3 is by far the dominant enzyme of A431 cells...
March 8, 2018: Biochimica et Biophysica Acta
Beatriz Lagunas, Liam Walker, Rana Muhammad Fraz Hussain, Ian Hands-Portman, Kate Woolley-Allen, Miriam L Gifford
Cell type-specific marker lines expressing fluorophores such as GFP or GUS can be used as starting material from which single cell types can be isolated by fluorescence-activated cell sorting (FACS) and/or for the study of root development. Establishing the stability of these lines is an essential step prior to further study to ensure that marker expression and localization is stable over time and during environmental perturbations of interest to researchers applying these lines as treatments. Here, we detail the use of root cross sectioning to investigate marker expression throughout the length and width of the root using the model legume Medicago truncatula as an example...
2018: Methods in Molecular Biology
Julius Müller, Domenik Prozeller, Artur Ghazaryan, Maria Kokkinopoulou, Volker Mailänder, Svenja Morsbach, Katharina Landfester
The interaction of nanocarriers with blood plasma components influences the biological response, and therefore, it needs to be controlled. Whereas protein adsorption to nanocarriers has been investigated to a large extent, the role of lipid interaction for drug delivery and its biological effect is not yet clear. However, lipids represent an important constituent of blood plasma and are usually bound in the form of lipoproteins. Because already for many nanocarrier systems an enrichment of apolipoproteins in their protein corona was reported, we examine the interaction of lipoproteins with nanocarriers...
March 7, 2018: Acta Biomaterialia
Michael A Beuwer, Bas van Hoof, Peter Zijlstra
The high sensitivity of localized surface plasmon resonance sensors to the local refractive index allows for the detection of single-molecule binding events. Though binding events of single objects can be detected by their induced plasmon shift, the broad distribution of observed shifts remains poorly understood. Here, we perform a single-particle study wherein single nanospheres bind to a gold nanorod, and relate the observed plasmon shift to the binding location using correlative microscopy. To achieve this we combine atomic force microscopy to determine the binding location, and single-particle spectroscopy to determine the corresponding plasmon shift...
March 1, 2018: Journal of Physical Chemistry. C, Nanomaterials and Interfaces
David E Timm, Valorie Bowman, Russell Madsen, Charles Rauch
Protein arginine methyl transferase 5 (PRMT5) is a signaling protein and histone modifying enzyme that is important in many cellular processes, including regulation of eukaryotic gene transcription. Reported here is a 3.7 Å structure of PRMT5, solved in complex with regulatory binding subunit MEP50 (methylosome associated protein 50, WDR77, p44), by single particle (SP) cryo-Electron Microscopy (cryo-EM) using micrographs of particles that are visibly crowded and aggregated. Despite suboptimal micrograph appearance, this cryo-EM structure is in good agreement with previously reported crystal structures of the complex, which revealed a 450 kDa hetero-octameric assembly having internal D2 symmetry...
2018: PloS One
Susovan Roy Chowdhury, H Peter Lu
Interrogating protein structure and function inter-relationship under a picoNewton force manipulation has been highly promising and informative. Although protein conformational changes under pulling force manipulations have been extensively studied, protein conformational changes under a compressive force have not been explored in detail. Using our home-modified sensitive and high signal-to-noise AFM microscopy approach, we have applied a picoNewton compressive force, manipulating a CaM molecule to characterize two different forms of Calmodulin, the Ca2+-ligated activated form, and the Ca2+ free non-activated form (Apo-Calmodulin)...
March 8, 2018: Biochemistry
Nicolas Pavillon, Alison J Hobro, Shizuo Akira, Nicholas I Smith
We present a method enabling the noninvasive study of minute cellular changes in response to stimuli, based on the acquisition of multiple parameters through label-free microscopy. The retrieved parameters are related to different attributes of the cell. Morphological variables are extracted from quantitative phase microscopy and autofluorescence images, while molecular indicators are retrieved via Raman spectroscopy. We show that these independent parameters can be used to build a multivariate statistical model based on logistic regression, which we apply to the detection at the single-cell level of macrophage activation induced by lipopolysaccharide (LPS) exposure and compare their respective performance in assessing the individual cellular state...
March 6, 2018: Proceedings of the National Academy of Sciences of the United States of America
Ling Zhang, Kasper P Kepp, Jens Ulstrup, Jingdong Zhang
Metalloporphyrins are active sites in metalloproteins and synthetic catalysts. They have also been studied extensively by electrochemistry as well as being prominent targets in electrochemical scanning tunneling microscopy (STM). Previous studies of FePPIX adsorbed on graphite and alkylthiol modified Au electrodes showed a pair of reversible Fe(III/II)PPIX peaks at about -0.41 V (vs. NHE) at high solution pH. We recently used iron protoporphyrin IX (FeP-PIX) as an intercalating probe for long-range electrochemical electron transfer through a G-quadruplex oligonucleo-tide (DNAzyme); this study disclosed two, rather than a single pair of voltammetric peaks with a new and dominating peak, shifted 200 mV positive relative to the  -0...
March 6, 2018: Langmuir: the ACS Journal of Surfaces and Colloids
Yuxi Liu, Shane Gonen, Tamir Gonen, Todd O Yeates
Current single-particle cryo-electron microscopy (cryo-EM) techniques can produce images of large protein assemblies and macromolecular complexes at atomic level detail without the need for crystal growth. However, proteins of smaller size, typical of those found throughout the cell, are not presently amenable to detailed structural elucidation by cryo-EM. Here we use protein design to create a modular, symmetrical scaffolding system to make protein molecules of typical size suitable for cryo-EM. Using a rigid continuous alpha helical linker, we connect a small 17-kDa protein (DARPin) to a protein subunit that was designed to self-assemble into a cage with cubic symmetry...
March 5, 2018: Proceedings of the National Academy of Sciences of the United States of America
Luděk Vyšín, Tomáš Burian, Egor Ukraintsev, Marie Davídková, Michael E Grisham, Scott Heinbuch, Jorge J Rocca, Libor Juha
In this study, we examined dose-rate effects on strand break formation in plasmid DNA induced by pulsed extreme ultraviolet (XUV) radiation. Dose delivered to the target molecule was controlled by attenuating the incident photon flux using aluminum filters as well as by changing the DNA/buffer-salt ratio in the irradiated sample. Irradiated samples were examined using agarose gel electrophoresis. Yields of single- and double-strand breaks (SSBs and DSBs) were determined as a function of the incident photon fluence...
March 5, 2018: Radiation Research
Linwen Zhang, Christopher J Sevinsky, Brian M Davis, Akos Vertes
Specific subpopulations in a heterogeneous collection of cells, e.g., cancer stem cells in a tumor, are often associated with biological or medical conditions. Fluorescence microscopy, based on biomarkers labelled with fluorescent probes, is a widely used technique for the visualization and selection of such cells. Phenotypic differences for these subpopulations at the molecular level can be identified by their untargeted analysis using single cell mass spectrometry (MS). Here, we combine capillary microsampling MS with fluorescence microscopy for the analysis of metabolite and lipid levels in single cells to discern the heterogeneity of subpopulations corresponding to mitotic stages...
March 5, 2018: Analytical Chemistry
Pranesh Kumar, Ashok K Singh, Vinit Raj, Amit Rai, Amit K Keshari, Dinesh Kumar, Biswanath Maity, Anand Prakash, Sabyasachi Maiti, Sudipta Saha
Purpose: The application of betulinic acid (B), a potent antineoplastic agent, is limited due to poor bioavailability, short plasma half-life and inappropriate tissue distribution. Thus, we aimed to prepare novel 50:50 poly(lactic- co -glycolic acid) (PLGA)-loaded B nanoparticles (BNP) and to compare its anti-hepatocellular carcinoma (HCC) activity with parent B. Methods: BNP were synthesized and characterized using different methods such as scanning electron microscopy (SEM), fourier-transform infrared (FTIR) spectrometry and particle size analyses...
2018: International Journal of Nanomedicine
Laurent Potvin-Trottier, Scott Luro, Johan Paulsson
Bacteria have molecules present in low and fluctuating numbers that randomize cell behaviors. Understanding these stochastic processes and their impact on cells has, until recently, been limited by the lack of single-cell measurement methods. Here, we review recent developments in microfluidics that enable following individual cells over long periods of time under precisely controlled conditions, and counting individual fluorescent molecules in many cells. We showcase discoveries that were made possible using these devices in various aspects of microbiology, such as antibiotic tolerance/persistence, cell-size control, cell-fate determination, DNA damage response, and synthetic biology...
February 26, 2018: Current Opinion in Microbiology
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