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Single molecule microscopy

Jonathan B Grimm, Brian P English, Heejun Choi, Anand K Muthusamy, Brian P Mehl, Peng Dong, Timothy A Brown, Jennifer Lippincott-Schwartz, Zhe Liu, Timothée Lionnet, Luke D Lavis
Small-molecule fluorophores are important tools for advanced imaging experiments. We previously reported a general method to improve small, cell-permeable fluorophores which resulted in the azetidine-containing 'Janelia Fluor' (JF) dyes. Here, we refine and extend the utility of these dyes by synthesizing photoactivatable derivatives that are compatible with live-cell labeling strategies. Once activated, these derived compounds retain the superior brightness and photostability of the JF dyes, enabling improved single-particle tracking and facile localization microscopy experiments...
October 24, 2016: Nature Methods
Christian Helbing, Robert Stößel, Dominik A Hering, Matthias Michael Lothar Arras, Jörg Bossert, Klaus D Jandt
Nanostructured surfaces have the potential to influence the assembly as well as the orientation of adsorbed proteins and may, thus, strongly influence the biomaterials performance. For the class of polymeric (bio)materials a reproducible and well characterized nanostructure is the ordered chain folded surface of a polyethylene single crystal (PE-SC). We tested the hypothesis that the trinodal-rod shaped protein human plasma fibrinogen (HPF) adsorbs on the (001) surface of PE-SCs along specific crystallographic directions...
October 24, 2016: Langmuir: the ACS Journal of Surfaces and Colloids
Akio Hayashi, Hiromi Ota, Xavier López, Norihito Hiyoshi, Nao Tsunoji, Tsuneji Sano, Masahiro Sadakane
Dipotassium cation (K(+))-encapsulated Preyssler-type phosphotungstate, [P5W30O110K2](13-), was prepared by heating monobismuth (Bi(3+))-encapsulated Preyssler-type phosphotungstate, [P5W30O110Bi(H2O)](12-), in acetate buffer in the presence of an excess amount of potassium cations. Characterization of the isolated potassium salt, K13[P5W30O110K2] (1a), and its acid form, H13[P5W30O110K2] (1b), by single crystal X-ray structure analysis, (31)P and (183)W nuclear magnetic resonance (NMR), Fourier transform infrared (FT-IR) spectroscopy, cyclic voltammetry (CV), high-resolution electrospray ionization mass spectroscopy (HR-ESI-MS), and elemental analysis revealed that two potassium cations are encapsulated in the Preyssler-type phosphotungstate molecule with formal D5h symmetry, which is the first example of a Preyssler-type compound with two encapsulated cations...
October 24, 2016: Inorganic Chemistry
ChunSik Choe, Jürgen Lademann, Maxim E Darvin
Confocal Raman microscopy has been used to measure depth-dependent profiles of human SC in vivo in the high wavenumber (HWN) region. In order to keep the linearity of HWN region boundaries and to not remove an informative signal from Raman spectra, a new baseline subtraction procedure has been introduced. After baseline subtraction, the HWN spectrum was deconvoluted using 10 Gaussian functions with individual chemical meanings. The results show that the hydrogen bound water molecule types contributed differently to the water diffusion process in the SC...
October 24, 2016: Analyst
Younes Hanifehpour, Ali Morsali, Behzad Soltani, Babak Mirtamizdoust, Sang Woo Joo
Ultrasound was used to synthesize nano-structures of [Ni(bpzB)2]2(1), a new two-nuclear discrete-coordination compound of divalent nickel with bis-pyrazolyl borate(bpzB). The nanostructure was characterized by scanning electron microscopy, X-ray powder diffraction, infrared, and elemental analysis. The single-crystal X-ray data show that the coordination number of Ni(II) ions is four (Ni1N4 and Ni2N4) with square planar geometry. The supramolecular features in these complexes are guided and controlled by weak directional intermolecular interactions...
January 2017: Ultrasonics Sonochemistry
Janghyun Yoo, Tae-Sun Lee, Byungsan Choi, Min Ju Shon, Tae-Young Yoon
Extremely weak protein-protein interactions (PPIs), signified by micromolar or even millimolar dissociation constants, are one of the keys to understanding the rapid responses of cellular systems. Although single-molecule methods are particularly useful in determining kinetics of biological processes, their application is largely limited to rather strong interactions because of the diffraction-limited observation volume. In this study, we report a single-molecule method that allows the characterization of PPIs using a prey concentration four orders of magnitude lower than the dissociation constant...
October 19, 2016: Journal of the American Chemical Society
Anna Mularski, Jonathan Wilksch, Eric Hanssen, Jian Li, Takehiro Tomita, Sacha James Pidot, Tim Stinear, Frances Separovic, Dick Strugnell
Atomic force microscopy measurements of capsule thickness revealed that that the wild-type Klebsiella pneumoniae AJ218 capsular polysaccharides were rearranged by exposure to colistin. The increase in capsule thickness measured near minimum inhibitory/bactericidal concentration (MIC/MBC) is consistent with the idea that colistin displaces the divalent cations that cross-bridge adjacent lipopolysaccharide (LPS) molecules through the capsule network. Cryo-electron microscopy demonstrated that the measured capsule thickness at near MIC/MBC of 1...
October 17, 2016: European Biophysics Journal: EBJ
Alexey L Chernobrovkin, Roman A Zubarev
While the focus of attempts to classify cell death programs has finally shifted in 2010s from microscopy-based morphological characteristics to biochemical assays, more recent discoveries have put the underlying assumptions of many such assays under severe stress, mostly because of the limited specificity of the assays. On the other hand, proteomics can quantitatively measure the abundances of thousands of proteins in a single experiment. Thus proteomics could develop a modern alternative to both semiquantitative morphology assessment as well as single-molecule biochemical assays...
2016: Cell Death Discovery
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
Jing Liu, Xiangyu Fu, Qiwei Chen, Yajie Zhang, Yongfeng Wang, Dahui Zhao, Wei Chen, Guo Qin Xu, Peilin Liao, Kai Wu
Ordered two-dimensional arrays of silver adatoms with tunable metal atom density stabilized by 1,4-diethynyl-2,5-dimethylbenzene, a terminal alkyne, were prepared on Ag(111) and scrutinized by scanning tunneling microscopy and density functional theory calculations. Stabilization of the adatom arrays was attributed to the substrate-mediated electron localizations of the Ag adatom and terminal alkynyl in the molecule.
October 17, 2016: Chemical Communications: Chem Comm
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
Aida Llauró, Pablo Guerra, Ravi Kant, Brian Bothner, Núria Verdaguer, Pedro J de Pablo
Vault particles are naturally occurring proteinaceous cages with promising application as molecular containers. The use of vaults as functional transporters requires a profound understanding of their structural stability to guarantee the protection and controlled payload delivery. Previous results performed with bulk techniques or at non-physiological conditions have suggested pH as a parameter to control vault dynamics. Here we use Atomic Force Microscopy (AFM) to monitor the structural evolution of individual vault particles while changing the pH in real time...
October 14, 2016: Scientific Reports
Andrés M Vera, Mariano Carrión-Vázquez
Single-molecule force spectroscopy based on atomic force microscopy (AFM-SMFS) has allowed the measurement of the intermolecular forces involved in protein-protein interactions at the molecular level. While intramolecular interactions are routinely identified directly by the use of polyprotein fingerprinting, there is a lack of a general method to directly identify single-molecule intermolecular unbinding events. Here, we have developed an internally controlled strategy to measure protein-protein interactions by AFM-SMFS that allows the direct identification of dissociation force peaks while ensuring single-molecule conditions...
October 13, 2016: Angewandte Chemie
Suman Rana, Neena G Shetake, K C Barick, B N Pandey, H G Salunke, P A Hassan
The interfacial engineering of magnetic nanoparticles (MNPs) with specific functional groups or targeting ligands is important for their in vivo applications. We report here the preparation and characterization of bifunctional magnetic nanoparticles (BMNPs) which contain a carboxylic moiety for drug binding and an amine moiety for folate mediated drug targeting. BMNPs were prepared by introducing bioactive cysteine molecules onto the surface of undecenoic acid coated Fe3O4 magnetic nanoparticles (UMNPs) via a thiol-ene click reaction and then, folic acid was conjugated with these BMNPs through an EDC-NHS coupling reaction...
October 12, 2016: Dalton Transactions: An International Journal of Inorganic Chemistry
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
Chandan K Mishra, A K Sood, Rajesh Ganapathy
The monomer surface mobility is the single most important parameter that decides the nucleation density and morphology of islands during thin-film growth. During template-assisted surface growth in particular, low surface mobilities can prevent monomers from reaching target sites and this results in a partial to complete loss of nucleation control. Whereas in atomic systems a broad range of surface mobilities can be readily accessed, for colloids, owing to their large size, this window is substantially narrow and therefore imposes severe restrictions in extending template-assisted growth techniques to steer their self-assembly...
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
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
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