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Single molecule imaging

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https://www.readbyqxmd.com/read/28317888/direct-evidence-of-lack-of-colocalisation-of-fluorescently-labelled-gold-labels-used-in-correlative-light-electron-microscopy
#1
Benjamin T Miles, Alexander B Greenwood, David Benito-Alifonso, Hugh Tanner, M Carmen Galan, Paul Verkade, Henkjan Gersen
Fluorescently labelled nanoparticles are routinely used in Correlative Light Electron Microscopy (CLEM) to combine the capabilities of two separate microscope platforms: fluorescent light microscopy (LM) and electron microscopy (EM). The inherent assumption is that the fluorescent label observed under LM colocalises well with the electron dense nanoparticle observed in EM. Herein we show, by combining single molecule fluorescent imaging with optical detection of the scattering from single gold nanoparticles, that for a commercially produced sample of 10 nm gold nanoparticles tagged to Alexa-633 there is in fact no colocalisation between the fluorescent signatures of Alexa-633 and the scattering associated with the gold nanoparticle...
March 20, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28315485/quantifying-transcription-factor-binding-dynamics-at-the-single-molecule-level-in-live-cells
#2
Diego M Presman, David A Ball, Ville Paakinaho, Jonathan B Grimm, Luke D Lavis, Tatiana S Karpova, Gordon L Hager
Progressive, technological achievements in the quantitative fluorescence microscopy field are allowing researches from many different areas to start unraveling the dynamic intricacies of biological processes inside living cells. From super-resolution microscopy techniques to tracking of individual proteins, fluorescence microscopy is changing our perspective on how the cell works. Fortunately, a growing number of research groups are exploring single-molecule studies in living cells. However, no clear consensus exists on several key aspects of the technique such as image acquisition conditions, or analysis of the obtained data...
March 14, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28314119/a-combined-in-vitro-assay-for-evaluation-of-neurotrophic-activity-and-cytotoxicity
#3
Narayan D Chaurasiya, Surabhi Shukla, Babu L Tekwani
Neurotrophic assays are phenotypic methods to identify molecules that stimulate differentiation of neuronal cells. Bioactive small molecules with neurotrophic actions hold great promise as therapeutic agents for the treatment of neurodegenerative diseases and neuronal injuries by virtue of their ability to stimulate neuritic outgrowth. A combined in vitro method, which measures neurotrophic activity and cytotoxicity in a single assay, has been described. This assay, performed in 96-well microplates with PC12 and Neuroscreen-1 (NS-1; a subclone of PC12) cells, is a simple tool for identification of new neurotrophic agents...
March 1, 2017: SLAS Discov
https://www.readbyqxmd.com/read/28306243/optical-super-resolution-imaging-of-surface-reactions
#4
Tao Chen, Bin Dong, Kuangcai Chen, Fei Zhao, Xiaodong Cheng, Changbei Ma, Seungah Lee, Peng Zhang, Seong Ho Kang, Ji Won Ha, Weilin Xu, Ning Fang
Optical super-resolution imaging has gained momentum in investigations of heterogeneous and homogeneous chemical reactions at the single-molecule level. Thanks to its exceptional spatial resolution and ability to monitor dynamic systems, much detailed information on single-molecule reaction/adsorption processes and single-particle catalytic processes has been revealed, including chemical kinetics and reaction dynamics; active-site distributions on single-particle surfaces; and size-, shape-, and facet-dependent catalytic activities of individual nanocatalysts...
March 17, 2017: Chemical Reviews
https://www.readbyqxmd.com/read/28303166/single-molecule-studies-of-high-mobility-group-b-architectural-dna-bending-proteins
#5
REVIEW
Divakaran Murugesapillai, Micah J McCauley, L James Maher, Mark C Williams
Protein-DNA interactions can be characterized and quantified using single molecule methods such as optical tweezers, magnetic tweezers, atomic force microscopy, and fluorescence imaging. In this review, we discuss studies that characterize the binding of high-mobility group B (HMGB) architectural proteins to single DNA molecules. We show how these studies are able to extract quantitative information regarding equilibrium binding as well as non-equilibrium binding kinetics. HMGB proteins play critical but poorly understood roles in cellular function...
February 2017: Biophysical Reviews
https://www.readbyqxmd.com/read/28299937/non-covalent-protein-and-peptide-functionalization-of-single-walled-carbon-nanotubes-for-bio-delivery-and-optical-sensing-applications
#6
Alessandra Antonucci, Justyna Kupis-Rozmyslowicz, Ardemis Boghossian
The exquisite structural and optical characteristics of single-walled carbon nanotubes (SWCNTs), combined with the tunable specificities of proteins and peptides, can be exploited to strongly benefit technologies with applications in fields ranging from biomedicine to industrial biocatalysis. The key to exploiting the synergism of these materials is designing protein/peptide-SWCNT conjugation schemes that preserve biomolecule activity while keeping the near-infrared optical and electronic properties of SWCNTs intact...
March 16, 2017: ACS Applied Materials & Interfaces
https://www.readbyqxmd.com/read/28299282/extracellular-proton-concentrations-impacts-ln229-glioblastoma-tumor-cell-fate-via-differential-modulation-of-surface-lipids
#7
Sebastian John, K C Sivakumar, Rashmi Mishra
BACKGROUND: Glioblastoma multiforme (GBM) is a highly aggressive form of brain cancer with marginal survival rates. GBM extracellular acidosis can profoundly impact its cell fate heterogeneities and progression. However, the molecules and mechanisms that enable GBM tumor cells acid adaptation and consequent cell fate competencies are weakly understood. Since extracellular proton concentrations (pHe) directly intercept the tumor cell plasma membrane, surface lipids must play a crucial role in pHe-dependent tumor cell fate dynamics...
2017: Frontiers in Oncology
https://www.readbyqxmd.com/read/28295806/proteins-mediating-dna-loops-effectively-block-transcription
#8
Zsuzsanna Vörös, Yan Yan, Daniel T Kovari, Laura Finzi, David Dunlap
Loops are ubiquitous topological elements formed when proteins simultaneously bind to two non-contiguous DNA sites. While a loop-mediating protein may regulate initiation at a promoter, the presence of the protein at the other site may be an obstacle for RNA polymerases (RNAP) transcribing a different gene. To test whether a DNA loop alters the extent to which a protein blocks transcription, the lac repressor (LacI) was used. The outcome of in vitro transcription along templates containing two LacI operators separated by 400 bp in the presence of LacI concentrations that produced both looped and unlooped molecules was visualized with scanning force microscopy (SFM)...
March 14, 2017: Protein Science: a Publication of the Protein Society
https://www.readbyqxmd.com/read/28295739/gold-and-hairpin-dna-functionalization-of-upconversion-nanocrystals-for-imaging-and-in-vivo-drug-delivery
#9
Sanyang Han, Animesh Samanta, Xiaoji Xie, Ling Huang, Juanjuan Peng, Sung Jin Park, Daniel Boon Loong Teh, Yongdoo Choi, Young-Tae Chang, Angelo Homayoun All, Yanmei Yang, Bengang Xing, Xiaogang Liu
Although multifunctional upconversion imaging probes have recently attracted considerable interest in biomedical research, there are currently few methods for stabilizing these luminescent nanoprobes with oligonucleotides in biological systems. Herein, a method to robustly disperse upconversion nanoprobes in physiological buffers based on rational design and synthesis of nanoconjugates comprising hairpin-DNA-modified gold nanoparticles is presented. This approach imparts the upconversion nanoprobes with excellent biocompatibility and circumvents the problem of particle agglomeration...
March 10, 2017: Advanced Materials
https://www.readbyqxmd.com/read/28295057/high-resolution-phenotypic-profiling-of-natural-products-induced-effects-on-the-single-cell-level
#10
Stephan Kremb, Christian R Voolstra
Natural products (NPs) are highly evolved molecules making them a valuable resource for new therapeutics. Here we demonstrate the usefulness of broad-spectrum phenotypic profiling of NP-induced perturbations on single cells with imaging-based High-Content Screening to inform on physiology, mechanisms-of-actions, and multi-level toxicity. Our technology platform aims at broad applicability using a comprehensive marker panel with standardized settings streamlined towards an easy implementation in laboratories dedicated to natural products research...
March 15, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28290531/mapping-cell-surface-adhesion-by-rotation-tracking-and-adhesion-footprinting
#11
Isaac T S Li, Taekjip Ha, Yann R Chemla
Rolling adhesion, in which cells passively roll along surfaces under shear flow, is a critical process involved in inflammatory responses and cancer metastasis. Surface adhesion properties regulated by adhesion receptors and membrane tethers are critical in understanding cell rolling behavior. Locally, adhesion molecules are distributed at the tips of membrane tethers. However, how functional adhesion properties are globally distributed on the individual cell's surface is unknown. Here, we developed a label-free technique to determine the spatial distribution of adhesive properties on rolling cell surfaces...
March 14, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28289540/bfptool-a-software-tool-for-analysis-of-biomembrane-force-probe-experiments
#12
Daniel Šmít, Coralie Fouquet, Mohamed Doulazmi, Frédéric Pincet, Alain Trembleau, Martin Zapotocky
BACKGROUND: The Biomembrane Force Probe is an approachable experimental technique commonly used for single-molecule force spectroscopy and experiments on biological interfaces. The technique operates in the range of forces from 0.1 pN to 1000 pN. Experiments are typically repeated many times, conditions are often not optimal, the captured video can be unstable and lose focus; this makes efficient analysis challenging, while out-of-the-box non-proprietary solutions are not freely available...
2017: BMC Biophysics
https://www.readbyqxmd.com/read/28287710/single-molecule-localization-microscopy-in-eukaryotes
#13
Markus Sauer, Mike Heilemann
Super-resolution fluorescence imaging by photoactivation or photoswitching of single fluorophores and position determination (single-molecule localization microscopy, SMLM) provides microscopic images with subdiffraction spatial resolution. This technology has enabled new insights into how proteins are organized in a cellular context, with a spatial resolution approaching virtually the molecular level. A unique strength of SMLM is that it delivers molecule-resolved information, along with super-resolved images of cellular structures...
March 13, 2017: Chemical Reviews
https://www.readbyqxmd.com/read/28287587/analyzing-synaptic-modulation-of-drosophila-melanogaster-photoreceptors-after-exposure-to-prolonged-light
#14
Atsushi Sugie, Christoph Möhl, Satoko Hakeda-Suzuki, Hideaki Matsui, Takashi Suzuki, Gaia Tavosanis
The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at the synaptic level. The Active Zone (AZ) is the region at the presynaptic membrane that mediates neurotransmitter release and is composed of a dense collection of scaffold proteins. AZs of Drosophila melanogaster (Drosophila) photoreceptors undergo molecular remodeling after prolonged exposure to natural ambient light. Thus the level of neuronal activity can rearrange the molecular composition of the AZ and contribute to the regulation of the functional output...
February 10, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28276025/single-molecule-tracking-and-localization-of-mitochondrial-protein-complexes-in-live-cells
#15
Timo Appelhans, Karin Busch
Mitochondria are the power plant of most non-green eukaryotic cells. An understanding of their function and regulation is only possible with the knowledge of the spatiotemporal dynamics of their proteins. Mitochondrial membrane proteins involved in diverse functions like protein import, cell respiration, metabolite transport, and mitochondrial morphology are mobile within membranes. Here, we provide a protocol for a superresolution fluorescence microscopy technique named tracking and localization microscopy (TALM) that allows for localization and diffusion analysis of single mitochondrial membrane proteins in situ in cell cultures...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28267259/the-axonal-transport-motor-kinesin-2-navigates-microtubule-obstacles-via-protofilament-switching
#16
Gregory J Hoeprich, Keith J Mickolajczyk, Shane R Nelson, William O Hancock, Christopher L Berger
Axonal transport involves kinesin motors trafficking cargo along microtubules that are rich in microtubule-associated proteins (MAPs). Much attention has focused on the behavior of kinesin-1 in the presence of MAPs, which has overshadowed understanding the contribution of other kinesins such as kinesin-2 in axonal transport. We have previously shown that, unlike kinesin-1, kinesin-2 in vitro motility is insensitive to the neuronal MAP Tau. However, the mechanism by which kinesin-2 efficiently navigates Tau on the microtubule surface is unknown...
March 7, 2017: Traffic
https://www.readbyqxmd.com/read/28263962/atomic-scale-sensing-of-the-magnetic-dipolar-field-from-single-atoms
#17
Taeyoung Choi, William Paul, Steffen Rolf-Pissarczyk, Andrew J Macdonald, Fabian D Natterer, Kai Yang, Philip Willke, Christopher P Lutz, Andreas J Heinrich
Spin resonance provides the high-energy resolution needed to determine biological and material structures by sensing weak magnetic interactions. In recent years, there have been notable achievements in detecting and coherently controlling individual atomic-scale spin centres for sensitive local magnetometry. However, positioning the spin sensor and characterizing spin-spin interactions with sub-nanometre precision have remained outstanding challenges. Here, we use individual Fe atoms as an electron spin resonance (ESR) sensor in a scanning tunnelling microscope to measure the magnetic field emanating from nearby spins with atomic-scale precision...
March 6, 2017: Nature Nanotechnology
https://www.readbyqxmd.com/read/28261749/intra-image-referencing-for-simplified-assessment-of-her2-expression-in-breast-cancer-metastases-using-the-affibody-molecule-aby-025-with-pet-and-spect
#18
Dan Sandberg, Vladimir Tolmachev, Irina Velikyan, Helena Olofsson, Anders Wennborg, Joachim Feldwisch, Jörgen Carlsson, Henrik Lindman, Jens Sörensen
PURPOSE: In phase I/II-studies radiolabelled ABY-025 Affibody molecules identified human epidermal growth factor receptor 2 (HER2) expression in breast cancer metastases using PET and SPECT imaging. Here, we wanted to investigate the utility of a simple intra-image normalization using tumour-to-reference tissue-ratio (T/R) as a HER2 status discrimination strategy to overcome potential issues related to cross-calibration of scanning devices. METHODS: Twenty-three women with pre-diagnosed HER2-positive/negative metastasized breast cancer were scanned with [(111)In]-ABY-025 SPECT/CT (n = 7) or [(68)Ga]-ABY-025 PET/CT (n = 16)...
March 6, 2017: European Journal of Nuclear Medicine and Molecular Imaging
https://www.readbyqxmd.com/read/28260612/monitoring-a-simple-hydrolysis-process-in-an-organic-solid-by-observing-methyl-group-rotation
#19
Peter A Beckmann, Joseph M Bohen, Jamie Ford, William P Malachowski, Clelia W Mallory, Frank B Mallory, Andrew R McGhie, Arnold L Rheingold, Gilbert J Sloan, Steven T Szewczyk, Xianlong Wang, Kraig A Wheeler
We report a variety of experiments and calculations and their interpretations regarding methyl group (CH3) rotation in samples of pure 3-methylglutaric anhydride (1), pure 3-methylglutaric acid (2), and samples where the anhydride is slowly absorbing water from the air and converting to the acid [C6H8O3(1) + H2O → C6H10O4(2)]. The techniques are solid state (1)H nuclear magnetic resonance (NMR) spin-lattice relaxation, single-crystal X-ray diffraction, electronic structure calculations in both isolated molecules and in clusters of molecules that mimic the crystal structure, field emission scanning electron microscopy, differential scanning calorimetry, and high resolution (1)H NMR spectroscopy...
January 29, 2017: Solid State Nuclear Magnetic Resonance
https://www.readbyqxmd.com/read/28258033/rnp-transport-in-cell-biology-the-long-and-winding-road
#20
REVIEW
Carolina Eliscovich, Robert H Singer
Regulation of gene expression is key determinant to cell structure and function. RNA localization, where specific mRNAs are transported to subcellular regions and then translated, is highly conserved in eukaryotes ranging from yeast to extremely specialized and polarized cells such as neurons. Messenger RNA and associated proteins (mRNP) move from the site of transcription in the nucleus to their final destination in the cytoplasm both passively through diffusion and actively via directed transport. Dysfunction of RNA localization, transport and translation machinery can lead to pathology...
February 28, 2017: Current Opinion in Cell Biology
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