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Single molecule imaging

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https://www.readbyqxmd.com/read/28820941/a-photoactivatable-probe-for-super-resolution-imaging-of-enzymatic-activity-in-live-cells
#1
Elias A Halabi, Zacharias Thiel, Nils Trapp, Dorothea Pinotsi, Pablo Rivera-Fuentes
A dual-activatable, fluorogenic probe was developed to sense esterase activity with single-molecule resolution. Without enzymatic pre-activation, the diazoindanone-based probe has an electron poor core and, upon irradiation, undergoes Wolff rearrangement to give a ring-expanded xanthene core that is non-emissive. If the probe is pre-activated by carboxylesterases, the tricyclic core becomes electron-rich and the photoinduced Wolff rearrangement produces a highly emissive rhodol dye. Live-cell and solution studies confirmed the selectivity of the probe and revealed that the photoactivated dye does not diffuse away from the original location of activation because the intermediate ketene forms a covalent bond with surrounding macromolecules...
August 18, 2017: Journal of the American Chemical Society
https://www.readbyqxmd.com/read/28819935/plasma-biomarkers-of-inflammation-reflect-seizures-and-hemorrhagic-activity-of-cerebral-cavernous-malformations
#2
Romuald Girard, Hussein A Zeineddine, Maged D Fam, Anoop Mayampurath, Ying Cao, Changbin Shi, Robert Shenkar, Sean P Polster, Michael Jesselson, Ryan Duggan, Abdul-Ghani Mikati, Gregory Christoforidis, Jorge Andrade, Kevin J Whitehead, Dean Y Li, Issam A Awad
The clinical course of cerebral cavernous malformations (CCMs) is highly variable. Based on recent discoveries implicating angiogenic and inflammatory mechanisms, we hypothesized that serum biomarkers might reflect chronic or acute disease activity. This single-site prospective observational cohort study included 85 CCM patients, in whom 24 a priori chosen plasma biomarkers were quantified and analyzed in relation to established clinical and imaging parameters of disease categorization and severity. We subsequently validated the positive correlations in longitudinal follow-up of 49 subjects...
August 17, 2017: Translational Stroke Research
https://www.readbyqxmd.com/read/28819924/dynamic-imaging-of-mitochondrial-membrane-proteins-in-specific-sub-organelle-membrane-locations
#3
REVIEW
Timo Appelhans, Karin B Busch
Mitochondria are cellular organelles with multifaceted tasks and thus composed of different sub-compartments. The inner mitochondrial membrane especially has a complex nano-architecture with cristae protruding into the matrix. Related to their function, the localization of mitochondrial membrane proteins is more or less restricted to specific sub-compartments. In contrast, it can be assumed that membrane proteins per se diffuse unimpeded through continuous membranes. Fluorescence recovery after photobleaching is a versatile technology used in mobility analyses to determine the mobile fraction of proteins, but it cannot provide data on subpopulations or on confined diffusion behavior...
August 17, 2017: Biophysical Reviews
https://www.readbyqxmd.com/read/28815503/imaging-neural-architecture-in-brainbow-samples
#4
Douglas H Roossien, Dawen Cai
The fluorescent protein revolution has made the light microscope the most widely used tool for studying biological structure from the single-molecule to whole organism scales. However, traditional approaches are limited in their ability to resolve components in highly complex structures, such as the brain. In recent years, this limitation has been circumvented by the development of multicolor labeling methods, termed Brainbow. Brainbow tools rely on site-specific recombinases to make stochastic "choices" between different combinations of fluorescent proteins so that structures in close proximity to one another can be resolved based on their color profile...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28814796/crispr-cas9-mediated-labelling-allows-for-single-molecule-imaging-and-resolution
#5
Abdullah O Khan, Victoria A Simms, Jeremy A Pike, Steven G Thomas, Neil V Morgan
Single molecule imaging approaches like dSTORM and PALM resolve structures at 10-20 nm, and allow for unique insights into protein stoichiometry and spatial relationships. However, key obstacles remain in developing highly accurate quantitative single molecule approaches. The genomic tagging of PALM fluorophores through CRISPR-Cas9 offers an excellent opportunity for generating stable cell lines expressing a defined single molecule probe at endogenous levels, without the biological disruption and variability inherent to transfection...
August 16, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28808323/stress-hormone-rapidly-tunes-synaptic-nmda-receptor-through-membrane-dynamics-and-mineralocorticoid-signalling
#6
Lenka Mikasova, Hui Xiong, Amber Kerkhofs, Delphine Bouchet, Harm J Krugers, Laurent Groc
Stress hormones, such as corticosteroids, modulate the transmission of hippocampal glutamatergic synapses and NMDA receptor (NMDAR)-dependent synaptic plasticity, favouring salient behavioural responses to the environment. The corticosterone-induced synaptic adaptations partly rely on changes in NMDAR signalling, although the cellular pathway underlying this effect remains elusive. Here, we demonstrate, using single molecule imaging and electrophysiological approaches in hippocampal neurons, that corticosterone specifically controls GluN2B-NMDAR surface dynamics and synaptic content through mineralocorticoid signalling...
August 14, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28806873/super-resolving-the-actual-position-of-single-fluorescent-molecules-coupled-to-a-plasmonic-nanoantenna
#7
Bing Fu, Benjamin P Isaacoff, Julie S Biteen
Plasmonic nanoparticles (NPs) enhance the radiative decay rate of adjacent dyes and can significantly increase fluorescence intensity for improved spectroscopy. However, the NP nanoantenna complicates super-resolution imaging by introducing a mislocalization between the emitter position and its super-resolved emission position. The mislocalization magnitude depends strongly on the dye/NP coupling geometry. It is therefore crucial to quantify mislocalization to recover the actual emitter position in a coupled system...
August 18, 2017: ACS Nano
https://www.readbyqxmd.com/read/28803781/myc-regulates-chromatin-decompaction-and-nuclear-architecture-during-b-cell-activation
#8
Kyong-Rim Kieffer-Kwon, Keisuke Nimura, Suhas S P Rao, Jianliang Xu, Seolkyoung Jung, Aleksandra Pekowska, Marei Dose, Evan Stevens, Ewy Mathe, Peng Dong, Su-Chen Huang, Maria Aurelia Ricci, Laura Baranello, Ying Zheng, Francesco Tomassoni Ardori, Wolfgang Resch, Diana Stavreva, Steevenson Nelson, Michael McAndrew, Adriel Casellas, Elizabeth Finn, Charles Gregory, Brian Glenn St Hilaire, Steven M Johnson, Wendy Dubois, Maria Pia Cosma, Eric Batchelor, David Levens, Robert D Phair, Tom Misteli, Lino Tessarollo, Gordon Hager, Melike Lakadamyali, Zhe Liu, Monique Floer, Hari Shroff, Erez Lieberman Aiden, Rafael Casellas
50 years ago, Vincent Allfrey and colleagues discovered that lymphocyte activation triggers massive acetylation of chromatin. However, the molecular mechanisms driving epigenetic accessibility are still unknown. We here show that stimulated lymphocytes decondense chromatin by three differentially regulated steps. First, chromatin is repositioned away from the nuclear periphery in response to global acetylation. Second, histone nanodomain clusters decompact into mononucleosome fibers through a mechanism that requires Myc and continual energy input...
August 17, 2017: Molecular Cell
https://www.readbyqxmd.com/read/28799330/imaging-dynamic-collision-and-oxidation-of-single-silver-nanoparticles-at-the-electrode-solution-interface
#9
Rui Hao, Yunshan Fan, Bo Zhang
The electrochemical interface is an ultrathin interfacial region between the electrode surface and the electrolyte solution and is often characterized by numerous dynamic processes, such as solvation and desolvation, heterogeneous electron transfer, molecular adsorption and desorption, diffusion, and surface rearrangement. Many of these processes are driven and modulated by the presence of a large interfacial potential gradient. The study and better understanding of the electrochemical interface is important for designing better electrochemical systems where their applications may include batteries, fuel cells, electrocatalytic water splitting, corrosion protection, and electroplating...
August 11, 2017: Journal of the American Chemical Society
https://www.readbyqxmd.com/read/28798968/stretching-of-surface-tethered-polymers-in-pressure-driven-flow-under-confinement
#10
Tamal Roy, Kai Szuttor, Jens Smiatek, Christian Holm, Steffen Hardt
We study the effect of pressure-driven flow on a single surface-tethered DNA molecule confined between parallel surfaces. The influence of flow and channel parameters as well as the length of the molecules on their extension and orientation is explored. In the experiments the chain conformations are imaged by laser scanning confocal microscopy. We find that the fractional extension of the tethered DNA molecules mainly depends on the wall shear stress, with effects of confinement being very weak. Experiments performed with molecules of different contour length show that the fractional extension is a universal function of the product of the wall shear stress and the contour length, a result that can be obtained from a simple scaling relation...
August 11, 2017: Soft Matter
https://www.readbyqxmd.com/read/28798045/global-mrna-polarization-regulates-translation-efficiency-in-the-intestinal-epithelium
#11
Andreas E Moor, Matan Golan, Efi E Massasa, Doron Lemze, Tomer Weizman, Rom Shenhav, Shaked Baydatch, Orel Mizrahi, Roni Winkler, Ofra Golani, Noam Stern-Ginossar, Shalev Itzkovitz
Asymmetric mRNA localization facilitates efficient translation in cells such as neurons and fibroblasts. However, the extent and significance of mRNA polarization in epithelial tissues are unclear. Here, we used single molecule transcript imaging and subcellular transcriptomics to uncover global apical-basal intracellular polarization of mRNA in the mouse intestinal epithelium. The localization of mRNAs did not generally overlap protein localization. Instead, ribosomes were more abundant on the apical sides, and apical transcripts were consequently more efficiently translated...
August 10, 2017: Science
https://www.readbyqxmd.com/read/28792749/hide-probes-a-new-toolkit-for-visualizing-organelle-dynamics-longer-and-at-super-resolution
#12
Alexander D Thompson, Joerg Bewersdorf, Derek Toomre, Alanna Schepartz
Living cells are complex and dynamic assemblies that carefully sequester and orchestrate multiple diverse processes that enable growth, division, regulation, movement, and communication. Membrane-bound organelles such as the endoplasmic reticulum, mitochondria, plasma membrane, and others are integral to these processes, and their functions demand dynamic reorganization in both space and time. Visualizing these dynamics in live cells over long time periods demands probes that label discrete organelles specifically, at high density, and withstand long-term irradiation...
August 9, 2017: Biochemistry
https://www.readbyqxmd.com/read/28792742/sequential-release-of-proteins-from-structured-multishell-microcapsules
#13
Ulyana Shimanovich, Thomas C T Michaels, Erwin J de Genst, Dijana Matak-Vinkovic, Christopher M Dobson, Tuomas P J Knowles
In nature, a wide range of functional materials is based on proteins. Increasing attention is also turning to the use of proteins as artificial biomaterials in the form of films, gels, particles and fibrils that offer great potential for applications in areas ranging from molecular medicine to materials science. To date, however, most such applications have been limited to single component materials despite the fact that their natural analogues are composed of multiple types of proteins with a variety of functionalities that are co-assembled in a highly organized manner on the micron scale, a process which is currently challenging to achieve in the laboratory...
August 9, 2017: Biomacromolecules
https://www.readbyqxmd.com/read/28789884/single-molecule-imaging-and-manipulation-of-biomolecular-machines-and-systems
#14
REVIEW
Ryota Iino, Tatsuya Iida, Akihiko Nakamura, Ei-Ichiro Saita, Huijuan You, Yasushi Sako
Biological molecular machines support various activities and behaviors of cells, such as energy production, signal transduction, growth, differentiation, and migration. Scope of Review We provide an overview of single-molecule imaging methods involving both small and large probes used to monitor the dynamic motions of molecular machines in vitro (purified proteins) and in living cells, and single-molecule manipulation methods used to measure the forces, mechanical properties and responses of biomolecules. We also introduce several examples of single-molecule analysis, focusing primarily on motor proteins and signal transduction systems...
August 5, 2017: Biochimica et Biophysica Acta
https://www.readbyqxmd.com/read/28782052/real-time-sensing-of-single-ligand-delivery-with-nanoaperture-integrated-microfluidic-devices
#15
W Elliott Martin, Ning Ge, Bernadeta R Srijanto, Emily Furnish, C Patrick Collier, Christine A Trinkle, Christopher I Richards
The measurement of biological events on the surface of live cells at the single-molecule level is complicated by several factors including high protein densities that are incompatible with single-molecule imaging, cellular autofluorescence, and protein mobility on the cell surface. Here, we fabricated a device composed of an array of nanoscale apertures coupled with a microfluidic delivery system to quantify single-ligand interactions with proteins on the cell surface. We cultured live cells directly on the device and isolated individual epidermal growth factor receptors (EGFRs) in the apertures while delivering fluorescently labeled epidermal growth factor...
July 31, 2017: ACS Omega
https://www.readbyqxmd.com/read/28782000/particle-segmentation-algorithm-for-flexible-single-particle-reconstruction
#16
Qiang Zhou, Niyun Zhou, Hong-Wei Wang
As single particle cryo-electron microscopy has evolved to a new era of atomic resolution, sample heterogeneity still imposes a major limit to the resolution of many macromolecular complexes, especially those with continuous conformational flexibility. Here, we describe a particle segmentation algorithm towards solving structures of molecules composed of several parts that are relatively flexible with each other. In this algorithm, the different parts of a target molecule are segmented from raw images according to their alignment information obtained from a preliminary 3D reconstruction and are subjected to single particle processing in an iterative manner...
2017: Biophysics Reports
https://www.readbyqxmd.com/read/28780967/cell-and-single-molecule-based-methods-to-detect-anti-n-methyl-d-aspartate-receptor-autoantibodies-in-patients-with-first-episode-psychosis-from-the-optimise-project
#17
Julie Jézéquel, Véronique Rogemond, Thomas Pollak, Marilyn Lepleux, Leslie Jacobson, Hélène Gréa, Conrad Iyegbe, Rene Kahn, Philip McGuire, Angela Vincent, Jérôme Honnorat, Marion Leboyer, Laurent Groc
Circulating autoantibodies against glutamatergic N-methyl-D-aspartate receptor (NMDAR) have been reported in a proportion of patients with psychotic disorders, raising hopes for more appropriate treatment for these antibody-positive patients. However, the prevalence of circulating autoantibodies against glutamatergic NMDAR in psychotic disorders remains controversial, with detection prevalence rates and immunoglobulin classes varying considerably between studies, perhaps because of different detection methods...
July 6, 2017: Biological Psychiatry
https://www.readbyqxmd.com/read/28779837/single-molecule-fluorescence-studies-of-membrane-transporters-using-total-internal-reflection-microscopy
#18
Joris M H Goudsmits, Antoine M van Oijen, Dirk J Slotboom
Cells are delineated by a lipid bilayer that physically separates the inside from the outer environment. Most polar, charged, or large molecules require proteins to reduce the energetic barrier for passage across the membrane and to achieve transport rates that are relevant for life. Here, we describe techniques to visualize the functioning of membrane transport proteins with fluorescent probes at the single-molecule level. First, we explain how to produce membrane-reconstituted transporters with fluorescent labels...
2017: Methods in Enzymology
https://www.readbyqxmd.com/read/28777580/a-first-step-towards-a-universal-fluorescent-probe-unravelling-the-photodynamics-of-an-amino-maleimide-fluorophore
#19
Michael Staniforth, Wen-Dong Quan, Tolga N V Karsili, Lewis A Baker, Rachel K O'Reilly, Vasilios G Stavros
Continuous advancements in biophysics and medicine at the molecular level make the requirements to image structure-function processes in living cells ever more acute. While fluorophores such as the green fluorescent protein have proven instrumental towards such efforts, the advent of non-diffraction limited microscopy limits the utility of such fluorescent tags. Monoaminomaleimides are small, single molecule fluorophores that have been shown to possess stark variations in their emission spectra in different solvent environments, making them a potentially powerful tool for myriad applications...
August 4, 2017: Journal of Physical Chemistry. A
https://www.readbyqxmd.com/read/28777539/stoichiometry-and-dispersity-of-dna-nanostructures-using-photobleaching-pair-correlation-analysis
#20
Amani A Hariri, Graham D Hamblin, Jack S Hardwick, Robert Godin, Jean-Francois Desjardins, Paul W Wiseman, Hanadi F Sleiman, Gonzalo Cosa
A wide variety of approaches have become available for fabricating nanomaterials with increasing degrees of complexity, precision, and speed while minimizing cost. Their quantitative characterization however remains a challenge. Analytical methods to better inspect and validate the structure/composition of large nanoscale objects are required to optimize their applications in diverse technologies. Here we describe single molecule fluorescence-based strategies relying on photobleaching and multiple color colocalization features toward the characterization of supramolecular structures...
August 4, 2017: Bioconjugate Chemistry
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