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https://www.readbyqxmd.com/read/28900238/limitations-of-qdot-labelling-compared-to-directly-conjugated-probes-for-single-particle-tracking-of-b-cell-receptor-mobility
#1
Libin Abraham, Henry Y Lu, Rebeca Cardim Falcão, Joshua Scurll, Timothy Jou, Brian Irwin, Reza Tafteh, Michael R Gold, Daniel Coombs
Single-particle tracking (SPT) is a powerful method for exploring single-molecule dynamics in living cells with nanoscale spatiotemporal resolution. Photostability and bright fluorescence make quantum dots (Qdots) a popular choice for SPT. However, their large size could potentially alter the mobility of the molecule of interest. To test this, we labelled B cell receptors on the surface of B-lymphocytes with monovalent Fab fragments of antibodies that were either linked to Qdots via streptavidin or directly conjugated to the small organic fluorophore Cy3...
September 12, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28875184/applications-of-vibrational-tags-in-biological-imaging-by-raman-microscopy
#2
REVIEW
Zhilun Zhao, Yihui Shen, Fanghao Hu, Wei Min
As a superb tool to visualize and study the spatial-temporal distribution of chemicals, Raman microscopy has made a big impact in many disciplines of science. While label-free imaging has been the prevailing strategy in Raman microscopy, recent development and applications of vibrational/Raman tags, particularly when coupled with stimulated Raman scattering (SRS) microscopy, have generated intense excitement in biomedical imaging. SRS imaging of vibrational tags has enabled researchers to study a wide range of small biomolecules with high specificity, sensitivity and multiplex capability, at a single live cell level, tissue level or even in vivo...
September 6, 2017: Analyst
https://www.readbyqxmd.com/read/28869217/single-molecule-techniques-in-biophysics-a-review-of-the-progress-in-methods-and-applications
#3
Helen Miller, Zhaokun Zhou, Jack Shepherd, Adam Wollman, Mark Leake
Single-molecule biophysics has transformed our understanding of biology, but also of the physics of life. More exotic than simple soft matter, biomatter lives far from thermal equilibrium, covering multiple lengths from the nanoscale of single molecules to up several orders of magnitude to higher in cells, tissues and organisms. Biomolecules are often characterized by underlying instability: multiple metastable free energy states exist, separated by levels of just a few multiples of the thermal energy scale kBT, where kB is the Boltzmann constant and T absolute temperature, implying complex inter-conversion kinetics in the relatively hot, wet environment of active biological matter...
September 4, 2017: Reports on Progress in Physics
https://www.readbyqxmd.com/read/28855374/differential-mast-cell-outcomes-are-sensitive-to-fc%C3%AE%C2%B5ri-syk-binding-kinetics
#4
Samantha L Schwartz, Cédric Cleyrat, Mark J Olah, Peter K Relich, Genevieve K Phillips, William S Hlavacek, Keith A Lidke, Bridget S Wilson, Diane S Lidke
Crosslinking of IgE-bound FcεRI triggers multiple cellular responses, including degranulation and cytokine production. Signaling is dependent on recruitment of Syk via docking of its dual SH2 domains to phosphorylated tyrosines within the FcεRI immunoreceptor tyrosine-based activation motifs. Using single molecule imaging in live cells, we directly visualized and quantified the binding of individual mNeonGreen-tagged Syk molecules as they associated with the plasma membrane after FcεRI activation. We found that Syk colocalizes transiently to FcεRI and that Syk-FcεRI binding dynamics are independent of receptor aggregate size...
August 30, 2017: Molecular Biology of the Cell
https://www.readbyqxmd.com/read/28820941/a-photoactivatable-probe-for-super-resolution-imaging-of-enzymatic-activity-in-live-cells
#5
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 nonemissive. 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...
September 5, 2017: Journal of the American Chemical Society
https://www.readbyqxmd.com/read/28819924/dynamic-imaging-of-mitochondrial-membrane-proteins-in-specific-sub-organelle-membrane-locations
#6
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/28814796/crispr-cas9-mediated-labelling-allows-for-single-molecule-imaging-and-resolution
#7
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/28792749/hide-probes-a-new-toolkit-for-visualizing-organelle-dynamics-longer-and-at-super-resolution
#8
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/28789884/single-molecule-imaging-and-manipulation-of-biomolecular-machines-and-systems
#9
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
#10
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/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
#11
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/28777580/first-step-toward-a-universal-fluorescent-probe-unravelling-the-photodynamics-of-an-amino-maleimide-fluorophore
#12
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 toward such efforts, the advent of nondiffraction 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 a myriad of applications...
August 23, 2017: Journal of Physical Chemistry. A
https://www.readbyqxmd.com/read/28765585/multi-color-single-molecule-tracking-and-subtrajectory-analysis-for-quantification-of-spatiotemporal-dynamics-and-kinetics-upon-t-cell-activation
#13
Yuma Ito, Kumiko Sakata-Sogawa, Makio Tokunaga
The dynamic properties of molecules in living cells are attracting increasing interest. We propose a new method, moving subtrajectory analysis using single-molecule tracking, and demonstrate its utility in the spatiotemporal quantification of not only dynamics but also the kinetics of interactions using single-color images. Combining this technique with three-color simultaneous single-molecule imaging, we quantified the dynamics and kinetics of molecules in spatial relation to T cell receptor (TCR) microclusters, which trigger TCR signaling...
August 1, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28741941/efficient-delivery-of-quantum-dots-into-the-cytosol-of-cells-using-cell-penetrating-poly-disulfide-s
#14
Emmanuel Derivery, Eline Bartolami, Stefan Matile, Marcos Gonzalez-Gaitan
Quantum dots (QDs) are extremely bright, photostable, nanometer particles broadly used to investigate single molecule dynamics in vitro. However, the use of QDs in vivo to investigate single molecule dynamics is impaired by the absence of an efficient way to chemically deliver them into the cytosol of cells. Indeed, current methods (using cell-penetrating peptides for instance) provide very low yields: QDs stay at the plasma membrane or are trapped in endosomes. Here, we introduce a technology based on cell-penetrating poly(disulfide)s that solves this problem: we deliver about 70 QDs per cell, and 90% appear to freely diffuse in the cytosol...
August 2, 2017: Journal of the American Chemical Society
https://www.readbyqxmd.com/read/28734966/development-of-new-ganglioside-probes-and-unraveling-of-raft-domain-structure-by-single-molecule-imaging
#15
REVIEW
Kenichi G N Suzuki, Hiromune Ando, Naoko Komura, Takahiro K Fujiwara, Makoto Kiso, Akihiro Kusumi
Gangliosides are involved in a variety of biological roles and are a component of lipid rafts found in cell plasma membranes (PMs). Gangliosides are especially abundant in neuronal PMs and are essential to their physiological functions. However, the dynamic behaviors of gangliosides have not been investigated in living cells due to a lack of fluorescent probes that behave like their parental molecules. We have recently developed, using an entirely chemical method, four new ganglioside probes (GM1, GM2, GM3, and GD1b) that act similarly to their parental molecules in terms of raft partitioning and binding affinity...
October 2017: Biochimica et Biophysica Acta
https://www.readbyqxmd.com/read/28722423/generation-of-a-cgmp-indicator-with-an-expanded-dynamic-range-by-optimization-of-amino-acid-linkers-between-a-fluorescent-protein-and-pde5%C3%AE
#16
Shogo Matsuda, Kazuki Harada, Motoki Ito, Mai Takizawa, Devina Wongso, Takashi Tsuboi, Tetsuya Kitaguchi
Here we describe the development of a single fluorescent protein (FP)-based cGMP indicator, Green cGull, based on the cGMP binding domain from mouse phosphodiesterase 5α. The dynamic range of Green cGull was enhanced to a 7.5-fold fluorescence change upon cGMP binding by optimization of the amino acid linkers between the cGMP binding domain and FP. Green cGull has excitation and emission peaks at 498 and 522 nm, respectively, and specifically responds to cGMP in a dose-dependent manner. Live cell imaging analysis revealed that addition of a nitric oxide (NO) donor induced different cGMP kinetics and was cell-type dependent...
January 27, 2017: ACS Sensors
https://www.readbyqxmd.com/read/28716648/applications-of-high-speed-atomic-force-microscopy-to-real-time-visualization-of-dynamic-biomolecular-processes
#17
REVIEW
Takayuki Uchihashi, Simon Scheuring
BACKGROUND: Many biological processes in a living cell are consequences of sequential and hierarchical dynamic events of biological macromolecules such as molecular interactions and conformational changes. Hence, knowledge of structures, assembly and dynamics of proteins is the foundation for understanding how biological molecules work. Among several techniques to analyze dynamics of proteins, high-speed atomic force microscopy (HS-AFM) is unique to provide direct information about both structure and dynamics of single proteins at work...
July 14, 2017: Biochimica et Biophysica Acta
https://www.readbyqxmd.com/read/28710754/the-fast-halo-assay-for-the-detection-of-dna-damage
#18
Piero Sestili, Cinzia Calcabrini, Anna Rita Diaz, Carmela Fimognari, Vilberto Stocchi
The need for express screening of the DNA damaging potential of chemicals has progressively increased over the past 20 years due to the wide number of new synthetic molecules to be evaluated, as well as the adoption of more stringent chemical regulations such as the EU REACH and risk reduction politics. In this regard, DNA diffusion assays such as the microelectrophoretic comet assay paved the way for rapid genotoxicity testing. A more significant simplification and speeding up of the experimental processes was achieved with the fast halo assay (FHA) described in the present chapter...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28708098/combining-primed-photoconversion-and-uv-photoactivation-for-aberration-free-live-cell-compliant-multi-color-single-molecule-localization-microscopy-imaging
#19
David Virant, Bartosz Turkowyd, Alexander Balinovic, Ulrike Endesfelder
Super-resolution fluorescence microscopy plays a major role in revealing the organization and dynamics of living cells. Nevertheless, single-molecule localization microscopy imaging of multiple targets is still limited by the availability of suitable fluorophore combinations. Here, we introduce a novel imaging strategy which combines primed photoconversion (PC) and UV-photoactivation for imaging different molecular species tagged by suitable fluorescent protein combinations. In this approach, the fluorescent proteins can be specifically photoactivated/-converted by different light wavelengths using PC and UV-activation modes but emit fluorescence in the same spectral emission channel...
July 14, 2017: International Journal of Molecular Sciences
https://www.readbyqxmd.com/read/28694303/single-molecule-fluorescence-microscopy-review-shedding-new-light-on-old-problems
#20
REVIEW
Sviatlana Shashkova, Mark C Leake
Fluorescence microscopy is an invaluable tool in the biosciences, a genuine workhorse technique offering exceptional contrast in conjunction with high specificity of labelling with relatively minimal perturbation to biological samples compared with many competing biophysical techniques. Improvements in detector and dye technologies coupled to advances in image analysis methods have fuelled recent development towards single-molecule fluorescence microscopy, which can utilize light microscopy tools to enable the faithful detection and analysis of single fluorescent molecules used as reporter tags in biological samples...
August 31, 2017: Bioscience Reports
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