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live cell single molecule imaging

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https://www.readbyqxmd.com/read/28792749/hide-probes-a-new-toolkit-for-visualizing-organelle-dynamics-longer-and-at-super-resolution
#1
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
#2
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
#3
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
#4
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/a-first-step-towards-a-universal-fluorescent-probe-unravelling-the-photodynamics-of-an-amino-maleimide-fluorophore
#5
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/28765585/multi-color-single-molecule-tracking-and-subtrajectory-analysis-for-quantification-of-spatiotemporal-dynamics-and-kinetics-upon-t-cell-activation
#6
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
#7
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
#8
REVIEW
Kenichi G N Suzuki, Hiromune Ando, Naoko Komura, Takahiro 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...
July 19, 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
#9
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
#10
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
#11
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
#12
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
#13
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 to 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 utilise light microscopy tools to enable the faithful detection and analysis of single fluorescent molecules used as reporter tags in biological samples...
July 10, 2017: Bioscience Reports
https://www.readbyqxmd.com/read/28690601/illuminating-messengers-an-update-and-outlook-on-rna-visualization-in-bacteria
#14
REVIEW
Lieke A van Gijtenbeek, Jan Kok
To be able to visualize the abundance and spatiotemporal features of RNAs in bacterial cells would permit obtaining a pivotal understanding of many mechanisms underlying bacterial cell biology. The first methods that allowed observing single mRNA molecules in individual cells were introduced by Bertrand et al. (1998) and Femino et al. (1998). Since then, a plethora of techniques to image RNA molecules with the aid of fluorescence microscopy has emerged. Many of these approaches are useful for the large eukaryotic cells but their adaptation to study RNA, specifically mRNA molecules, in bacterial cells progressed relatively slow...
2017: Frontiers in Microbiology
https://www.readbyqxmd.com/read/28684722/overview-of-single-molecule-speckle-sims-microscopy-and-its-electroporation-based-version-with-efficient-labeling-and-improved-spatiotemporal-resolution
#15
REVIEW
Sawako Yamashiro, Naoki Watanabe
Live-cell single-molecule imaging was introduced more than a decade ago, and has provided critical information on remodeling of the actin cytoskeleton, the motion of plasma membrane proteins, and dynamics of molecular motor proteins. Actin remodeling has been the best target for this approach because actin and its associated proteins stop diffusing when assembled, allowing visualization of single-molecules of fluorescently-labeled proteins in a state specific manner. The approach based on this simple principle is called Single-Molecule Speckle (SiMS) microscopy...
July 6, 2017: Sensors
https://www.readbyqxmd.com/read/28671584/an-infrared-actin-probe-for-deep-cell-electroporation-based-single-molecule-speckle-esims-microscopy
#16
Sawako Yamashiro, Naoki Watanabe
Single-molecule speckle (SiMS) microscopy is a powerful method to directly elucidate biochemical reactions in live cells. However, since the signal from an individual fluorophore is extremely faint, the observation area by epi-fluorescence microscopy is restricted to the thin cell periphery to reduce autofluorescence, or only molecules near the plasma membrane are visualized by total internal reflection fluorescence (TIRF) microscopy. Here, we introduce a new actin probe labeled with near infrared (NIR) emissive CF680R dye for easy-to-use, electroporation-based SiMS microscopy (eSiMS) for deep-cell observation...
July 1, 2017: Sensors
https://www.readbyqxmd.com/read/28639395/spatiotemporally-controllable-peptide-based-nanoassembly-in-single-living-cells-for-a-biological-self-portrait
#17
Yuanyuan Zhao, Xu Zhang, Zhipeng Li, Shuaidong Huo, Ke Zhang, Juntao Gao, Hao Wang, Xing-Jie Liang
Simultaneous precise localization and activity evaluation of a biomolecule in a single living cell is through an enzyme-specific signal-amplification process, which involves the localized, site-specific self-assembly, and activation of a presignaling molecule. The inactive presignaling tetraphenylethylene (TPE)-peptide derivative, TPE-YpYY, is nondetectable and highly biocompatible and these small molecules rapidly diffuse into living cells. Upon safely arriving at an active site, and accessing the catalytic pocket of an enzyme, TPE-YpYY immediately and quantitatively accumulates in situ in response to enzymatic activity, forms an enzyme anchor TPE-YYY nanoassembly, displays aggregation-induced emission behavior, and finally lights up the active enzyme, indicating its activity, and allowing its status in living cells to be tracked...
June 22, 2017: Advanced Materials
https://www.readbyqxmd.com/read/28635963/single-molecule-analysis-of-steroid-receptor-and-cofactor-action-in-living-cells
#18
Ville Paakinaho, Diego M Presman, David A Ball, Thomas A Johnson, R Louis Schiltz, Peter Levitt, Davide Mazza, Tatsuya Morisaki, Tatiana S Karpova, Gordon L Hager
Population-based assays have been employed extensively to investigate the interactions of transcription factors (TFs) with chromatin and are often interpreted in terms of static and sequential binding. However, fluorescence microscopy techniques reveal a more dynamic binding behaviour of TFs in live cells. Here we analyse the strengths and limitations of in vivo single-molecule tracking and performed a comprehensive analysis on the intranuclear dwell times of four steroid receptors and a number of known cofactors...
June 21, 2017: Nature Communications
https://www.readbyqxmd.com/read/28627118/optical-microresonators-for-sensing-and-transduction-a-materials-perspective
#19
REVIEW
Kevin D Heylman, Kassandra A Knapper, Erik H Horak, Morgan T Rea, Sudheer K Vanga, Randall H Goldsmith
Optical microresonators confine light to a particular microscale trajectory, are exquisitely sensitive to their microenvironment, and offer convenient readout of their optical properties. Taken together, this is an immensely attractive combination that makes optical microresonators highly effective as sensors and transducers. Meanwhile, advances in material science, fabrication techniques, and photonic sensing strategies endow optical microresonators with new functionalities, unique transduction mechanisms, and in some cases, unparalleled sensitivities...
August 2017: Advanced Materials
https://www.readbyqxmd.com/read/28618223/covalent-protein-labeling-and-improved-single-molecule-optical-properties-of-aqueous-cdse-cds-quantum-dots
#20
Sara M Wichner, Victor R Mann, Alexander S Powers, Maya A Segal, Mustafa Mir, Jigar N Bandaria, Mark A DeWitt, Xavier Darzacq, Ahmet Yildiz, Bruce E Cohen
Semiconductor quantum dots (QDs) have proven to be superior probes for single-molecule imaging compared to organic or genetically encoded fluorophores, but they are limited by difficulties in protein targeting, their larger size, and on-off blinking. Here, we report compact aqueous CdSe/CdS QDs with significantly improved bioconjugation efficiency and superior single-molecule optical properties. We have synthesized covalent protein labeling ligands (i.e., SNAP tags) that are optimized for nanoparticle use, and QDs functionalized with these ligands label SNAP-tagged proteins ∼10-fold more efficiently than existing SNAP ligands...
July 25, 2017: ACS Nano
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