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Single molecule imaging

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https://www.readbyqxmd.com/read/28933536/a-bimetallic-silver-neodymium-theranostic-nanoparticle-with-multimodal-nir-mri-ct-imaging-and-combined-chemo-photothermal-therapy
#1
Sandeep K Mishra, S Kannan
An engineered metallic nanostructure is an excellent candidate for "theranosis" of cancer, having intrinsic properties of multimodal imaging and therapy. Toward this target, the development of silver-neodymium bimetallic nanoparticles (Ag-Nd BNPs) via microwave-assisted polyol synthesis is presented. The resultant Ag-Nd BNPs exhibit good monodispersity with average size of 10 nm, fluorescence in the near-infrared (NIR) region, and magnetic properties. The Ag-Nd BNPs also validate MRI, CT, and NIR trimodal imaging ability and enunciate valuable temperature response upon irradiation under a NIR laser...
September 21, 2017: Inorganic Chemistry
https://www.readbyqxmd.com/read/28932696/an-automated-workflow-for-quantifying-rna-transcripts-in-individual-cells-in-large-data-sets
#2
Matthew C Pharris, Tzu-Ching Wu, Xinping Chen, Xu Wang, David M Umulis, Vikki M Weake, Tamara L Kinzer-Ursem
Advanced molecular probing techniques such as single molecule fluorescence in situ hybridization (smFISH) or RNAscope can be used to assess the quantity and spatial location of mRNA transcripts within cells. Quantifying mRNA expression in large image sets usually involves automated counting of fluorescent spots. Though conventional spot counting algorithms may suffice, they often lack high-throughput capacity and accuracy in cases of crowded signal or excessive noise. Automatic identification of cells and processing of many images is still a challenge...
2017: MethodsX
https://www.readbyqxmd.com/read/28931892/new-views-on-phototransduction-from-atomic-force-microscopy-and-single-molecule-force-spectroscopy-on-native-rods
#3
Sourav Maity, Nina Ilieva, Alessandro Laio, Vincent Torre, Monica Mazzolini
By combining atomic force microscopy (AFM) imaging and single-molecule force spectroscopy (SMFS), we analyzed membrane proteins of the rod outer segments (OS). With this combined approach we were able to study the membrane proteins in their natural environment. In the plasma membrane we identified native cyclic nucleotide-gated (CNG) channels which are organized in single file strings. We also identified rhodopsin located both in the discs and in the plasma membrane. SMFS reveals strikingly different mechanical properties of rhodopsin unfolding in the two environments...
September 20, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28927531/discriminating-rna-variants-with-single-molecule-allele-specific-fish
#4
REVIEW
Martyna O Urbanek, Wlodzimierz J Krzyzosiak
DNA mutations of various types often affect the cellular localization and function of gene products. The role of mutant transcripts in the pathogenesis of human disease is increasingly recognized. Among the pathogenic RNA variants are transcripts with single nucleotide substitutions, small insertions or deletions, aberrantly or alternatively spliced transcripts and RNAs derived from fused genes. To discriminate among transcripts, particularly those of low abundance, showing small or large sequence differences, a highly sensitive and specific RNA imaging method is required...
July 2017: Mutation Research
https://www.readbyqxmd.com/read/28926050/temperature-dependence-of-the-conversion-efficiency-of-photochromic-perylene-bisimide-dithienylcyclopentene-triads-embedded-in-a-polymer
#5
Johann Thurn, Johannes Maier, Martti Pärs, Katja Gräf, Mukundan Thelakkat, Jürgen Köhler
Photochromic molecules that are covalently linked to a strong fluorophore combine the requirements of external control and strong fluorescence, which will become increasingly important for super-resolution microscopy techniques based on single molecules. However, given the bulky structure of such constructs, steric hindrance might affect their photoconversion efficiencies upon immobilising them for imaging purposes. In this study the efficiencies of the photochromic conversion processes of molecular triads that are embedded in a polymer have been studied as a function of temperature...
September 19, 2017: Physical Chemistry Chemical Physics: PCCP
https://www.readbyqxmd.com/read/28924672/in-situ-super-resolution-imaging-of-genomic-dna-with-oligostorm-and-oligodna-paint
#6
Brian J Beliveau, Alistair N Boettiger, Guy Nir, Bogdan Bintu, Peng Yin, Xiaowei Zhuang, C-Ting Wu
OligoSTORM and OligoDNA-PAINT meld the Oligopaint technology for fluorescent in situ hybridization (FISH) with, respectively, Stochastic Optical Reconstruction Microscopy (STORM) and DNA-based Point Accumulation for Imaging in Nanoscale Topography (DNA-PAINT) to enable in situ single-molecule super-resolution imaging of nucleic acids. Both strategies enable ≤20 nm resolution and are appropriate for imaging nanoscale features of the genomes of a wide range of species, including human, mouse, and fruit fly (Drosophila)...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28924670/correlative-single-molecule-localization-microscopy-and-confocal-microscopy
#7
Christian Soeller, Yufeng Hou, Isuru D Jayasinghe, David Baddeley, David Crossman
Single-molecule localization microscopy allows the ability to image fluorescence labeled molecular targets at nanoscale resolution. However, for many biological questions the ability to provide tissue and cellular context in addition to these high resolution data is eminently informative. Here, we describe a procedure to achieve this aim by correlatively imaging human cardiac tissue first at the nanoscale with direct stochastic optical reconstruction microscopy (dSTORM) and then at the diffraction limit with conventional confocal microscopy...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28924669/measuring-nanometer-distances-between-fluorescent-labels-step-by-step
#8
Susanna Maria Früh, Ingmar Schoen
Super-resolution fluorescence microscopy methods are increasingly applied to study the structure of biological molecules within their natural context or at biomaterial interfaces. We here provide a protocol for Single-molecule High-Resolution Imaging with Photobleaching (SHRImP) that can be used to obtain information about the conformation of large proteins or other macromolecules at the single-molecule level. This procedure requires site-specific protein labeling with fluorescent dyes, immobilization and sample preparation, optimization of imaging buffer composition and microscope settings, and acquisition of short time-lapse movies that capture the stepwise bleaching behavior of individual molecules...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28924668/synthesis-of-janelia-fluor-halotag-and-snap-tag-ligands-and-their-use-in-cellular-imaging-experiments
#9
Jonathan B Grimm, Timothy A Brown, Brian P English, Timothée Lionnet, Luke D Lavis
The development of genetically encoded self-labeling protein tags such as the HaloTag and SNAP-tag has expanded the utility of chemical dyes in microscopy. Intracellular labeling using these systems requires small, cell-permeable dyes with high brightness and photostability. We recently discovered a general method to improve the properties of classic fluorophores by replacing N,N-dimethylamino groups with four-membered azetidine rings to create the "Janelia Fluor" dyes. Here, we describe the synthesis of the HaloTag and SNAP-tag ligands of Janelia Fluor 549 and Janelia Fluor 646 as well as standard labeling protocols for use in ensemble and single-molecule cellular imaging...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28924667/correlative-in-resin-super-resolution-fluorescence-and-electron-microscopy-of-cultured-cells
#10
Errin Johnson, Rainer Kaufmann
Correlative super-resolution light and electron microscopy (super-resolution CLEM) is a powerful and emerging tool in biological research. The practical realization of these two very different microscopy techniques with their individual requirements remains a challenging task. There is a broad range of approaches to choose from, each with their own advantages and limitations. Here, we present a detailed protocol for in-resin super-resolution CLEM of high-pressure frozen and freeze substituted cultured cells...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28924666/brain-slice-staining-and-preparation-for-three-dimensional-super-resolution-microscopy
#11
Christopher L German, Manasa V Gudheti, Annette E Fleckenstein, Erik M Jorgensen
Localization microscopy techniques-such as photoactivation localization microscopy (PALM), fluorescent PALM (FPALM), ground state depletion (GSD), and stochastic optical reconstruction microscopy (STORM)-provide the highest precision for single-molecule localization currently available. However, localization microscopy has been largely limited to cell cultures due to the difficulties that arise in imaging thicker tissue sections. Sample fixation and antibody staining, background fluorescence, fluorophore photoinstability, light scattering in thick sections, and sample movement create significant challenges for imaging intact tissue...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28924665/fully-automated-targeted-confocal-and-single-molecule-localization-microscopy
#12
Jan Philipp Eberle, Walter Muranyi, Holger Erfle, Manuel Gunkel
Single-molecule localization microscopy (SMLM) enables imaging of biological structures in the nanometre range. Long measurement times are the consequence of this kind of microscopy due to the need of acquiring thousands of images. We built a setup that automatically detects target structures using confocal microscopy and images them with SMLM. Utilizing the Konstanz Information Miner (KNIME), we were able to connect a confocal microscope with an SMLM unit for targeted screening. In this process, we developed KNIME plugins to communicate with the microscope components and combined them to a workflow...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28924663/quantitative-single-molecule-localization-microscopy-qsmlm-of-membrane-proteins-based-on-kinetic-analysis-of-fluorophore-blinking-cycles
#13
Franziska Fricke, Joel Beaudouin, Sebastian Malkusch, Roland Eils, Mike Heilemann
Photoswitchable or photoactivatable fluorophores are the key in single-molecule localization microscopy. Next to providing fluorescence images with subdiffraction spatial resolution, additional information is available from observing single fluorophores over time. This includes the characteristic photophysical phenomenon of "blinking" that is exhibited by single fluorescent proteins or fluorophores and follows well-defined kinetic laws. Analyzing the kinetics of "blinking" allows determining the number of fluorophores in a multi-molecular complex...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28920762/measuring-dynamics-of-eukaryotic-transcription-initiation-challenges-insights-and-opportunities
#14
Zhengjian Zhang, Robert Tjian
Transcription of protein-encoding genes in eukaryotic cells is a dynamically coordinated process. Many of the key transcription regulators contain functionally essential intrinsically disordered regions (IDRs), the dynamic nature of which creates extra challenges to traditional biochemical analyses. Recent advances in single-molecule fluorescence imaging technology have enabled direct visualization of these rapid, complex and dynamic molecular interactions in real time.
September 18, 2017: Transcription
https://www.readbyqxmd.com/read/28920426/methotrexate-camptothecin-prodrug-nanoassemblies-as-a-versatile-nanoplatform-for-biomodal-imaging-guided-self-active-targeted-and-synergistic-chemotherapy
#15
Yang Li, Jinyan Lin, Jinyuan Ma, Liang Song, Huirong Lin, Bowen Tang, Dengyue Chen, Guanghao Su, Shefang Ye, Xuan Zhu, Fang-Hong Luo, Zhenqing Hou
"All-in-one" carrier-free-based nano-multi-drug self-delivery system could combine triple advantages of small molecules, nanoscale characteristics, and synergistic combination therapy together. Researches have showed that dual-acting small molecular methotrexate (MTX) could target and kill the folate receptors-overexpressing cancer cells. Inspired by this mechanism, a novel collaborative "early-phase tumor-selective targeting and late-phase synergistic anticancer" approach was developed for the self-assembly of chemotherapeutic drug-drug conjugate, which showed various advantages of more simplicity, efficiency, and flexibility over the conventional approach that was only based on single or combination cancer chemotherapy...
September 18, 2017: ACS Applied Materials & Interfaces
https://www.readbyqxmd.com/read/28917122/structure-determination-based-on-continuous-diffraction-from-macromolecular-crystals
#16
REVIEW
Henry N Chapman, Petra Fromme
Bright and coherent X-ray sources, such free-electron lasers, have spurred large activities in developing new methods to obtain the structures of biological macromolecules. In particular, single-molecule diffraction is highly desired, as it would abolish the need for crystallization. It provides considerably more diffraction intensity information than needed to solve a structure, unlike crystal diffraction, which is usually insufficient for direct phasing. To overcome the challenge of weak scattering signals of single molecules, the direct phasing approaches in coherent diffractive imaging have been combined with crystals in several imaginative ways...
September 13, 2017: Current Opinion in Structural Biology
https://www.readbyqxmd.com/read/28911512/cardioprotective-effects-of-hsp72%C3%A2-administration-on-ischemia-reperfusion-injury
#17
Takashi Tanimoto, Missag H Parseghian, Takehiro Nakahara, Hideki Kawai, Navneet Narula, Dongbin Kim, Robert Nishimura, Richard H Weisbart, Grace Chan, Richard A Richieri, Nezam Haider, Farhan Chaudhry, Glenn T Reynolds, John Billimek, Francis G Blankenberg, Partho P Sengupta, Artiom D Petrov, Takashi Akasaka, H William Strauss, Jagat Narula
BACKGROUND: Although early reperfusion is the most desirable intervention after ischemic myocardial insult, it may add to damage through oxidative stress. OBJECTIVES: This study investigated the cardioprotective effects of a single intravenous dose of heat shock protein-72 (HSP72) coupled to a single-chain variable fragment (Fv) of monoclonal antibody 3E10 (3E10Fv) in a rabbit ischemia-reperfusion model. The Fv facilitates rapid transport of HSP72 into cells, even with intact membranes...
September 19, 2017: Journal of the American College of Cardiology
https://www.readbyqxmd.com/read/28904870/membrane-dynamics-of-resting-and-internalin-b-bound-met-receptor-tyrosine-kinase-studied-by-single-molecule-tracking
#18
Marie-Lena I E Harwardt, Phoebe Young, Willem M Bleymüller, Timo Meyer, Christos Karathanasis, Hartmut H Niemann, Mike Heilemann, Marina S Dietz
The human MET receptor tyrosine kinase contributes to vertebrate development and cell proliferation. As a proto-oncogene, it is a target in cancer therapies. MET is also relevant for bacterial infection by Listeria monocytogenes and is activated by the bacterial protein internalin B. The processes of ligand binding, receptor activation, and the diffusion behavior of MET within the plasma membrane as well as its interconnections with various cell components are not fully understood. We investigated the receptor diffusion dynamics using single-particle tracking and imaging fluorescence correlation spectroscopy and elucidated mobility states of resting and internalin B-bound MET...
September 2017: FEBS Open Bio
https://www.readbyqxmd.com/read/28904250/building-a-complete-image-of-genome-regulation-in-the-model-organism-escherichia-coli
#19
Akira Ishihama
The model organism, Escherichia coli, contains a total of more than 4,500 genes, but the total number of RNA polymerase (RNAP) core enzyme or the transcriptase is only about 2,000 molecules per genome. The regulatory targets of RNAP are, however, modulated by changing its promoter selectivity through two-steps of protein-protein interplay with 7 species of the sigma factor in the first step, and then 300 species of the transcription factor (TF) in the second step. Scientists working in the field of prokaryotic transcription in Japan have made considerable contributions to the elucidation of genetic frameworks and regulatory modes of the genome transcription in E...
September 12, 2017: Journal of General and Applied Microbiology
https://www.readbyqxmd.com/read/28902853/the-human-bitter-taste-receptor-t2r38-is-broadly-tuned-for-bacterial-compounds
#20
Christophe Verbeurgt, Alex Veithen, Sébastien Carlot, Maxime Tarabichi, Jacques E Dumont, Sergio Hassid, Pierre Chatelain
T2R38 has been shown to be a specific bacterial detector implicated in innate immune defense mechanism of human upper airway. Several clinical studies have demonstrated that this receptor is associated with the development of chronic rhinosinusitis (CRS). T2R38 was previously reported to bind to homoserine lactones (HSL), quorum sensing molecules specific of Pseudomonas Aeruginosa and other gram negative species. Nevertheless, these bacteria are not the major pathogens found in CRS. Here we report on the identification of bacterial metabolites acting as new agonists of T2R38 based on a single cell calcium imaging study...
2017: PloS One
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