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High throughput microscopy

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https://www.readbyqxmd.com/read/28527910/radiative-decay-engineering-8-coupled-emission-microscopy-for-lens-free-high-throughput-fluorescence-detection
#1
Liangfu Zhu, Ramachandram Badugu, Douguo Zhang, Ruxue Wang, Emiliano Descrovi, Joseph R Lakowicz
Fluorescence spectroscopy and imaging are now used throughout the biosciences. Fluorescence microscopes, spectrofluorometers, microwell plate readers and microarray imagers all use multiple optical components to collect, redirect and focus the emission onto single point or array imaging detectors. For almost all biological samples, except those with regular nanoscale features, emission occurs in all directions. With the exception of complex microscope objectives with large collection angles (NA ≤ 0.5), all these instruments collect only a small fraction of the total emission...
May 17, 2017: Analytical Biochemistry
https://www.readbyqxmd.com/read/28518171/fish-flow-a-protocol-for-the-concurrent-detection-of-mrna-and-protein-in-single-cells-using-fluorescence-in-situ-hybridization-and-flow-cytometry
#2
Riccardo Arrigucci, Yuri Bushkin, Felix Radford, Karim Lakehal, Pooja Vir, Richard Pine, December Martin, Jeffrey Sugarman, Yanlin Zhao, George S Yap, Alfred A Lardizabal, Sanjay Tyagi, Maria Laura Gennaro
We describe a flow-cytometry-based protocol for intracellular mRNA measurements in nonadherent mammalian cells using fluorescence in situ hybridization (FISH) probes. The method, which we call FISH-Flow, allows for high-throughput multiparametric measurements of gene expression, a task that was not feasible with earlier, microscopy-based approaches. The FISH-Flow protocol involves cell fixation, permeabilization and hybridization with a set of fluorescently labeled oligonucleotide probes. In this protocol, surface and intracellular protein markers can also be stained with fluorescently labeled antibodies for simultaneous protein and mRNA measurement...
June 2017: Nature Protocols
https://www.readbyqxmd.com/read/28503924/complete-prevention-of-dendrite-formation-in-zn-metal-anodes-by-means-of-pulsed-charging-protocols
#3
Grecia Garcia, Edgar Ventosa, Wolfgang Schuhmann
Zn metal as anode in rechargeable batteries such as Zn/air or Zn/Ni batteries suffers from poor cyclability. The formation of Zn dendrites upon cycling is the key limiting step. We report a systematic study of the influence of pulsed electroplating protocols on the formation of Zn dendrites and in turn on strategies to completely prevent Zn dendrite formation. Due to the large number of variables in electroplating protocols, a scanning droplet cell technique was adapted as high-throughput methodology in which a descriptor of the surface roughness can be in-situ derived by means of electrochemical impedance spectroscopy...
May 15, 2017: ACS Applied Materials & Interfaces
https://www.readbyqxmd.com/read/28502181/rapid-nano-gram-scale-screening-method-of-micro-arrays-to-evaluate-drug-polymer-blends-using-high-throughput-printing-technology
#4
Vincenzo Taresco, Iria Louzao, David Scurr, Jonathan Booth, Kevin Treacher, James McCabe, Eleanor Turpin, Charles A Laughton, Cameron Alexander, Jonathan C Burley, Martin C Garnett
A miniaturized, high-throughput assay was optimized to screen polymer-drug solid dispersions using a 2-D Ink-jet printer. By simply printing nanoliter amounts of polymer and drug solutions onto an inert surface, drug:polymer micro-dots of tunable composition were produced in an easily-addressable micro-array format. The amount of material printed for each dried spot ranged from 25 ng to 650 ng. These arrays were used to assess the stability of drug:polymer dispersions with respect to recrystallization, using polarized light microscopy...
May 14, 2017: Molecular Pharmaceutics
https://www.readbyqxmd.com/read/28501082/a-high-content-screening-technology-for-quantitatively-studying-podocyte-dynamics
#5
REVIEW
Jochen Reiser, Ha Won Lee, Vineet Gupta, Mehmet M Altintas
Podocytes form the visceral layer of a kidney glomerulus and express a characteristic octopus-like cellular architecture specialized for the ultrafiltration of blood. The cytoskeletal dynamics and structural elasticity of podocytes rely on the self-organization of highly interconnected actin bundles, and the maintenance of these features is important for the intact glomerular filtration. Development of more differentiated podocytes in culture has dramatically increased our understanding of the molecular mechanisms regulating podocyte actin dynamics...
May 2017: Advances in Chronic Kidney Disease
https://www.readbyqxmd.com/read/28495957/resolving-the-complex-genetic-basis-of-phenotypic-variation-and-variability-of-cellular-growth
#6
Naomi Ziv, Bentley M Shuster, Mark L Siegal, David Gresham
In all organisms, many traits vary continuously between individuals. Explaining the genetic basis of quantitative trait variation requires comprehensively accounting for genetic and non-genetic factors as well as their interactions. The growth of microbial cells can be characterized by a lag duration, an exponential growth phase and a stationary phase. Parameters that characterize these growth phases can vary among genotypes (phenotypic variation), environmental conditions (phenotypic plasticity) and among isogenic cells in a given environment (phenotypic variability)...
May 11, 2017: Genetics
https://www.readbyqxmd.com/read/28490437/high-throughput-crispri-phenotyping-identifies-new-essential-genes-in-streptococcus-pneumoniae
#7
Xue Liu, Clement Gallay, Morten Kjos, Arnau Domenech, Jelle Slager, Sebastiaan P van Kessel, Kèvin Knoops, Robin A Sorg, Jing-Ren Zhang, Jan-Willem Veening
Genome-wide screens have discovered a large set of essential genes in the opportunistic human pathogen Streptococcus pneumoniae However, the functions of many essential genes are still unknown, hampering vaccine development and drug discovery. Based on results from transposon sequencing (Tn-seq), we refined the list of essential genes in S. pneumoniae serotype 2 strain D39. Next, we created a knockdown library targeting 348 potentially essential genes by CRISPR interference (CRISPRi) and show a growth phenotype for 254 of them (73%)...
May 10, 2017: Molecular Systems Biology
https://www.readbyqxmd.com/read/28484480/single-event-resolution-of-plant-plasma-membrane-protein-endocytosis-by-tirf-microscopy
#8
Alexander Johnson, Grégory Vert
Endocytosis is a key process in the internalization of extracellular materials and plasma membrane proteins, such as receptors and transporters, thereby controlling many aspects of cell signaling and cellular homeostasis. Endocytosis in plants has an essential role not only for basic cellular functions but also for growth and development, nutrient delivery, toxin avoidance, and pathogen defense. The precise mechanisms of endocytosis in plants remain quite elusive. The lack of direct visualization and examination of single events of endocytosis has greatly hampered our ability to precisely monitor the cell surface lifetime and the recruitment profile of proteins driving endocytosis or endocytosed cargos in plants...
2017: Frontiers in Plant Science
https://www.readbyqxmd.com/read/28476626/nile-red-fluorescence-spectrum-decomposition-enables-rapid-screening-of-large-protein-aggregates-in-complex-biopharmaceutical-formulations-like-influenza-vaccines
#9
Ziya Sahin, Senem Akkoc, Ronald Neeleman, Jonathan Haines, Veysel Kayser
The extensive presence of large (high molecular weight) protein aggregates in biopharmaceutical formulations is a concern for formulation stability and possibly safety. Tests to screen large aggregate content in such bioformulations are therefore needed for rapid and reliable quality control in industrial settings. Herein, non-commercial seasonal influenza split-virus vaccine samples, produced using various strains and extracted from selected industrial processing steps, were used as model complex bioformulations...
May 2, 2017: Vaccine
https://www.readbyqxmd.com/read/28474754/ultrasensitive-measurement-of-ca-2-influx-into-lipid-vesicles-induced-by-protein-aggregates
#10
Patrick Flagmeier, Suman De, David C Wirthensohn, Steven F Lee, Cécile Vincke, Serge Muyldermans, Tuomas P J Knowles, Sonia Gandhi, Christopher M Dobson, David Klenerman
To quantify and characterize the potentially toxic protein aggregates associated with neurodegenerative diseases, a high-throughput assay based on measuring the extent of aggregate-induced Ca(2+) entry into individual lipid vesicles has been developed. This approach was implemented by tethering vesicles containing a Ca(2+) sensitive fluorescent dye to a passivated surface and measuring changes in the fluorescence as a result of membrane disruption using total internal reflection microscopy. Picomolar concentrations of Aβ42 oligomers could be observed to induce Ca(2+) influx, which could be inhibited by the addition of a naturally occurring chaperone and a nanobody designed to bind to the Aβ peptide...
May 5, 2017: Angewandte Chemie
https://www.readbyqxmd.com/read/28471460/using-hyperlopit-to-perform-high-resolution-mapping-of-the-spatial-proteome
#11
Claire M Mulvey, Lisa M Breckels, Aikaterini Geladaki, Nina Kočevar Britovšek, Daniel J H Nightingale, Andy Christoforou, Mohamed Elzek, Michael J Deery, Laurent Gatto, Kathryn S Lilley
The organization of eukaryotic cells into distinct subcompartments is vital for all functional processes, and aberrant protein localization is a hallmark of many diseases. Microscopy methods, although powerful, are usually low-throughput and dependent on the availability of fluorescent fusion proteins or highly specific and sensitive antibodies. One method that provides a global picture of the cell is localization of organelle proteins by isotope tagging (LOPIT), which combines biochemical cell fractionation using density gradient ultracentrifugation with multiplexed quantitative proteomics mass spectrometry, allowing simultaneous determination of the steady-state distribution of hundreds of proteins within organelles...
June 2017: Nature Protocols
https://www.readbyqxmd.com/read/28468325/a-large-area-nanoplasmonic-sensor-fabricated-by-rapid-thermal-annealing-treatment-for-label-free-and-multi-point-immunoglobulin-sensing
#12
Hana Tzu-Han Lin, Chuan-Kai Yang, Chi-Chen Lin, Albert Meng-Hsin Wu, Lon A Wang, Nien-Tsu Huang
Immunoglobulins are important biomarkers to evaluate the immune status or development of infectious diseases. To provide timely clinical treatments, it is important to continuously monitor the level of multiple immunoglobulins. Localized surface plasmon resonance (LSPR)-based nanoplasmonic sensors have been demonstrated for multiplex immunoglobulins detection. However, the sensor fabrication process is usually slow and complicated, so it is not accessible for large-area and batch fabrication. Herein, we report a large-area (2 cm × 2 cm) nanofabrication method using physical vapor deposition followed by a rapid thermal annealing treatment...
May 2, 2017: Nanomaterials
https://www.readbyqxmd.com/read/28464514/cell-density-modulates-intracellular-mass-transport-in-neural-networks
#13
Patricia Cintora, Jyothi Arikkath, Mikhail Kandel, Gabriel Popescu, Catherine Best-Popescu
In order to fully understand brain connectivity and elucidate the mechanisms involved in central nervous system disease, the field of neuroscience depends on quantitative studies of neuronal structure and function. Cell morphology and neurite (axonal and dendritic) arborization are typically studied by immunohistochemical and fluorescence techniques. However, dry mass content and intracellular mass transport rates have largely been under-investigated given the inherent difficulties in their measurement. Here, spatial light interference microscopy (SLIM) and dispersion-relation phase spectroscopy (DPS) were used to measure pathlength fluctuations that report on the dry mass and transport within cultured primary neurons across low, medium, and high cell density conditions...
May 2, 2017: Cytometry. Part A: the Journal of the International Society for Analytical Cytology
https://www.readbyqxmd.com/read/28458744/combinatorial-study-of-fe-co-v-hard-magnetic-thin-films
#14
Sean W Fackler, Vasileios Alexandrakis, Dennis König, A Gilad Kusne, Tieren Gao, Matthew J Kramer, Drew Stasak, Kenny Lopez, Brad Zayac, Apurva Mehta, Alfred Ludwig, Ichiro Takeuchi
Thin film libraries of Fe-Co-V were fabricated by combinatorial sputtering to study magnetic and structural properties over wide ranges of composition and thickness by high-throughput methods: synchrotron X-ray diffraction, magnetometry, composition, and thickness were measured across the Fe-Co-V libraries. In-plane magnetic hysteresis loops were shown to have a coercive field of 23.9 kA m(-1) (300 G) and magnetization of 1000 kA m(-1). The out-of-plane direction revealed enhanced coercive fields of 207 kA m(-1) (2...
2017: Science and Technology of Advanced Materials
https://www.readbyqxmd.com/read/28455334/validation-of-the-hirst-type-spore-trap-for-simultaneous-monitoring-of-prokaryotic-and-eukaryotic-biodiversity-in-urban-air-samples-by-ngs
#15
Andrés Núñez, Guillermo Amo de Paz, Zuzana Ferencova, Alberto Rastrojo, Raúl Guantes, Ana M García, Antonio Alcamí, A Montserrat Gutiérrez-Bustillo, Diego A Moreno
Pollen, fungi and bacteria are the main microscopic biological entities present in the air outdoor causing allergy symptoms, disease transmission and having a significant role in atmosphere dynamics. Despite their relevance, a method for monitoring simultaneously these biological particles in metropolitan environments has not been developed yet. Here, we assessed the use of the Hirst-type spore trap to characterize the global airborne biota by high-throughput DNA sequencing, selecting regions of the genes 16S ribosomal RNA gene and internal transcribed spacer for the taxonomic assignment...
April 28, 2017: Applied and Environmental Microbiology
https://www.readbyqxmd.com/read/28450034/microbubble-enzyme-linked-immunosorbent-assay-for-the-detection-of-targeted-microbubbles-in-in%C3%A2-vitro-static-binding%C3%A2-assays
#16
Jennifer Wischhusen, Frederic Padilla
Targeted microbubbles (MBs) are ultrasound contrast agents that are functionalized with a ligand for ultrasound molecular imaging of endothelial markers. Novel targeted MBs are characterized in vitro by incubation in protein-coated wells, followed by binding quantification by microscopy or ultrasound imaging. Both methods provide operator-dependent results: Between 3 and 20 fields of view from a heterogeneous sample are typically selected for analysis by microscopy, and in ultrasound imaging, different acoustic settings affect signal intensities...
April 24, 2017: Ultrasound in Medicine & Biology
https://www.readbyqxmd.com/read/28448005/open-source-single-particle-analysis-for-super-resolution-microscopy-with-virusmapper
#17
Robert D M Gray, Jason Mercer, Ricardo Henriques
Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm allows for the routine imaging of nanoscale biological complexes and processes. This increase in resolution also means that methods popular in electron microscopy, such as single-particle analysis, can readily be applied to super-resolution fluorescence microscopy. By combining this analytical approach with super-resolution optical imaging, it becomes possible to take advantage of the molecule-specific labeling capacity of fluorescence microscopy to generate structural maps of molecular elements within a metastable structure...
April 9, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28447785/graphene-microcapsule-arrays-for-combinatorial-electron-microscopy-and-spectroscopy-in-liquids
#18
Alexander Yulaev, Hongxuan Guo, Evgheni Strelcov, Lei Chen, Ivan V Vlassiouk, Andrei Kolmakov
Atomic-scale thickness, molecular impermeability, low atomic number, and mechanical strength make graphene an ideal electron-transparent membrane for material characterization in liquids and gases with scanning electron microscopy and spectroscopy. Here, we present a novel sample platform made of an array of thousands of identical isolated graphene-capped micro-channels with high aspect ratio. A combination of a global wide field of view with high resolution local imaging of the array allows for high throughput in situ studies as well as for combinatorial screening of solutions, liquid interfaces and immersed samples...
April 27, 2017: ACS Applied Materials & Interfaces
https://www.readbyqxmd.com/read/28447132/high-throughput-analysis-of-sub-visible-mab-aggregate-particles-using-automated-fluorescence-microscopy-imaging
#19
Albert Jesuran Paul, Fabian Bickel, Martina Röhm, Lisa Hospach, Bettina Halder, Nina Rettich, René Handrick, Eva Maria Herold, Hans Kiefer, Friedemann Hesse
Aggregation of therapeutic proteins is a major concern as aggregates lower the yield and can impact the efficacy of the drug as well as the patient's safety. It can occur in all production stages; thus, it is essential to perform a detailed analysis for protein aggregates. Several methods such as size exclusion high-performance liquid chromatography (SE-HPLC), light scattering, turbidity, light obscuration, and microscopy-based approaches are used to analyze aggregates. None of these methods allows determination of all types of higher molecular weight (HMW) species due to a limited size range...
April 27, 2017: Analytical and Bioanalytical Chemistry
https://www.readbyqxmd.com/read/28446788/resolution-enhanced-fourier-ptychographic-microscopy-based-on-high-numerical-aperture-illuminations
#20
Jiasong Sun, Chao Zuo, Liang Zhang, Qian Chen
High-resolution and wide field-of-view (FOV) microscopic imaging plays a central role in diverse applications such as high-throughput screening and digital pathology. However, conventional microscopes face inherent trade-offs between the spatial resolution and FOV, which are fundamental limited by the space-bandwidth product (SBP) of the optical system. The resolution-FOV tradeoff can be effectively decoupled in Fourier ptychography microscopy (FPM), however, to date, the effective imaging NA achievable with a typical FPM system is still limited to the range of 0...
April 26, 2017: Scientific Reports
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