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High throughput microscopy

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https://www.readbyqxmd.com/read/28344036/focus-the-interface-between-data-collection-and-data-processing-in-cryo-em
#1
Nikhil Biyani, Ricardo D Righetto, Robert McLeod, Daniel Caujolle-Bert, Daniel Castano-Diez, Kenneth N Goldie, Henning Stahlberg
We present a new software package called Focus that interfaces cryo-transmission electron microscopy (cryo-EM) data collection with computer image processing. Focus creates a user-friendly environment to import and manage data recorded by direct electron detectors and perform elemental image processing tasks in a high-throughput manner while new data is being acquired at the microscope. It provides the functionality required to remotely monitor the progress of data collection and data processing, which is essential now that automation in cryo-EM allows a steady flow of images of single particles, two-dimensional crystals, or electron tomography data to be recorded in overnight sessions...
March 23, 2017: Journal of Structural Biology
https://www.readbyqxmd.com/read/28341988/unexpected-differences-between-planar-and-column-liquid-chromatographic-retention-of-1-acenaphthenol-enantiomers-controlled-by-supramolecular-interactions-involving-%C3%AE-cyclodextrin-at-subambient-temperatures
#2
Hatsuichi Ohta, Elżbieta Włodarczyk, Krzysztof Piaskowski, Aleksandra Kaleniecka, Lucyna Lewandowska, Michał J Baran, Mariusz Wojnicz, Kiyokatsu Jinno, Yoshihiro Saito, Paweł K Zarzycki
We report the results of experimental work focusing on host-guest supramolecular complex creation between macrocyclic compound (β-cyclodextrin) and 1-acenaphthenol enantiomers (racemic mixture) in liquid phase composed of 35% acetonitrile in water (v/v) at different temperatures ranging from 0 to 90 °C. Experimental setup involved several analytical protocols based on classical non-forced flow planar chromatography (RP-18 TLC plates), micro-TLC (RP-18 W HPTLC plates), column chromatography (HPLC with C-18 and C-30 stationary phases), as well as UV-Vis spectrophotometry and optical microscopy...
March 24, 2017: Analytical and Bioanalytical Chemistry
https://www.readbyqxmd.com/read/28340485/an-approach-for-liposome-immobilization-using-sterically-stabilized-micelles-ssms-as-a-precursor-for-bio-layer-interferometry-based-interaction-studies
#3
Jakob Wallner, Gabriele Lhota, Markus Schosserer, Karola Vorauer-Uhl
Non-fluidic bio-layer interferometry (BLI) has rapidly become a standard tool for monitoring almost all biomolecular interactions in a label-free, real-time and high-throughput manner. High-efficiency screening methods which measure the kinetics of liposomes with a variety of compounds require the immobilization of liposomes. In this work, a method is described for immobilizing liposomes for interaction studies, based on the biophysical principles of this biosensor platform. The immobilization approach includes the loading of DSPE-PEG(2000)-biotin containing sterically stabilized micelles (SSMs) which are restructured in a buffer change step, resulting in an accessible substrate for liposome immobilization...
March 12, 2017: Colloids and Surfaces. B, Biointerfaces
https://www.readbyqxmd.com/read/28336360/gain-a-graph-based-method-to-automatically-analyze-neurite-outgrowth-in-cell-cultures
#4
B L Long, H Li, A Mahadevan, T Tang, K Balotin, N Grandel, J Soto, S Y Wong, A Abrego, S Li, A A Qutub
BACKGROUND: Neurite outgrowth is a metric widely used to assess the success of in vitro neural stem cell differentiation or neuron reprogramming protocols and of high-content screening assays for neural regenerative drug discovery. However, neurite measurements are tedious to perform manually, and there is a paucity of freely available, fully automated software to determine neurite measurements and neuron counting. To provide such a tool to the neurobiology, stem cell, cell engineering, and neuroregenerative communities, we developed an algorithm for performing high-throughput neurite analysis in immunofluorescence images...
March 20, 2017: Journal of Neuroscience Methods
https://www.readbyqxmd.com/read/28328318/toward-discovery-of-novel-microtubule-targeting-agents-a-snap-tag-based-high-content-screening-assay-for-the-analysis-of-microtubule-dynamics-and-cell-cycle-progression
#5
Nina Berges, Katharina Arens, Verena Kreusch, Rainer Fischer, Stefano Di Fiore
Microtubule targeting agents (MTAs) are used for the treatment of cancer. Novel MTAs could provide additional and beneficial therapeutic options. To improve the sensitivity and throughput of standard immunofluorescence assays for the characterization of MTAs, we used SNAP-tag technology to produce recombinant tubulin monomers. To visualize microtubule filaments, A549 cells transfected with SNAP-tubulin were stained with a membrane-permeable, SNAP-reactive dye. The treatment of SNAP-tubulin cells with stabilizing MTAs such as paclitaxel resulted in the formation of coarsely structured microtubule filaments, whereas depolymerizing MTAs such as nocodazole resulted in diffuse staining patterns in which the tubulin filaments were no longer distinguishable...
April 2017: SLAS Discov
https://www.readbyqxmd.com/read/28328316/a-high-throughput-screening-assay-using-a-photoconvertable-protein-for-identifying-inhibitors-of-transcription-translation-or-proteasomal-degradation
#6
David K Heidary, Ashley Fox, Chris I Richards, Edith C Glazer
Dysregulated transcription, translation, and protein degradation are common features of cancer cells, regardless of specific genetic profiles. Several clinical anticancer agents take advantage of this characteristic vulnerability and interfere with the processes of transcription and translation or inhibit protein degradation. However, traditional assays that follow the process of protein production and removal require multistep processing and are not easily amenable to high-throughput screening. The use of recombinant fluorescent proteins provides a convenient solution to this problem, and moreover, photoconvertable fluorescent proteins allow for ratiometric detection of both new protein production and removal of existing proteins...
April 2017: SLAS Discov
https://www.readbyqxmd.com/read/28323930/%C3%AE-cells-persist-in-t1d-pancreata-without-evidence-for-ongoing-%C3%AE-cell-turnover-or-neogenesis
#7
Carol J Lam, Daniel R Jacobson, Matthew M Rankin, Aaron R Cox, Jake A Kushner
Context: The cellular basis of persistent β-cell function in T1D remains enigmatic. No extensive quantitative β-cell studies of T1D pancreata have been performed to test for ongoing β-cell regeneration or neogenesis. Objective: We sought to determine the mechanism of β-cell persistence in T1D pancreata. Design: We studied T1D (n=47) and non-diabetic control (n=59) pancreata over a wide range of ages from the JDRF Network of Pancreatic Organ donors with Diabetes (nPOD) via high throughput microscopy...
February 27, 2017: Journal of Clinical Endocrinology and Metabolism
https://www.readbyqxmd.com/read/28319716/multiple-stressors-in-sediments-impact-adjacent-hard-substrate-habitats-and-across-biological-domains
#8
Jasmin C Lawes, Katherine A Dafforn, Graeme F Clark, Mark V Brown, Emma L Johnston
Coastal systems are increasingly impacted by human activities. While the direct effects of individual contaminants have been investigated, the potential for multiple contaminants to impact adjacent hard substrate habitats is poorly understood. Sediment-bound contaminants pose a risk to water column organisms through resuspension and the fluxing of dissolved nutrients and metals. This study experimentally manipulated contaminated coastal sediments in mesocosms with additions of a common fertiliser to investigate the impact on both bacterial biofilms and macrofouling communities on nearby hard substrates...
March 16, 2017: Science of the Total Environment
https://www.readbyqxmd.com/read/28301074/high-throughput-inclusion-body-sizing-nano-particle-tracking-analysis
#9
Wieland N Reichelt, Andreas Kaineder, Markus Brillmann, Lukas Neutsch, Alexander Taschauer, Hans Lohninger, Christoph Herwig
The expression of pharmaceutical relevant proteins in E. coli frequently triggers inclusion body (IB) formation caused by protein aggregation. In literature, substantial effort has been invested into the quantification of IB size. But up to this date particle based methods to analyze physical IB properties of representative numbers of IBs lack sensitivity and/or lack orthogonal verification. Using high pressure freezing and automated freeze substitution for transmission electron microscopy (TEM) the cytosolic inclusion body structure was preserved within the cells...
March 16, 2017: Biotechnology Journal
https://www.readbyqxmd.com/read/28288168/a-novel-flow-cytometry-based-assay-for-the-quantification-of-antibody-dependent-pneumococcal-agglutination
#10
Marrit N Habets, Saskia van Selm, Christa E van der Gaast-de Jongh, Dimitri A Diavatopoulos, Marien I de Jonge
The respiratory pathogen Streptococcus pneumoniae is a major cause of diseases such as otitis media, pneumonia, sepsis and meningitis. The first step towards infection is colonization of the nasopharynx. Recently, it was shown that agglutinating antibodies play an important role in the prevention of mucosal colonization with S. pneumoniae. Here, we present a novel method to quantify antibody-dependent pneumococcal agglutination in a high-throughput manner using flow cytometry. We found that the concentration of agglutinating antibodies against pneumococcal capsule are directly correlated with changes in the size and complexity of bacterial aggregates, as measured by flow cytometry and confirmed by light microscopy...
2017: PloS One
https://www.readbyqxmd.com/read/28287589/a-high-throughput-cell-microarray-platform-for-correlative-analysis-of-cell-differentiation-and-traction-forces
#11
Kerim B Kaylan, Andreas P Kourouklis, Gregory H Underhill
Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly controlled, high-throughput engineered system for measuring the impact of arrayed biochemical signals on cell differentiation. Recent efforts using cell microarrays have demonstrated their utility for combinatorial studies in which many microenvironmental factors are presented in parallel. However, these efforts have focused primarily on investigating the effects of biochemical cues on cell responses...
March 1, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28282404/characterization-of-bacterial-community-associated-with-phytoplankton-bloom-in-a-eutrophic-lake-in-south-norway-using-16s-rrna-gene-amplicon-sequence-analysis
#12
Niranjan Nitin Parulekar, Pandurang Kolekar, Andrew Jenkins, Synne Kleiven, Hans Utkilen, Anette Johansen, Sangeeta Sawant, Urmila Kulkarni-Kale, Mohan Kale, Mona Sæbø
Interactions between different phytoplankton taxa and heterotrophic bacterial communities within aquatic environments can differentially support growth of various heterotrophic bacterial species. In this study, phytoplankton diversity was studied using traditional microscopic techniques and the bacterial communities associated with phytoplankton bloom were studied using High Throughput Sequencing (HTS) analysis of 16S rRNA gene amplicons from the V1-V3 and V3-V4 hypervariable regions. Samples were collected from Lake Akersvannet, a eutrophic lake in South Norway, during the growth season from June to August 2013...
2017: PloS One
https://www.readbyqxmd.com/read/28271016/x-ray-phase-microtomography-with-a-single-grating-for-high-throughput-investigations-of-biological-tissue
#13
Marie-Christine Zdora, Joan Vila-Comamala, Georg Schulz, Anna Khimchenko, Alexander Hipp, Andrew C Cook, Daniel Dilg, Christian David, Christian Grünzweig, Christoph Rau, Pierre Thibault, Irene Zanette
The high-throughput 3D visualisation of biological specimens is essential for studying diseases and developmental disorders. It requires imaging methods that deliver high-contrast, high-resolution volumetric information at short sample preparation and acquisition times. Here we show that X-ray phase-contrast tomography using a single grating can provide a powerful alternative to commonly employed techniques, such as high-resolution episcopic microscopy (HREM). We present the phase tomography of a mouse embryo in paraffin obtained with an X-ray single-grating interferometer at I13-2 Beamline at Diamond Light Source and discuss the results in comparison with HREM measurements...
February 1, 2017: Biomedical Optics Express
https://www.readbyqxmd.com/read/28270973/time-stretch-microscopy-on-a-dvd-for-high-throughput-imaging-cell-based-assay
#14
Anson H L Tang, P Yeung, Godfrey C F Chan, Barbara P Chan, Kenneth K Y Wong, Kevin K Tsia
Cell-based assay based on time-stretch imaging is recognized to be well-suited for high-throughput phenotypic screening. However, this ultrafast imaging technique has primarily been limited to suspension-cell assay, leaving a wide range of solid-substrate assay formats uncharted. Moreover, time-stretch imaging is generally restricted to intrinsic biophysical phenotyping, but lacks the biomolecular signatures of the cells. To address these challenges, we develop a spinning time-stretch imaging assay platform based on the functionalized digital versatile disc (DVD)...
February 1, 2017: Biomedical Optics Express
https://www.readbyqxmd.com/read/28270966/label-free-fingerprinting-of-tumor-cells-in-bulk-flow-using-inline-digital-holographic-microscopy
#15
Dhananjay Kumar Singh, Caroline C Ahrens, Wei Li, Siva A Vanapalli
Large-scale and label-free phenotyping of cells holds great promise in medicine, especially in cancer diagnostics and prognosis. Here, we introduce inline digital holography microscopy for volumetric imaging of cells in bulk flow and fingerprinting of flowing tumor cells based on two metrics, in-focus scattered intensity and cell diameter. Using planar distribution of immobilized particles, we identify the optimal recording distance and microscope objective magnification that minimizes the error in measurement of particle position, size and scattered intensity...
February 1, 2017: Biomedical Optics Express
https://www.readbyqxmd.com/read/28270582/bacteria-getting-into-shape-genetic-determinants-of-e-%C3%A2-coli-morphology
#16
Shawn French, Jean-Philippe Côté, Jonathan M Stokes, Ray Truant, Eric D Brown
Perturbation of cellular processes is a prevailing approach to understanding biology. To better understand the complicated biology that defines bacterial shape, a sensitive, high-content platform was developed to detect multiple morphological defect phenotypes using microscopy. We examined morphological phenotypes across the Escherichia coli K-12 deletion (Keio) collection at the mid-exponential growth phase, revealing 111 deletions perturbing shape. Interestingly, 64% of these were uncharacterized mutants, illustrating the complex nature of shape maintenance and regulation in bacteria...
March 7, 2017: MBio
https://www.readbyqxmd.com/read/28267565/automating-cell-detection-and-classification-in-human-brain-fluorescent-microscopy-images-using-dictionary-learning-and-sparse-coding
#17
Maryana Alegro, Panagiotis Theofilas, Austin Nguy, Patricia A Castruita, William Seeley, Helmut Heinsen, Daniela M Ushizima, Lea T Grinberg
BACKGROUND: Immunofluorescence (IF) plays a major role in quantifying protein expression in situ and understanding cell function. It is widely applied in assessing disease mechanisms and in drug discovery research. Automation of IF analysis can transform studies using experimental cell models. However, IF analysis of postmortem human tissue relies mostly on manual interaction, often subjected to low-throughput and prone to error, leading to low inter and intra-observer reproducibility...
March 4, 2017: Journal of Neuroscience Methods
https://www.readbyqxmd.com/read/28258008/high-content-screening-of-clinically-tested-anticancer-drugs-identifies-novel-inhibitors-of-human-mrp1-abcc1
#18
Brian G Peterson, Kee W Tan, Bremansu Osa-Andrews, Surtaj H Iram
Multidrug resistance protein 1 (MRP1/ABCC1), an integral transmembrane efflux transporter, belongs to the ATP-binding cassette (ABC) protein superfamily. MRP1 governs the absorption and disposition of a wide variety of endogenous and xenobiotic substrates including various drugs across organs and physiological barriers. Additionally, its overexpression has been implicated in multidrug resistance in chemotherapy of multiple cancers. Here, we describe the development of a high content imaging-based screening assay for MRP1 activity...
February 28, 2017: Pharmacological Research: the Official Journal of the Italian Pharmacological Society
https://www.readbyqxmd.com/read/28255014/macrothrombocytopenia-and-dense-granule-deficiency-associated-with-fli1-variants-ultrastructural-and-pathogenic-features
#19
Paul Saultier, Léa Vidal, Matthias Canault, Denis Bernot, Céline Falaise, Catherine Pouymayou, Jean-Claude Bordet, Noémie Saut, Agathe Rostan, Véronique Baccini, Franck Peiretti, Marie Favier, Pauline Lucca, Jean-François Deleuze, Robert Olaso, Anne Boland, Pierre Emmanuel Morange, Christian Gachet, Fabrice Malergue, Sixtine Fauré, Anita Eckly, David-Alexandre Trégouët, Marjorie Poggi, Marie-Christine Alessi
Congenital macrothrombocytopenia is a family of rare diseases, of which a significant fraction remains to be genetically characterized. To analyze cases of unexplained thrombocytopenia, 27 individuals from a patient cohort of the Bleeding and Thrombosis Exploration Center of the University Hospital of Marseille were recruited for a high-throughput gene sequencing study. This strategy led to the identification of two novel FLI1 variants (c.1010G>A and c.1033A>G) responsible for macrothrombocytopenia. The FLI1 variant carriers platelets exhibited a defect in aggregation induced by low dose ADP, collagen and TRAP, a defect in ATP secretion, a reduced mepacrine uptake and release and a reduced CD63 expression upon TRAP stimulation...
March 2, 2017: Haematologica
https://www.readbyqxmd.com/read/28253977/high-throughput-quantification-of-gfp-lc3-dots-by-automated-fluorescence-microscopy
#20
J M Bravo-San Pedro, F Pietrocola, V Sica, V Izzo, A Sauvat, O Kepp, M C Maiuri, G Kroemer, L Galluzzi
Macroautophagy is a specific variant of autophagy that involves a dedicated double-membraned organelle commonly known as autophagosome. Various methods have been developed to quantify the size of the autophagosomal compartment, which is an indirect indicator of macroautophagic responses, based on the peculiar ability of microtubule-associated protein 1 light chain 3 beta (MAP1LC3B; best known as LC3) to accumulate in forming autophagosomes upon maturation. One particularly convenient method to monitor the accumulation of mature LC3 within autophagosomes relies on a green fluorescent protein (GFP)-tagged variant of this protein and fluorescence microscopy...
2017: Methods in Enzymology
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