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https://www.readbyqxmd.com/read/28528643/a-new-method-for-cryo-sectioning-cell-monolayers-using-a-correlative-workflow
#1
Androniki Kolovou, Martin Schorb, Abul Tarafder, Carsten Sachse, Yannick Schwab, Rachel Santarella-Mellwig
Cryo-electron microscopy (cryo-EM) techniques have made a huge advancement recently, providing close to atomic resolution of the structure of protein complexes. Interestingly, this imaging technique can be performed in cells, giving access to the molecular machines in their natural context, therefore bridging structural and cell biology. However, in situ structural electron microscopy faces one major challenge, which is the ability to focus on specific subcellular regions to capture the objects of interest...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/28528642/minimal-resin-embedding-of-multicellular-specimens-for-targeted-fib-sem-imaging
#2
Nicole L Schieber, Pedro Machado, Sebastian M Markert, Christian Stigloher, Yannick Schwab, Anna M Steyer
Correlative light and electron microscopy (CLEM) is a powerful tool to perform ultrastructural analysis of targeted tissues or cells. The large field of view of the light microscope (LM) enables quick and efficient surveys of the whole specimen. It is also compatible with live imaging, giving access to functional assays. CLEM protocols take advantage of the features to efficiently retrace the position of targeted sites when switching from one modality to the other. They more often rely on anatomical cues that are visible both by light and electron microscopy...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/28528641/preserving-the-photoswitching-ability-of-standard-fluorescent-proteins-for-correlative-in-resin-super-resolution-and-electron-microscopy
#3
Errin Johnson, Rainer Kaufmann
There are many different correlative light and electron microscopy (CLEM) techniques available. The use of super-resolution microscopy in CLEM is an emerging application and while offering the obvious advantages of improved resolution in the fluorescence image, and therefore more precise correlation to electron microscopy (EM) ultrastructure, it also presents new challenges. Choice of fluorophore, method of fixation, and timing of the fluorescence imaging are critical to the success of super-resolution CLEM and the relative importance, and technical difficulty, of each of these factors depends on the type of super-resolution microscopy being employed...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/28528640/ec-clem-a-multidimension-multimodel-software-to-correlate-intermodal-images-with-a-focus-on-light-and-electron-microscopy
#4
Xavier Heiligenstein, Perrine Paul-Gilloteaux, Graça Raposo, Jean Salamero
Correlative light and electron microscopy (CLEM) is a scientific method covered by a broad range of techniques. The path taken to explore a scientific question is often driven both by the question and the technology available. Yet, one common step to all CLEM workflows is the registration of the multimodal images to assign a fluorescent signal to an ultrastructure. The manual relocation and registration of light microscopy and electron microscopy images can be challenging and time-consuming (Muller-Reichert & Verkade, 2014)...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/28528639/matrix-maps-an-intuitive-software-to-acquire-analyze-and-annotate-light-microscopy-data-for-clem
#5
Martin Schorb, Frank Sieckmann
Matrix MAPS provides an intuitive interface for acquiring light microscopy data during a correlative light and electron microscopy experiment either at room or cryogenic temperatures. First, the user graphically defines the geometry of the acquisition region on top of preview images. Multiple independent regions can then be imaged in an automated way, each with individual settings. The same interface allows the user to mark and select points or regions of interest whose coordinates can subsequently be transferred directly to the electron microscope...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/28528638/triclem-combining-high-precision-room-temperature-clem-with%C3%A2-cryo-fluorescence-microscopy-to-identify-very-rare-events
#6
Nicholas R Ader, Wanda Kukulski
Fiducial-based correlation of fluorescence and electron microscopy data from high-pressure frozen and resin-embedded samples allows for high-precision localization of fluorescent signals to subcellular ultrastructure. Here we introduce the triCLEM procedure to facilitate the identification of very rare events for high-precision correlation. We present a detailed protocol to screen high-pressure frozen cell monolayers on sapphire disks for very rare signals by cryo-fluorescence microscopy, relocate the cells of interest after freeze substitution and Lowicryl embedding, and perform fiducial-based correlation of the identified fluorescent signals to high-magnification electron tomograms...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/28528633/correlative-light-electron-microscopy-in-liquid-using%C3%A2-an-inverted-sem-asem
#7
Chikara Sato, Takaaki Kinoshita, Nassirhadjy Memtily, Mari Sato, Shoko Nishihara, Toshiko Yamazawa, Shinya Sugimoto
In atmospheric scanning electron microscope (ASEM), the inverted scanning electron microscope (SEM) observes the wet sample from below, while an optical microscope observes it from above simultaneously. The ASEM sample holder has a disposable dish shape with a silicon nitride film window at the bottom. It can be coated variously for the primary-culture of substrate-sensitive cells; primary cells were cultured in a few milliliters of culture medium in a stable incubator environment. For the inverted SEM observation, cells and the excised tissue blocks were aldehyde-fixed, immersed in radical scavenger solution, and observed at minimum electron dose...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/28528631/a-fully-integrated-three-dimensional-fluorescence-to-electron-microscopy-correlative-workflow
#8
Claudia S López, Cedric Bouchet-Marquis, Christopher P Arthur, Jessica L Riesterer, Gregor Heiss, Guillaume Thibault, Lee Pullan, Sunjong Kwon, Joe W Gray
While fluorescence microscopy provides tools for highly specific labeling and sensitive detection, its resolution limit and lack of general contrast has hindered studies of cellular structure and protein localization. Recent advances in correlative light and electron microscopy (CLEM), including the fully integrated CLEM workflow instrument, the FEI CorrSight with MAPS, have allowed for a more reliable, reproducible, and quicker approach to correlate three-dimensional time-lapse confocal fluorescence data, with three-dimensional focused ion beam-scanning electron microscopy data...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/28508074/pressure-compression-of-cdse-nanoparticles-into-luminescent-nanowires
#9
Binsong Li, Kaifu Bian, Xiaowang Zhou, Ping Lu, Sheng Liu, Igal Brener, Michael Sinclair, Ting Luk, Hattie Schunk, Leanne Alarid, Paul G Clem, Zhongwu Wang, Hongyou Fan
Oriented attachment (OA) of synthetic nanocrystals is emerging as an effective means of fabricating low-dimensional nanoscale materials. However, OA relies on energetically favorable nanocrystal facets to grow nanostructured materials. Consequently, nanostructures synthesized through OA are generally limited to a specific crystal facet in their final morphology. We report our discovery that high-pressure compression can induce consolidation of spherical CdSe nanocrystal arrays, leading to unexpected one-dimensional semiconductor nanowires that do not exhibit the typical crystal facet...
May 2017: Science Advances
https://www.readbyqxmd.com/read/28481640/multiple-breath-washout-outcomes-are-sensitive-to-inflammation-and-infection-in-children-with-cystic-fibrosis
#10
Kathryn A Ramsey, Rachel E Foong, Jasmine Grdosic, Alana Harper, Billy Skoric, Charles Clem, Miriam Davis, Lidija Turkovic, Stephen M Stick, Stephanie D Davis, Sarath C Ranganathan, Graham L Hall
RATIONALE: The lung clearance index is a measure of ventilation distribution from the multiple breath washout technique. The lung clearance index is increased in the presence of lower respiratory tract inflammation and infection in infants with cystic fibrosis, however the associations during the preschool years are unknown. OBJECTIVES: We assessed the ability of the lung clearance index to detect the presence and extent of lower respiratory tract inflammation and infection in preschool children with cystic fibrosis...
May 8, 2017: Annals of the American Thoracic Society
https://www.readbyqxmd.com/read/28472592/enhanced-neuronal-synchrony-during-skilled-reaching-at-high-altitude
#11
Matt Gaidica, Jenna Clem
No abstract text is available yet for this article.
May 4, 2017: High Altitude Medicine & Biology
https://www.readbyqxmd.com/read/28470743/correlative-light-and-electron-microscopy-reveals-discrepancy-between-gold-and-fluorescence-labelling
#12
D M VAN Elsland, E Bos, J B Pawlak, H S Overkleeft, A J Koster, S I VAN Kasteren
Electron microscopy (EM) is traditionally employed as a follow-up to fluorescence microscopy (FM) to resolve the cellular ultrastructures wherein fluorescently labelled biomolecules reside. In order to translate the information derived from FM studies to EM analysis, biomolecules of interest must be identified in a manner compatible with EM. Although fluorescent signals can serve this purpose when FM is combined with EM in correlative light and electron microscopy (CLEM), the traditional immunogold labelling remains commonly used in this context...
May 4, 2017: Journal of Microscopy
https://www.readbyqxmd.com/read/28450447/fair-play-a-study-of-scientific-workforce-trainers-experience-playing-an-educational-video-game-about-racial-bias
#13
Anna Kaatz, Molly Carnes, Belinda Gutierrez, Julia Savoy, Clem Samuel, Amarette Filut, Christine Maidl Pribbenow
Explicit racial bias has decreased in the United States, but racial stereotypes still exist and conspire in multiple ways to perpetuate the underparticipation of Blacks in science careers. Capitalizing on the potential effectiveness of role-playing video games to promote the type of active learning required to increase awareness of and reduce subtle racial bias, we developed the video game Fair Play, in which players take on the role of Jamal, a Black male graduate student in science, who experiences discrimination in his PhD program...
2017: CBE Life Sciences Education
https://www.readbyqxmd.com/read/28435429/antibacterial-and-cytotoxic-activities-of-naphthoquinone-pigments-from-onosma-visianii-clem
#14
Milena D Vukic, Nenad L Vukovic, Gorica T Djelic, Suzana Lj Popovic, Milan M Zaric, Dejan D Baskic, Gordana B Krstic, Vele V Tesevic, Miroslava M Kacaniova
In this study, the antibacterial and cytotoxic activities of isolated compounds from the roots of Onosma visianii were investigated. By using different chromatographic techniques and appropriate spectroscopic methods, the seven naphthoquinones were described: deoxyshikonin ( 1 ), isobutyrylshikonin ( 2 ), α-methylbutyrylshikonin ( 3 ), acetylshikonin ( 4 ), β-hydroxyisovalerylshikonin ( 5 ), 5,8-O-dimethyl isobutyrylshikonin ( 6 ) and 5,8-O-dimethyl deoxyshikonin ( 7 ). Among the tested compounds, 3 and 4 exhibited the highest antibacterial activities toward all tested bacterial species (MIC50 and MIC90 for gram positive bacteria: 6...
2017: EXCLI journal
https://www.readbyqxmd.com/read/28430270/atomistic-non-adiabatic-dynamics-of-the-lh2-complex-with-a-gpu-accelerated-ab-initio-exciton-model
#15
Aaron Sisto, Clem Stross, Marc W van der Kamp, Michael O'Connor, Simon McIntosh-Smith, Graham T Johnson, Edward G Hohenstein, Fred R Manby, David R Glowacki, Todd J Martinez
We recently outlined an efficient multi-tiered parallel ab initio excitonic framework that utilizes time dependent density functional theory (TDDFT) to calculate ground and excited state energies and gradients of large supramolecular complexes in atomistic detail - enabling us to undertake non-adiabatic simulations which explicitly account for the coupled anharmonic vibrational motion of all the constituent atoms in a supramolecular system. Here we apply that framework to the 27 coupled bacterio-chlorophyll-a chromophores which make up the LH2 complex, using it to compute an on-the-fly nonadiabatic surface-hopping (SH) trajectory of electronically excited LH2...
April 21, 2017: Physical Chemistry Chemical Physics: PCCP
https://www.readbyqxmd.com/read/28429122/quantifying-golgi-structure-using-em-combining-volume-sem-and-stereology-for-higher-throughput
#16
REVIEW
Sophie Ferguson, Anna M Steyer, Terry M Mayhew, Yannick Schwab, John Milton Lucocq
Investigating organelles such as the Golgi complex depends increasingly on high-throughput quantitative morphological analyses from multiple experimental or genetic conditions. Light microscopy (LM) has been an effective tool for screening but fails to reveal fine details of Golgi structures such as vesicles, tubules and cisternae. Electron microscopy (EM) has sufficient resolution but traditional transmission EM (TEM) methods are slow and inefficient. Newer volume scanning EM (volume-SEM) methods now have the potential to speed up 3D analysis by automated sectioning and imaging...
April 20, 2017: Histochemistry and Cell Biology
https://www.readbyqxmd.com/read/28425495/the-biglen-gpr171-peptide-receptor-system-within-the-basolateral-amygdala-regulates-anxiety-like-behavior-and-contextual-fear-conditioning
#17
Erin N Bobeck, Ivone Gomes, Darlene Pena, Kirstie A Cummings, Roger L Clem, Mihaly Mezei, Lakshmi A Devi
Studies show that neuropeptide-receptor systems in the basolateral amygdala (BLA) play an important role in the pathology of anxiety and other mood disorders. Since GPR171, a recently deorphanized receptor for the abundant neuropeptide BigLEN, is expressed in the BLA, we investigated its role in fear and anxiety-like behaviors. To carry out these studies we identified small molecule ligands using a homology model of GPR171 to virtually screen a library of compounds. One of the hits, MS0021570_1, was identified as a GPR171 antagonist based on its ability to block (i) BigLEN-mediated activation of GPR171 in heterologous cells, (ii) BigLEN-mediated hyperpolarization of BLA pyramidal neurons, and (iii) feeding induced by DREADD-mediated activation of BigLEN containing AgRP neurons in the arcuate nucleus...
April 20, 2017: Neuropsychopharmacology: Official Publication of the American College of Neuropsychopharmacology
https://www.readbyqxmd.com/read/28384138/diverse-protocols-for-correlative-super-resolution-fluorescence-imaging-and-electron-microscopy-of-chemically-fixed-samples
#18
Benjamin G Kopek, Maria G Paez-Segala, Gleb Shtengel, Kem A Sochacki, Mei G Sun, Yalin Wang, C Shan Xu, Schuyler B van Engelenburg, Justin W Taraska, Loren L Looger, Harald F Hess
Our groups have recently developed related approaches for sample preparation for super-resolution imaging within endogenous cellular environments using correlative light and electron microscopy (CLEM). Four distinct techniques for preparing and acquiring super-resolution CLEM data sets for aldehyde-fixed specimens are provided, including Tokuyasu cryosectioning, whole-cell mount, cell unroofing and platinum replication, and resin embedding and sectioning. The choice of the best protocol for a given application depends on a number of criteria that are discussed in detail...
May 2017: Nature Protocols
https://www.readbyqxmd.com/read/28361485/the-use-of-correlative-light-electron-microscopy-clem-to-study-pink1-parkin-mediated-mitophagy
#19
Chieko Kishi-Itakura, Folma Buss
In this chapter we describe the use of correlative light-electron microscopy (CLEM) to study, in cultured cells, the turnover of damaged mitochondria by PINK1/Parkin-dependent mitophagy. CLEM combines the advantages of light microscopy, which allows to image and rapidly screen a large number of the cells, while electron microscopy provides high-resolution imaging of these selected cells and a detailed structural analysis of their cellular organelles. We describe in detail how to prepare the cell cultures for optimum preservation of their cellular ultrastructure for CLEM using the most suitable buffers, fixatives, and embedding resins...
March 31, 2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28348028/examination-of-e-faecalis-toxin-antitoxin-ta-toxin-fst-function-utilizing-a-pheromone-inducible-expression-vector-with-tight-repression-and-large-dynamic-range
#20
Keith E Weaver, Yuqing Chen, Elly M Miiller, Jake N Johnson, Alex A Dangler, Dawn A Manias, Aaron M Clem, Daniel J Schjodt, Gary M Dunny
Tools for regulated gene expression in E. faecalis are extremely limited. In this study we describe the construction of an expression vector for E. faecalis, designated pCIE, utilizing the PQ pheromone-responsive promoter of plasmid pCF10. We demonstrate that this promoter is tightly repressed, responds to nanogram quantities of the peptide pheromone, and has a large dynamic range. To demonstrate its utility, the promoter was used to control expression of the toxic peptides of two par family toxin-antitoxin loci present in E...
March 27, 2017: Journal of Bacteriology
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