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Journal of Visualized Experiments: JoVE

Meena Dhankhar, Marek Vaňatka, Michal Urbanek
Electron and x-ray magnetic microscopies allow for high-resolution magnetic imaging down to tens of nanometers. However, the samples need to be prepared on transparent membranes which are very fragile and difficult to manipulate. We present processes for the fabrication of samples with magnetic micro- and nanostructures with spin configurations forming magnetic vortices suitable for Lorentz transmission electron microscopy and magnetic transmission x-ray microscopy studies. The samples are prepared on silicon nitride membranes and the fabrication consists of a spin coating, UV and electron-beam lithography, the chemical development of the resist, and the evaporation of the magnetic material followed by a lift-off process forming the final magnetic structures...
July 2, 2018: Journal of Visualized Experiments: JoVE
Lars Michel, Pia Stock, Christos Rammos, Matthias Totzeck, Tienush Rassaf, Ulrike B Hendgen-Cotta
Acute myocardial infarction can lead to acute heart failure and cardiogenic shock. The evaluation of hemodynamics is critical for the evaluation of any potential therapeutic approach directed against acute left ventricular (LV) dysfunction. Current imaging modalities (e.g., echocardiography and magnetic resonance imaging) have several limitations since data on LV pressure cannot directly be measured. LV catheterization in mice undergoing coronary artery occlusion could serve as a novel method for a real-time evaluation of LV function...
July 2, 2018: Journal of Visualized Experiments: JoVE
Yanglei Yi, Zhibo Li, Oscar P Kuipers
Beneficial plant-associated bacteria play an important role in promoting growth and preventing disease in plants. The application of plant growth-promoting rhizobacteria (PGPR) as biofertilizers or biocontrol agents has become an effective alternative to the use of conventional fertilizers and can increase crop productivity at low cost. Plant-microbe interactions depend upon host plant-secreted signals and a reaction hereon by their associated bacteria. However, the molecular mechanisms of how beneficial bacteria respond to their associated plant-derived signals are not fully understood...
July 2, 2018: Journal of Visualized Experiments: JoVE
N David Bethoney, Kevin D E Stokesbury
Underwater imaging has long been used in the field of marine ecology but decreasing costs of high-resolution cameras and data storage have made the approach more practical than in the past. Image-based surveys allow for initial samples to be revisited and are non-invasive compared to traditional survey methods that typically involve nets or dredges. Protocols for image-based surveys can vary greatly but should be driven by target species behavior and survey objectives. To demonstrate this, we describe our most recent methods for an Atlantic sea scallop (Placopecten magellanicus) drop camera survey to provide a procedural example and representative results...
July 2, 2018: Journal of Visualized Experiments: JoVE
Tao Jiang, Jose Munguia-Lopez, Salvador Flores-Torres, Joel Grant, Sanahan Vijayakumar, Antonio De Leon-Rodriguez, Joseph M Kinsella
The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell lines using conventional two-dimensional (2D) cell culture. These challenges can be overcome by using bioprinting techniques to build heterogeneous three-dimensional (3D) tumor models whereby different types of cells are embedded. Alginate and gelatin are two of the most common biomaterials employed in bioprinting due to their biocompatibility, biomimicry, and mechanical properties...
July 2, 2018: Journal of Visualized Experiments: JoVE
Junhwan Kwon, Myunghwan Choi
In a mammalian nervous system, myelin provides an electrical insulation by enwrapping the axon fibers in a multilayered spiral. Inspired by its highly-organized subcellular architecture, we recently developed a new imaging modality, named spectral reflectometry (SpeRe), which enables unprecedented label-free nanoscale imaging of the live myelinated axons in situ. The underlying principle is to obtain nanostructural information by analyzing the reflectance spectrum of the multilayered subcellular structure. In this article, we describe a detailed step-by-step protocol for performing a basic SpeRe imaging of the nervous tissues using a commercial confocal microscopic system, equipped with a white-light laser and a tunable filter...
July 2, 2018: Journal of Visualized Experiments: JoVE
Xiaomin Peng, Guitao Zhong, Hao Wang
Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent proteins and generation of fluorescent fusion proteins have been wildly used as an effective tool to directly visualize the protein localization and dynamics in cells. It is especially useful to compare them with well-known organelle markers after co-expression with the protein of interest. Nevertheless, classical approaches for protein co-expression in plants usually involve multiple independent expression plasmids, and therefore have drawbacks that include low co-expression efficiency, expression-level variation, and high time expenditure in genetic crossing and screening...
July 1, 2018: Journal of Visualized Experiments: JoVE
Quinton L Anderson, Alexey V Revtovich, Natalia V Kirienko
The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to combat multiple drug resistance. This has led to the conclusion that researchers do not only need to find new drugs, but also need to develop new ways of finding them. Amongst the most promising candidate methods are whole-organism, in vivo assays that use high-throughput, phenotypic readouts and hosts that range from Caenorhabditis elegans to Danio rerio. These hosts have several powerful advantages, including dramatic reductions in false positive hits, as compounds that are toxic to the host and/or biounavailable are typically dropped in the initial screen, prior to costly follow up...
July 1, 2018: Journal of Visualized Experiments: JoVE
Jonas-Frederic Sauer, Michael Strüber, Marlene Bartos
The local field potential (LFP) emerges from ion movements across neural membranes. Since the voltage recorded by LFP electrodes reflects the summed electrical field of a large volume of brain tissue, extracting information about local activity is challenging. Studying neuronal microcircuits, however, requires a reliable distinction between truly local events and volume-conducted signals originating in distant brain areas. Current source density (CSD) analysis offers a solution for this problem by providing information about current sinks and sources in the vicinity of the electrodes...
July 1, 2018: Journal of Visualized Experiments: JoVE
Dae Hwan Kim, Long-Hui Cui, Ha-Rim Seo, Hyung Joon Joo, Seung-Cheol Choi, Do-Sun Lim, Kyu Back Lee
Nanotopography can be found in various extracellular matrices (ECMs) around the body and is known to have important regulatory actions upon cellular reactions. However, it is difficult to determine the relation between the size of a nanostructure and the responses of cells owing to the lack of proper screening tools. Here, we show the development of reproducible and cost-effective gradient nanopattern plates for the manipulation of cellular responses. Using anodic aluminum oxide (AAO) as a master mold, gradient nanopattern plates with nanopillars of increasing diameter ranges [120-200 nm (GP 120/200), 200-280 nm (GP 200/280), and 280-360 nm (GP 280/360)] were fabricated by a thermal imprinting technique...
July 1, 2018: Journal of Visualized Experiments: JoVE
Shouming Zhang, Xueping Gao, Genhui Xiao, Chengping Lu, Huochun Yao, Hongjie Fan, Zongfu Wu
Streptococcus suis is not only a major bacterial pathogen of pigs worldwide but also an emerging zoonotic agent. In humans and pigs, meningitis is a major manifestation of S. suis infections. A suitable infection model is an essential tool to understand the mechanisms of diseases caused by pathogens. Several routes of infection in mice have been developed to study the pathogenesis of S. suis infection. However, the intraperitoneal, intranasal, and intravenous routes of infection are not suitable for studying the roles of S...
July 1, 2018: Journal of Visualized Experiments: JoVE
Maiken W Rosenstierne, Christopher E Jensen, Anders Fomsgaard
The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood...
June 30, 2018: Journal of Visualized Experiments: JoVE
Mohammadali Masigol, Niloy Barua, Bradley S Lokitz, Ryan R Hansen
In this paper, fabrication methods that generate novel surfaces using the azlactone-based block co-polymer, poly (glycidyl methacrylate)-block-poly (vinyl dimethyl azlactone) (PGMA-b-PVDMA), are presented. Due to the high reactivity of azlactone groups towards amine, thiol, and hydroxyl groups, PGMA-b-PVDMA surfaces can be modified with secondary molecules to create chemically or biologically functionalized interfaces for a variety of applications. Previous reports of patterned PGMA-b-PVDMA interfaces have used traditional top-down patterning techniques that generate non-uniform films and poorly controlled background chemistries...
June 30, 2018: Journal of Visualized Experiments: JoVE
Daniel Andrén, Pawel Karpinski, Mikael Käll
The possibility to generate and measure rotation and torque at the nanoscale is of fundamental interest to the study and application of biological and artificial nanomotors and may provide new routes towards single cell analysis, studies of non-equilibrium thermodynamics, and mechanical actuation of nanoscale systems. A facile way to drive rotation is to use focused circularly polarized laser light in optical tweezers. Using this approach, metallic nanoparticles can be operated as highly efficient scattering-driven rotary motors spinning at unprecedented rotation frequencies in water...
June 30, 2018: Journal of Visualized Experiments: JoVE
Thinh Nguyen, Thomas Potter, Christof Karmonik, Robert Grossman, Yingchun Zhang
Electroencephalography (EEG) and functional magnetic resonance imaging (fMRI) are two of the fundamental noninvasive methods for identifying brain activity. Multimodal methods have sought to combine the high temporal resolution of EEG with the spatial precision of fMRI, but the complexity of this approach is currently in need of improvement. The protocol presented here describes the recently developed spatiotemporal fMRI-constrained EEG source imaging method, which seeks to rectify source biases and improve EEG-fMRI source localization through the dynamic recruitment of fMRI sub-regions...
June 30, 2018: Journal of Visualized Experiments: JoVE
Timo Appelhans, Felix R M Beinlich, Christian P Richter, Rainer Kurre, Karin B Busch
Knowledge about the localization of proteins in cellular subcompartments is crucial to understand their specific function. Here, we present a super-resolution technique that allows for the determination of the microcompartments that are accessible for proteins by generating localization and tracking maps of these proteins. Moreover, by multi-color localization microscopy, the localization and tracking profiles of proteins in different subcompartments are obtained simultaneously. The technique is specific for live cells and is based on the repetitive imaging of single mobile membrane proteins...
June 30, 2018: Journal of Visualized Experiments: JoVE
Megan Stevens, Sebastian Oltean
The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects...
June 30, 2018: Journal of Visualized Experiments: JoVE
Ceres Kosuta, Kate Daniel, Devon L Johnstone, Kevin Mongeon, Kevin Ban, Sophie LeBlanc, Stuart MacLeod, Karim Et-Tahiry, Marc Ekker, Alex MacKenzie, Izabella Pena
Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short generation time, are easy to handle, display a high reproductive rate, low cost, and are easily amenable to genetic manipulations by microinjection of DNA in embryos. Recent advances in gene editing tools are enabling precise introduction of mutations and transgenes in zebrafish. Disease modeling in zebrafish often leads to larval phenotypes and early death which can be challenging to interpret if genotypes are unknown...
June 29, 2018: Journal of Visualized Experiments: JoVE
Yahui Kong, Pantea Ebrahimpour, Yu Liu, Chaoxing Yang, Laura C Alonso
Experiments using isolated pancreatic islets are important for diabetes research, but islets are expensive and of limited abundance. Islets contain a mixed cell population in a structured architecture that impacts function, and human islets are widely variable in cell type composition. Current frequently used methods to study cultured islets include molecular studies performed on whole islets, lumping disparate islet cell types together, or microscopy or molecular studies on dispersed islet cells, disrupting islet architecture...
June 29, 2018: Journal of Visualized Experiments: JoVE
Adam B Cornwell, Jesse R Llop, Peter Salzman, Juilee Thakar, Andrew V Samuelson
The Replica Set method is an approach to quantitatively measure lifespan or survival of Caenorhabditis elegans nematodes in a high-throughput manner, thus allowing a single investigator to screen more treatments or conditions over the same amount of time without loss of data quality. The method requires common equipment found in most laboratories working with C. elegans and is thus simple to adopt. The approach centers on assaying independent samples of a population at each observation point, rather than a single sample over time as with traditional longitudinal methods...
June 29, 2018: Journal of Visualized Experiments: JoVE
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