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Pubmed Article
Seasonally-Dynamic Presence-Only Species Distribution Models for a Cryptic Migratory Bat Impacted by Wind Energy Development.
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PLoS ONE
PUBLISHED: 07-25-2015
Understanding seasonal distribution and movement patterns of animals that migrate long distances is an essential part of monitoring and conserving their populations. Compared to migratory birds and other more conspicuous migrants, we know very little about the movement patterns of many migratory bats. Hoary bats (Lasiurus cinereus), a cryptic, wide-ranging, long-distance migrant, comprise a substantial proportion of the tens to hundreds of thousands of bat fatalities estimated to occur each year at wind turbines in North America. We created seasonally-dynamic species distribution models (SDMs) from 2,753 museum occurrence records collected over five decades in North America to better understand the seasonal geographic distributions of hoary bats. We used 5 SDM approaches: logistic regression, multivariate adaptive regression splines, boosted regression trees, random forest, and maximum entropy and consolidated outputs to generate ensemble maps. These maps represent the first formal hypotheses for sex- and season-specific hoary bat distributions. Our results suggest that North American hoary bats winter in regions with relatively long growing seasons where temperatures are moderated by proximity to oceans, and then move to the continental interior for the summer. SDMs suggested that hoary bats are most broadly distributed in autumn-the season when they are most susceptible to mortality from wind turbines; this season contains the greatest overlap between potentially suitable habitat and wind energy facilities. Comparing wind-turbine fatality data to model outputs could test many predictions, such as 'risk from turbines is highest in habitats between hoary bat summering and wintering grounds'. Although future field studies are needed to validate the SDMs, this study generated well-justified and testable hypotheses of hoary bat migration patterns and seasonal distribution.
ABSTRACT
The mammalian integument includes sebaceous glands that secrete an oily material onto the skin surface. Sebum production is part of the innate immune system that is protective against pathogenic microbes. Abnormal sebum production and chemical composition are also a clinical symptom of specific skin diseases. Sebum contains a complex mixture of lipids, including triacylglycerides, which is species-specific. The broad chemical properties exhibited by diverse lipid classes hinder the specific determination of sebum composition. Analytical techniques for lipids typically require chemical derivatizations that are labor-intensive and increase sample preparation costs. This paper describes how to extract lipids from mammalian integument, separate broad lipid classes by thin-layer chromatography, and profile the triacylglyceride contents using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. This robust method enables a direct determination of the triacylglyceride profiles among species and individuals, and it can be readily applied to any taxonomic group of mammals.
19 Related JoVE Articles!
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Exploring the Effects of Atmospheric Forcings on Evaporation: Experimental Integration of the Atmospheric Boundary Layer and Shallow Subsurface
Authors: Kathleen Smits, Victoria Eagen, Andrew Trautz.
Institutions: Colorado School of Mines.
Evaporation is directly influenced by the interactions between the atmosphere, land surface and soil subsurface. This work aims to experimentally study evaporation under various surface boundary conditions to improve our current understanding and characterization of this multiphase phenomenon as well as to validate numerical heat and mass transfer theories that couple Navier-Stokes flow in the atmosphere and Darcian flow in the porous media. Experimental data were collected using a unique soil tank apparatus interfaced with a small climate controlled wind tunnel. The experimental apparatus was instrumented with a suite of state of the art sensor technologies for the continuous and autonomous collection of soil moisture, soil thermal properties, soil and air temperature, relative humidity, and wind speed. This experimental apparatus can be used to generate data under well controlled boundary conditions, allowing for better control and gathering of accurate data at scales of interest not feasible in the field. Induced airflow at several distinct wind speeds over the soil surface resulted in unique behavior of heat and mass transfer during the different evaporative stages.
Environmental Sciences, Issue 100, Bare-soil evaporation, Land-atmosphere interactions, Heat and mass flux, Porous media, Wind tunnel, Soil thermal properties, Multiphase flow
52704
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Use of Chironomidae (Diptera) Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies
Authors: Petra Kranzfelder, Alyssa M. Anderson, Alexander T. Egan, Jane E. Mazack, R. William Bouchard, Jr., Moriya M. Rufer, Leonard C. Ferrington, Jr..
Institutions: University of Minnesota, Northern State University, Minnesota Pollution Control Agency, RMB Environmental Laboratories, Inc..
Rapid bioassessment protocols using benthic macroinvertebrate assemblages have been successfully used to assess human impacts on water quality. Unfortunately, traditional benthic larval sampling methods, such as the dip-net, can be time-consuming and expensive. An alternative protocol involves collection of Chironomidae surface-floating pupal exuviae (SFPE). Chironomidae is a species-rich family of flies (Diptera) whose immature stages typically occur in aquatic habitats. Adult chironomids emerge from the water, leaving their pupal skins, or exuviae, floating on the water’s surface. Exuviae often accumulate along banks or behind obstructions by action of the wind or water current, where they can be collected to assess chironomid diversity and richness. Chironomids can be used as important biological indicators, since some species are more tolerant to pollution than others. Therefore, the relative abundance and species composition of collected SFPE reflect changes in water quality. Here, methods associated with field collection, laboratory processing, slide mounting, and identification of chironomid SFPE are described in detail. Advantages of the SFPE method include minimal disturbance at a sampling area, efficient and economical sample collection and laboratory processing, ease of identification, applicability in nearly all aquatic environments, and a potentially more sensitive measure of ecosystem stress. Limitations include the inability to determine larval microhabitat use and inability to identify pupal exuviae to species if they have not been associated with adult males.
Environmental Sciences, Issue 101, biological monitoring, aquatic systems, aquatic ecology, water quality, macroinvertebrates, midge, Chironomid Pupal Exuviae Technique, rapid bioassessment protocol
52558
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Cerenkov Luminescence Imaging of Interscapular Brown Adipose Tissue
Authors: Xueli Zhang, Chaincy Kuo, Anna Moore, Chongzhao Ran.
Institutions: Massachusetts General Hospital/Harvard Medical School, China Pharmaceutical University, Perkin Elmer.
Brown adipose tissue (BAT), widely known as a “good fat” plays pivotal roles for thermogenesis in mammals. This special tissue is closely related to metabolism and energy expenditure, and its dysfunction is one important contributor for obesity and diabetes. Contrary to previous belief, recent PET/CT imaging studies indicated the BAT depots are still present in human adults. PET imaging clearly shows that BAT has considerably high uptake of 18F-FDG under certain conditions. In this video report, we demonstrate that Cerenkov luminescence imaging (CLI) with 18F-FDG can be used to optically image BAT in small animals. BAT activation is observed after intraperitoneal injection of norepinephrine (NE) and cold treatment, and depression of BAT is induced by long anesthesia. Using multiple-filter Cerenkov luminescence imaging, spectral unmixing and 3D imaging reconstruction are demonstrated. Our results suggest that CLI with 18F-FDG is a practical technique for imaging BAT in small animals, and this technique can be used as a cheap, fast, and alternative imaging tool for BAT research.
Medicine, Issue 92, Cerenkov luminescence imaging, brown adipose tissue, 18F-FDG, optical imaging, in vivo imaging, spectral unmixing
51790
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Flying Insect Detection and Classification with Inexpensive Sensors
Authors: Yanping Chen, Adena Why, Gustavo Batista, Agenor Mafra-Neto, Eamonn Keogh.
Institutions: University of California, Riverside, University of California, Riverside, University of São Paulo - USP, ISCA Technologies.
An inexpensive, noninvasive system that could accurately classify flying insects would have important implications for entomological research, and allow for the development of many useful applications in vector and pest control for both medical and agricultural entomology. Given this, the last sixty years have seen many research efforts devoted to this task. To date, however, none of this research has had a lasting impact. In this work, we show that pseudo-acoustic optical sensors can produce superior data; that additional features, both intrinsic and extrinsic to the insect’s flight behavior, can be exploited to improve insect classification; that a Bayesian classification approach allows to efficiently learn classification models that are very robust to over-fitting, and a general classification framework allows to easily incorporate arbitrary number of features. We demonstrate the findings with large-scale experiments that dwarf all previous works combined, as measured by the number of insects and the number of species considered.
Bioengineering, Issue 92, flying insect detection, automatic insect classification, pseudo-acoustic optical sensors, Bayesian classification framework, flight sound, circadian rhythm
52111
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Human Brown Adipose Tissue Depots Automatically Segmented by Positron Emission Tomography/Computed Tomography and Registered Magnetic Resonance Images
Authors: Aliya Gifford, Theodore F. Towse, Ronald C. Walker, Malcolm J. Avison, E. Brian Welch.
Institutions: Vanderbilt University, Vanderbilt University School of Medicine, Vanderbilt University Medical Center, Vanderbilt University.
Reliably differentiating brown adipose tissue (BAT) from other tissues using a non-invasive imaging method is an important step toward studying BAT in humans. Detecting BAT is typically confirmed by the uptake of the injected radioactive tracer 18F-Fluorodeoxyglucose (18F-FDG) into adipose tissue depots, as measured by positron emission tomography/computed tomography (PET-CT) scans after exposing the subject to cold stimulus. Fat-water separated magnetic resonance imaging (MRI) has the ability to distinguish BAT without the use of a radioactive tracer. To date, MRI of BAT in adult humans has not been co-registered with cold-activated PET-CT. Therefore, this protocol uses 18F-FDG PET-CT scans to automatically generate a BAT mask, which is then applied to co-registered MRI scans of the same subject. This approach enables measurement of quantitative MRI properties of BAT without manual segmentation. BAT masks are created from two PET-CT scans: after exposure for 2 hr to either thermoneutral (TN) (24 °C) or cold-activated (CA) (17 °C) conditions. The TN and CA PET-CT scans are registered, and the PET standardized uptake and CT Hounsfield values are used to create a mask containing only BAT. CA and TN MRI scans are also acquired on the same subject and registered to the PET-CT scans in order to establish quantitative MRI properties within the automatically defined BAT mask. An advantage of this approach is that the segmentation is completely automated and is based on widely accepted methods for identification of activated BAT (PET-CT). The quantitative MRI properties of BAT established using this protocol can serve as the basis for an MRI-only BAT examination that avoids the radiation associated with PET-CT.
Medicine, Issue 96, magnetic resonance imaging, brown adipose tissue, cold-activation, adult human, fat water imaging, fluorodeoxyglucose, positron emission tomography, computed tomography
52415
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Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT
Authors: Xukui Wang, Laurie J. Minze, Zheng-Zheng Shi.
Institutions: The Methodist Hospital Research Institute, Houston, The Methodist Hospital Research Institute, Houston.
Brown adipose tissue (BAT) differs from white adipose tissue (WAT) by its discrete location and a brown-red color due to rich vascularization and high density of mitochondria. BAT plays a major role in energy expenditure and non-shivering thermogenesis in newborn mammals as well as the adults 1. BAT-mediated thermogenesis is highly regulated by the sympathetic nervous system, predominantly via β adrenergic receptor 2, 3. Recent studies have shown that BAT activities in human adults are negatively correlated with body mass index (BMI) and other diabetic parameters 4-6. BAT has thus been proposed as a potential target for anti-obesity/anti-diabetes therapy focusing on modulation of energy balance 6-8. While several cold challenge-based positron emission tomography (PET) methods are established for detecting human BAT 9-13, there is essentially no standardized protocol for imaging and quantification of BAT in small animal models such as mice. Here we describe a robust PET/CT imaging method for functional assessment of BAT in mice. Briefly, adult C57BL/6J mice were cold treated under fasting conditions for a duration of 4 hours before they received one dose of 18F-Fluorodeoxyglucose (FDG). The mice were remained in the cold for one additional hour post FDG injection, and then scanned with a small animal-dedicated micro-PET/CT system. The acquired PET images were co-registered with the CT images for anatomical references and analyzed for FDG uptake in the interscapular BAT area to present BAT activity. This standardized cold-treatment and imaging protocol has been validated through testing BAT activities during pharmacological interventions, for example, the suppressed BAT activation by the treatment of β-adrenoceptor antagonist propranolol 14, 15, or the enhanced BAT activation by β3 agonist BRL37344 16. The method described here can be applied to screen for drugs/compounds that modulate BAT activity, or to identify genes/pathways that are involved in BAT development and regulation in various preclinical and basic studies.
Molecular Biology, Issue 69, Neuroscience, Anatomy, Physiology, Medicine, Brown adipose tissue, mice, 18F-Fluorodeoxyglucose, micro-PET, PET, CT, CT scan, tomography, imaging
4060
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Automated Quantification of Hematopoietic Cell – Stromal Cell Interactions in Histological Images of Undecalcified Bone
Authors: Sandra Zehentmeier, Zoltan Cseresnyes, Juan Escribano Navarro, Raluca A. Niesner, Anja E. Hauser.
Institutions: German Rheumatism Research Center, a Leibniz Institute, German Rheumatism Research Center, a Leibniz Institute, Max-Delbrück Center for Molecular Medicine, Wimasis GmbH, Charité - University of Medicine.
Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. However, the analysis and quantification of cellular localization is difficult, as in many cases it relies on manual counting, thus bearing the risk of introducing a rater-dependent bias and reducing interrater reliability. Moreover, it is often difficult to judge whether the co-localization between two cells results from random positioning, especially when cell types differ strongly in the frequency of their occurrence. Here, a method for unbiased quantification of cellular co-localization in the bone marrow is introduced. The protocol describes the sample preparation used to obtain histological sections of whole murine long bones including the bone marrow, as well as the staining protocol and the acquisition of high-resolution images. An analysis workflow spanning from the recognition of hematopoietic and non-hematopoietic cell types in 2-dimensional (2D) bone marrow images to the quantification of the direct contacts between those cells is presented. This also includes a neighborhood analysis, to obtain information about the cellular microenvironment surrounding a certain cell type. In order to evaluate whether co-localization of two cell types is the mere result of random cell positioning or reflects preferential associations between the cells, a simulation tool which is suitable for testing this hypothesis in the case of hematopoietic as well as stromal cells, is used. This approach is not limited to the bone marrow, and can be extended to other tissues to permit reproducible, quantitative analysis of histological data.
Developmental Biology, Issue 98, Image analysis, neighborhood analysis, bone marrow, stromal cells, bone marrow niches, simulation, bone cryosectioning, bone histology
52544
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Vision Training Methods for Sports Concussion Mitigation and Management
Authors: Joseph F. Clark, Angelo Colosimo, James K. Ellis, Robert Mangine, Benjamin Bixenmann, Kimberly Hasselfeld, Patricia Graman, Hagar Elgendy, Gregory Myer, Jon Divine.
Institutions: University of Cincinnati, University of Cincinnati, University of Cincinnati, University of Cincinnati, University of Cincinnati, Cincinnati Children's Hospital Medical Center.
There is emerging evidence supporting the use vision training, including light board training tools, as a concussion baseline and neuro-diagnostic tool and potentially as a supportive component to concussion prevention strategies. This paper is focused on providing detailed methods for select vision training tools and reporting normative data for comparison when vision training is a part of a sports management program. The overall program includes standard vision training methods including tachistoscope, Brock’s string, and strobe glasses, as well as specialized light board training algorithms. Stereopsis is measured as a means to monitor vision training affects. In addition, quantitative results for vision training methods as well as baseline and post-testing *A and Reaction Test measures with progressive scores are reported. Collegiate athletes consistently improve after six weeks of training in their stereopsis, *A and Reaction Test scores. When vision training is initiated as a team wide exercise, the incidence of concussion decreases in players who participate in training compared to players who do not receive the vision training. Vision training produces functional and performance changes that, when monitored, can be used to assess the success of the vision training and can be initiated as part of a sports medical intervention for concussion prevention.
Behavior, Issue 99, Vision training, peripheral vision, functional peripheral vision, concussion, concussion management, diagnosis, rehabilitation, eyes, sight, seeing, sight
52648
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Laboratory-determined Phosphorus Flux from Lake Sediments as a Measure of Internal Phosphorus Loading
Authors: Mary E. Ogdahl, Alan D. Steinman, Maggie E. Weinert.
Institutions: Grand Valley State University.
Eutrophication is a water quality issue in lakes worldwide, and there is a critical need to identify and control nutrient sources. Internal phosphorus (P) loading from lake sediments can account for a substantial portion of the total P load in eutrophic, and some mesotrophic, lakes. Laboratory determination of P release rates from sediment cores is one approach for determining the role of internal P loading and guiding management decisions. Two principal alternatives to experimental determination of sediment P release exist for estimating internal load: in situ measurements of changes in hypolimnetic P over time and P mass balance. The experimental approach using laboratory-based sediment incubations to quantify internal P load is a direct method, making it a valuable tool for lake management and restoration. Laboratory incubations of sediment cores can help determine the relative importance of internal vs. external P loads, as well as be used to answer a variety of lake management and research questions. We illustrate the use of sediment core incubations to assess the effectiveness of an aluminum sulfate (alum) treatment for reducing sediment P release. Other research questions that can be investigated using this approach include the effects of sediment resuspension and bioturbation on P release. The approach also has limitations. Assumptions must be made with respect to: extrapolating results from sediment cores to the entire lake; deciding over what time periods to measure nutrient release; and addressing possible core tube artifacts. A comprehensive dissolved oxygen monitoring strategy to assess temporal and spatial redox status in the lake provides greater confidence in annual P loads estimated from sediment core incubations.
Environmental Sciences, Issue 85, Limnology, internal loading, eutrophication, nutrient flux, sediment coring, phosphorus, lakes
51617
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Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport
Authors: Yves Molino, Françoise Jabès, Emmanuelle Lacassagne, Nicolas Gaudin, Michel Khrestchatisky.
Institutions: VECT-HORUS SAS, CNRS, NICN UMR 7259.
The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm·cm2 on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 ± 0.11 x 10-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.
Medicine, Issue 88, rat brain endothelial cells (RBEC), mouse, spinal cord, tight junction (TJ), receptor-mediated transport (RMT), low density lipoprotein (LDL), LDLR, transferrin, TfR, P-glycoprotein (P-gp), transendothelial electrical resistance (TEER),
51278
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Using Informational Connectivity to Measure the Synchronous Emergence of fMRI Multi-voxel Information Across Time
Authors: Marc N. Coutanche, Sharon L. Thompson-Schill.
Institutions: University of Pennsylvania.
It is now appreciated that condition-relevant information can be present within distributed patterns of functional magnetic resonance imaging (fMRI) brain activity, even for conditions with similar levels of univariate activation. Multi-voxel pattern (MVP) analysis has been used to decode this information with great success. FMRI investigators also often seek to understand how brain regions interact in interconnected networks, and use functional connectivity (FC) to identify regions that have correlated responses over time. Just as univariate analyses can be insensitive to information in MVPs, FC may not fully characterize the brain networks that process conditions with characteristic MVP signatures. The method described here, informational connectivity (IC), can identify regions with correlated changes in MVP-discriminability across time, revealing connectivity that is not accessible to FC. The method can be exploratory, using searchlights to identify seed-connected areas, or planned, between pre-selected regions-of-interest. The results can elucidate networks of regions that process MVP-related conditions, can breakdown MVPA searchlight maps into separate networks, or can be compared across tasks and patient groups.
Neuroscience, Issue 89, fMRI, MVPA, connectivity, informational connectivity, functional connectivity, networks, multi-voxel pattern analysis, decoding, classification, method, multivariate
51226
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Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study
Authors: Johannes Felix Buyel, Rainer Fischer.
Institutions: RWTH Aachen University, Fraunhofer Gesellschaft.
Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems.
Bioengineering, Issue 83, design of experiments (DoE), transient protein expression, plant-derived biopharmaceuticals, promoter, 5'UTR, fluorescent reporter protein, model building, incubation conditions, monoclonal antibody
51216
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Monitoring Intraspecies Competition in a Bacterial Cell Population by Cocultivation of Fluorescently Labelled Strains
Authors: Lorena Stannek, Richard Egelkamp, Katrin Gunka, Fabian M. Commichau.
Institutions: Georg-August University.
Many microorganisms such as bacteria proliferate extremely fast and the populations may reach high cell densities. Small fractions of cells in a population always have accumulated mutations that are either detrimental or beneficial for the cell. If the fitness effect of a mutation provides the subpopulation with a strong selective growth advantage, the individuals of this subpopulation may rapidly outcompete and even completely eliminate their immediate fellows. Thus, small genetic changes and selection-driven accumulation of cells that have acquired beneficial mutations may lead to a complete shift of the genotype of a cell population. Here we present a procedure to monitor the rapid clonal expansion and elimination of beneficial and detrimental mutations, respectively, in a bacterial cell population over time by cocultivation of fluorescently labeled individuals of the Gram-positive model bacterium Bacillus subtilis. The method is easy to perform and very illustrative to display intraspecies competition among the individuals in a bacterial cell population.
Cellular Biology, Issue 83, Bacillus subtilis, evolution, adaptation, selective pressure, beneficial mutation, intraspecies competition, fluorophore-labelling, Fluorescence Microscopy
51196
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Investigating the Three-dimensional Flow Separation Induced by a Model Vocal Fold Polyp
Authors: Kelley C. Stewart, Byron D. Erath, Michael W. Plesniak.
Institutions: The George Washington University, Clarkson University.
The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. Polyps and nodules, which are geometric abnormalities that form on the medial surface of the vocal folds, can disrupt vocal fold dynamics and thus can have devastating consequences on a patient's ability to communicate. Our laboratory has reported particle image velocimetry (PIV) measurements, within an investigation of a model polyp located on the medial surface of an in vitro driven vocal fold model, which show that such a geometric abnormality considerably disrupts the glottal jet behavior. This flow field adjustment is a likely reason for the severe degradation of the vocal quality in patients with polyps. A more complete understanding of the formation and propagation of vortical structures from a geometric protuberance, such as a vocal fold polyp, and the resulting influence on the aerodynamic loadings that drive the vocal fold dynamics, is necessary for advancing the treatment of this pathological condition. The present investigation concerns the three-dimensional flow separation induced by a wall-mounted prolate hemispheroid with a 2:1 aspect ratio in cross flow, i.e. a model vocal fold polyp, using an oil-film visualization technique. Unsteady, three-dimensional flow separation and its impact of the wall pressure loading are examined using skin friction line visualization and wall pressure measurements.
Bioengineering, Issue 84, oil-flow visualization, vocal fold polyp, three-dimensional flow separation, aerodynamic pressure loadings
51080
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Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy
Authors: Nikki M. Curthoys, Michael J. Mlodzianoski, Dahan Kim, Samuel T. Hess.
Institutions: University of Maine.
Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.
Basic Protocol, Issue 82, Microscopy, Super-resolution imaging, Multicolor, single molecule, FPALM, Localization microscopy, fluorescent proteins
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Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells
Authors: Ulrike Liisberg Aune, Lauren Ruiz, Shingo Kajimura.
Institutions: University of California, San Francisco , University of Copenhagen, Denmark, National Institute of Nutrition and Seafood Research, Bergen, Norway.
Brown adipocytes have the ability to uncouple the respiratory chain in mitochondria and dissipate chemical energy as heat. Development of UCP1-positive brown adipocytes in white adipose tissues (so called beige or brite cells) is highly induced by a variety of environmental cues such as chronic cold exposure or by PPARγ agonists, therefore, this cell type has potential as a therapeutic target for obesity treatment. Although most immortalized adipocyte lines cannot recapitulate the process of "browning" of white fat in culture, primary adipocytes isolated from stromal vascular fraction in subcutaneous white adipose tissue (WAT) provide a reliable cellular system to study the molecular control of beige/brite cell development. Here we describe a protocol for effective isolation of primary preadipocytes and for inducing differentiation to beige/brite cells in culture. The browning effect can be assessed by the expression of brown fat-selective markers such as UCP1.
Cellular Biology, Issue 73, Medicine, Anatomy, Physiology, Molecular Biology, Surgery, Adipose Tissue, Adipocytes, Transcription Factors, Cell Differentiation, Obesity, Diabetes, brown adipose tissue, beige/brite cells, primary adipocytes, stromal-vascular fraction, differentiation, uncoupling protein 1, rosiglitazone, differentiation, cells, isolation, fat, animal model
50191
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Trajectory Data Analyses for Pedestrian Space-time Activity Study
Authors: Feng Qi, Fei Du.
Institutions: Kean University, University of Wisconsin-Madison.
It is well recognized that human movement in the spatial and temporal dimensions has direct influence on disease transmission1-3. An infectious disease typically spreads via contact between infected and susceptible individuals in their overlapped activity spaces. Therefore, daily mobility-activity information can be used as an indicator to measure exposures to risk factors of infection. However, a major difficulty and thus the reason for paucity of studies of infectious disease transmission at the micro scale arise from the lack of detailed individual mobility data. Previously in transportation and tourism research detailed space-time activity data often relied on the time-space diary technique, which requires subjects to actively record their activities in time and space. This is highly demanding for the participants and collaboration from the participants greatly affects the quality of data4. Modern technologies such as GPS and mobile communications have made possible the automatic collection of trajectory data. The data collected, however, is not ideal for modeling human space-time activities, limited by the accuracies of existing devices. There is also no readily available tool for efficient processing of the data for human behavior study. We present here a suite of methods and an integrated ArcGIS desktop-based visual interface for the pre-processing and spatiotemporal analyses of trajectory data. We provide examples of how such processing may be used to model human space-time activities, especially with error-rich pedestrian trajectory data, that could be useful in public health studies such as infectious disease transmission modeling. The procedure presented includes pre-processing, trajectory segmentation, activity space characterization, density estimation and visualization, and a few other exploratory analysis methods. Pre-processing is the cleaning of noisy raw trajectory data. We introduce an interactive visual pre-processing interface as well as an automatic module. Trajectory segmentation5 involves the identification of indoor and outdoor parts from pre-processed space-time tracks. Again, both interactive visual segmentation and automatic segmentation are supported. Segmented space-time tracks are then analyzed to derive characteristics of one's activity space such as activity radius etc. Density estimation and visualization are used to examine large amount of trajectory data to model hot spots and interactions. We demonstrate both density surface mapping6 and density volume rendering7. We also include a couple of other exploratory data analyses (EDA) and visualizations tools, such as Google Earth animation support and connection analysis. The suite of analytical as well as visual methods presented in this paper may be applied to any trajectory data for space-time activity studies.
Environmental Sciences, Issue 72, Computer Science, Behavior, Infectious Diseases, Geography, Cartography, Data Display, Disease Outbreaks, cartography, human behavior, Trajectory data, space-time activity, GPS, GIS, ArcGIS, spatiotemporal analysis, visualization, segmentation, density surface, density volume, exploratory data analysis, modelling
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Manufacturing and Using Piggy-back Multibarrel Electrodes for In vivo Pharmacological Manipulations of Neural Responses
Authors: Anna Dondzillo, Jennifer L. Thornton, Daniel J. Tollin, Achim Klug.
Institutions: University of Colorado Medical Campus.
In vivo recordings from single neurons allow an investigator to examine the firing properties of neurons, for example in response to sensory stimuli. Neurons typically receive multiple excitatory and inhibitory afferent and/or efferent inputs that integrate with each other, and the ultimate measured response properties of the neuron are driven by the neural integrations of these inputs. To study information processing in neural systems, it is necessary to understand the various inputs to a neuron or neural system, and the specific properties of these inputs. A powerful and technically relatively simple method to assess the functional role of certain inputs that a given neuron is receiving is to dynamically and reversibly suppress or eliminate these inputs, and measure the changes in the neuron's output caused by this manipulation. This can be accomplished by pharmacologically altering the neuron's immediate environment with piggy-back multibarrel electrodes. These electrodes consist of a single barrel recording electrode and a multibarrel drug electrode that can carry up to 4 different synaptic agonists or antagonists. The pharmacological agents can be applied iontophoretically at desired times during the experiment, allowing for time-controlled delivery and reversible reconfiguration of synaptic inputs. As such, pharmacological manipulation of the microenvironment represents a powerful and unparalleled method to test specific hypotheses about neural circuit function. Here we describe how piggy-back electrodes are manufactured, and how they are used during in vivo experiments. The piggy-back system allows an investigator to combine a single barrel recording electrode of any arbitrary property (resistance, tip size, shape etc) with a multibarrel drug electrode. This is a major advantage over standard multi-electrodes, where all barrels have more or less similar shapes and properties. Multibarrel electrodes were first introduced over 40 years ago 1-3, and have undergone a number of design improvements 2,3 until the piggy-back type was introduced in the 1980s 4,5. Here we present a set of important improvements in the laboratory production of piggy-back electrodes that allow for deep brain penetration in intact in vivo animal preparations due to a relatively thin electrode shaft that causes minimal damage. Furthermore these electrodes are characterized by low noise recordings, and have low resistance drug barrels for very effective iontophoresis of the desired pharmacological agents.
Neuroscience, Issue 71, Biophysics, Physiology, Neurobiology, Medicine, Pharmacology, Mechanical Engineering, Electrical Engineering, Piggyback electrode, iontophoresis, iontophoresis pump, single cell recording, neural excitation, neural inhibition, in vivo electrophysiology
4358
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Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins
Authors: Savannah E. Sanchez, Daniel A. Cuevas, Jason E. Rostron, Tiffany Y. Liang, Cullen G. Pivaroff, Matthew R. Haynes, Jim Nulton, Ben Felts, Barbara A. Bailey, Peter Salamon, Robert A. Edwards, Alex B. Burgin, Anca M. Segall, Forest Rohwer.
Institutions: San Diego State University, San Diego State University, San Diego State University, San Diego State University, San Diego State University, Argonne National Laboratory, Broad Institute.
Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.
Immunology, Issue 100, phenomics, phage, viral metagenome, Multi-phenotype Assay Plates (MAPs), continuous culture, metabolomics
52854
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