University of Delaware View Institution's Website 33 articles published in JoVE Biology Whole Mount Imaging to Visualize and Quantify Peripheral Lens Structure, Cell Morphology, and Organization Grace Emin*1, Sadia T. Islam*1, Rylee E. King1, Velia M. Fowler1, Catherine Cheng2, Justin Parreno1,3 1Department of Biological Sciences, University of Delaware, 2School of Optometry and Vision Science Program, Indiana University, 3Department of Biomedical Engineering, University of Delaware The present protocols describe novel whole mount imaging for the visualization of peripheral structures in the ocular lens with methods for image quantification. These protocols can be used in studies to better understand the relationship between lens microscale structures and lens development/function. Medicine Measurement of Healthy and Injured Triceps Surae Morphology Hayley Powell Smitheman1, Kayla D. Seymore1, Morgan N. Potter1, Andy K. Smith1, Susanna Aufwerber2,3, Karin Grävare Silbernagel1 1Department of Physical Therapy, University of Delaware, 2Department of Molecular Medicine and Surgery, Karolinska Institute, 3Women’s Health and Allied Health Professionals Theme, Medical Unit of Occupational Therapy and Physiotherapy, Karolinska University Hospital Ultrasound imaging is becoming more accessible in clinical and research settings, and a consistent protocol will be beneficial for comparison between studies and for clinical interpretations. This protocol for ultrasound evaluation is a valid and reliable method to evaluate Achilles tendon morphology in healthy, tendinopathic, and ruptured tendons. Neuroscience Using the Chick Embryo Brain as a Model for In Vivo and Ex Vivo Analyses of Human Glioblastoma Cell Behavior Nicole G. Pastorino1, Saori Tomatsu1, Amy Lin1, Jackson Doerr1, Zachary Waterman1, Krisztina Sershen1, Pulak Ray2, Analiz Rodriguez3, Deni S. Galileo1 1Department of Biological Sciences, University of Delaware, 2Helen F. Graham Cancer Center and Research Institute, Christiana Care, 3Department of Neurosurgery, Winthrop P. Rockefeller Cancer Institute, University of Arkansas for Medical Sciences Chick embryos are used for studying human glioblastoma (GBM) brain tumors in ovo and in ex vivo brain slice co-cultures. GBM cell behavior can be recorded by time-lapse microscopy in ex vivo co-cultures, and both preparations can be analyzed at the experimental endpoint by detailed 3D confocal analysis. Developmental Biology Visualizing Early Infection Sites of Rice Blast Disease (Magnaporthe oryzae) on Barley (Hordeum vulgare) Using a Basic Microscope and a Smartphone Jessica G. Cooper1, Nicole M. Donofrio1, Jeffrey L. Caplan1, Timothy R. Chaya1 1Department of Plant and Soil Sciences, University of Delaware This is a straightforward protocol of a barley leaf sheath assay using minimal reagents and common laboratory equipment (including a basic smartphone). The purpose is to visualize the early infection process of blast disease in labs without access to advanced microscopy equipment. Genetics Measuring Embryonic Viability and Brood Size in Caenorhabditis elegans Ji Kent Kwah1, Aimee Jaramillo-Lambert1 1Department of Biological Sciences, University of Delaware Here, we present a general method to determine the embryonic viability and total number of embryos produced (brood) using the model organism C. elegans. Neuroscience Assessing Corticospinal Excitability During Goal-Directed Reaching Behavior Isaac N. Gomez1, Serena R. Orsinger1, Hyosub E. Kim2, Ian Greenhouse1 1Department of Human Physiology, University of Oregon, 2Department of Physical Therapy/Psychological & Brain Sciences, University of Delaware Reaching is a fundamental skill that allows humans to interact with the environment. Several studies have aimed to characterize reaching behavior using a variety of methodologies. This paper offers an open-source application of transcranial magnetic stimulation to assess the state of corticospinal excitability in humans during reaching task performance. Engineering Optimizing Magnetic Force Microscopy Resolution and Sensitivity to Visualize Nanoscale Magnetic Domains Audrey C. Parker*1, Olivia O. Maryon*1, Mojtaba T. Kaffash2, M. Benjamin Jungfleisch2, Paul H. Davis1,3 1Micron School of Materials Science & Engineering, Boise State University, 2Department of Physics and Astronomy, University of Delaware, 3Center for Advanced Energy Studies Magnetic force microscopy (MFM) employs a vertically magnetized atomic force microscopy probe to measure sample topography and local magnetic field strength with nanoscale resolution. Optimizing MFM spatial resolution and sensitivity requires balancing decreasing lift height against increasing drive (oscillation) amplitude, and benefits from operating in an inert atmosphere glovebox. Biology Combining 3D Magnetic Force Actuator and Multi-Functional Fluorescence Imaging to Study Nucleus Mechanobiology Miao Huang1,5, Heyang Wang1,5, Alfredo A. Delgado1, Tyler A. Reid1, Julian Long2, Shu Wang3,5, Hayley Sussman4, Juan Guan5,6,7, Hitomi Yamaguchi1, Xin Tang1,5,8,9 1Department of Mechanical and Aerospace Engineering, Herbert Wertheim College of Engineering, University of Florida, 2Department of Materials Science and Engineering, University of Florida, 3Department of Biostatistics, University of Florida, 4Department of Biomedical Engineering, College of Engineering (COE), University of Delaware (UD), 5UF Health Cancer Center, University of Florida, 6Department of Physics, College of Liberal Arts and Sciences, University of Florida, 7Department of Anatomy and Cell Biology, College of Medicine, University of Florida, 8J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, 9Department of Physiology and Functional Genomics, University of Florida This study presents a new protocol to directly apply mechanical force on the cell nucleus through magnetic microbeads delivered into the cytoplasm and to conduct simultaneous live-cell fluorescent imaging. Biology Isolation and Identification of Vascular Endothelial Cells from Distinct Adipose Depots for Downstream Applications Thanh Nguyen1, Sang Joon Ahn2, Richard West3, Ibra S. Fancher1 1Department of Kinesiology and Applied Physiology, College of Health Sciences, University of Delaware, 2Division of Pulmonary, Critical Care, Sleep and Allergy, Department of Medicine, University of Illinois at Chicago, 3University of Delaware Flow Cytometry Core, Delaware Biotechnology Institute, University of Delaware This protocol details a method for the dissection of mouse adipose depots and the isolation and digestion of respective arteries to liberate and then identify the endothelial cell population. Freshly isolated cells used in downstream applications will advance the understanding of vascular cell biology and the mechanisms of vascular dysfunction. Genetics Measuring Caenorhabditis elegans Sensitivity to the Acetylcholine Receptor Agonist Levamisole Allison N. Davis1, Jessica E. Tanis1 1Department of Biological Sciences, University of Delaware The present protocol describes an assay to determine response to levamisole, a pharmacological agonist of one class of Caenorhabditis elegans acetylcholine receptors. In this liquid levamisole swim assay, researchers visually observe and quantitate the time-dependent paralysis of animals cultivated in 24-well plates. Bioengineering Area-based Image Analysis Algorithm for Quantification of Macrophage-fibroblast Cocultures Tohn Borjigin1, Anuraag Boddupalli1, Millicent O. Sullivan1 1Department of Chemical and Biomolecular Engineering, University of Delaware We present a method, which utilizes a generalizable area-based image analysis approach to identify cell counts. Analysis of different cell populations exploited the significant cell height and structure differences between distinct cell types within an adaptive algorithm. Immunology and Infection Efficient SARS-CoV-2 Quantitative Reverse Transcriptase PCR Saliva Diagnostic Strategy utilizing Open-Source Pipetting Robots Rachel E. Ham*1, Austin R. Smothers*1,2, Kylie L. King1, Justin M. Napolitano1, Theodore J. Swann3, Lesslie G. Pekarek4, Mark A. Blenner*1,5,6, Delphine Dean*1,2 1Center for Innovative Medical Devices and Sensors (REDDI Lab), Clemson University, 2Department of Bioengineering, Clemson University, 3Swann Medicine, 4Student Health Services, Clemson University, 5Department of Chemical & Biomolecular Engineering, Clemson University, 6Department of Chemical & Biomolecular Engineering, University of Delaware The protocol describes a SARS-CoV-2 diagnostic method that utilizes open-source automation to perform RT-qPCR molecular testing of saliva samples. This scalable approach can be applied to clinical public health surveillance as well as to increase the capacity of smaller university laboratories. Environment A Method to Preserve Wetland Roots and Rhizospheres for Elemental Imaging Angelia L. Seyfferth1, Matt A. Limmer1, Ryan Tappero2 1Department of Plant and Soil Sciences, University of Delaware, 2National Synchrotron Radiation Lightsource-II, Brookhaven National Laboratory We describe a protocol to sample, preserve, and section intact roots and the surrounding rhizosphere soil from wetland environments using rice (Oryza sativa L.) as a model species. Once preserved, the sample can be analyzed using elemental imaging techniques, such as synchrotron X-ray fluorescence (XRF) chemical speciation imaging. Bioengineering Evaluating Regional Pulmonary Deposition using Patient-Specific 3D Printed Lung Models Emma L. Peterman1, Emily L. Kolewe1, Catherine A. Fromen1 1Department of Chemical and Biomolecular Engineering, University of Delaware We present a high-throughput, in vitro method for quantifying regional pulmonary deposition at the lobe level using CT scan-derived, 3D printed lung models with tunable air flow profiles. Neuroscience Measuring Statistical Learning Across Modalities and Domains in School-Aged Children Via an Online Platform and Neuroimaging Techniques Julie M. Schneider1, Anqi Hu1, Jennifer Legault1, Zhenghan Qi1 1Department of Linguistics and Cognitive Science, University of Delaware Presented here is a protocol introducing a set of child-friendly statistical learning tasks geared towards examining children’s learning of temporal statistical patterns across domains and sensory modalities. The developed tasks collect behavioral data using the web-based platform and task-based functional magnetic resonance imaging (fMRI) data for examining neural engagement during statistical learning. Bioengineering Fabrication and Implementation of a Reference-Free Traction Force Microscopy Platform Omar A. Banda1, John H. Slater1,2,3 1Department of Biomedical Engineering, University of Delaware, 2Department of Materials Science & Engineering, University of Delaware, 3Delaware Biotechnology Institute This protocol provides instructions for implementing multiphoton lithography to fabricate three-dimensional arrays of fluorescent fiducial markers embedded in poly(ethylene glycol)-based hydrogels for use as reference-free, traction force microscopy platforms. Using these instructions, measurement of 3D material strain and calculation of cellular tractions is simplified to promote high-throughput traction force measurements. Neuroscience Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain Brianna Kimmelmann-Shultz1, Negin Mohmammadmirzaei1, Jeffrey Caplan2, Dayan Knox1 1Department of Psychological and Brain Sciences, University of Delaware, 2Plant and Soil Sciences, Delaware Biotechnology Institute, University of Delaware Here, we present a protocol that uses near-infrared dyes in conjunction with immunohistochemistry and high-resolution scanning to assay proteins in brain regions. Behavior Using a Real-Time Locating System to Measure Walking Activity Associated with Wandering Behaviors Among Institutionalized Older Adults Mary E. Bowen1,2, William Kearns3, Jeremy R. Crenshaw4, Steven J. Stanhope4 1School of Nursing, University of Delaware, 2Corporeal Michael J. Crescenz VA Medical Center, 3Child and Family Studies, University of South Florida, 4Department of Kinesiology and Applied Physiology, University of Delaware This paper discusses the use of a continuous and objective real-time locating system to measure walking activity associated with wandering behaviors, focusing on older adults with cognitive impairment. Walking activity is measured by walking distance, sustained walking distance, and sustained gait speed. Also assessed are gait quality and balance ability. Neuroscience Neuroimaging Field Methods Using Functional Near Infrared Spectroscopy (NIRS) Neuroimaging to Study Global Child Development: Rural Sub-Saharan Africa Kaja K. Jasińska1,2, Sosthène Guei3 1Linguistics and Cognitive Science Department, University of Delaware, 2Haskins Laboratories, 3Centre de Recherche et d'Action pour la Paix Portable neuroimaging approaches (functional Near Infrared Spectroscopy) provide advances to the study of the brain in previously inaccessible regions; here, rural Côte d'Ivoire. Innovation in methods and development of culturally-appropriate neuroimaging protocols permits novel study of the brain's development and children's learning outcomes in environments with significant poverty and adversity. Genetics A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells Natalia Rivera-Torres1,2, Eric B. Kmiec1,2 1Gene Editing Institute, Helen F. Graham Cancer Center and Research Institute, Christiana Care Health Services, 2Department of Medical Sciences, University of Delaware This protocol outlines the workflow of a CRISPR/Cas9-based gene editing system for the repair of point mutations in mammalian cells. Here, we use a combinatorial approach to gene editing with a detailed follow-on experimental strategy for measuring indel formation at the target site—in essence, analyzing onsite mutagenesis. Bioengineering Predicting Gene Silencing Through the Spatiotemporal Control of siRNA Release from Photo-responsive Polymeric Nanocarriers Chad T. Greco1, Thomas H. Epps, III1,2, Millicent O. Sullivan1 1Department of Chemical and Biomolecular Engineering, University of Delaware, 2Department of Materials Science and Engineering, University of Delaware We present a novel method that uses photo-responsive block copolymers for more efficient spatiotemporal control of gene silencing with no detectable off-target effects. Additionally, changes in gene expression can be predicted using straightforward siRNA release assays and simple kinetic modeling. Environment Functionalization and Dispersion of Carbon Nanomaterials Using an Environmentally Friendly Ultrasonicated Ozonolysis Process Eudora S. Y. Yeo1, Gary I. Mathys2, Narelle Brack3, Erik T. Thostenson4,5,6, Andrew N. Rider1 1Aerospace Division, Defence Science and Technology Group, 2Maritime Division, Defence Science and Technology Group, 3Department of Chemistry and Physics, La Trobe Institute for Molecular Science, La Trobe University, 4Department of Mechanical Engineering, University of Delaware, 5Department of Materials Science and Engineering, University of Delaware, 6Center for Composite Materials, University of Delaware Here, a novel method for the functionalization and stable dispersion of carbon nanomaterials in aqueous environments is described. Ozone is injected directly into an aqueous dispersion of carbon nanomaterial that is continuously recirculated through a high-powered ultrasonic cell. Engineering Dielectric RheoSANS — Simultaneous Interrogation of Impedance, Rheology and Small Angle Neutron Scattering of Complex Fluids Jeffrey J. Richards1, Cedric V. L. Gagnon2, Jeffery R. Krzywon1, Norman J. Wagner3, Paul D. Butler1 1NIST Center for Neutron Research, National Institute of Standards and Technology, 2Department of Materials Science and Engineering, University of Maryland, 3Center for Neutron Science, Department of Chemical and Biomolecular Engineering, University of Delaware Here, we present a procedure for the measurement of simultaneous impedance, rheology and neutron scattering from soft matter materials under shear flow. Behavior Wheel Running and Environmental Complexity as a Therapeutic Intervention in an Animal Model of FASD Zachary H. Gursky1, Anna Y. Klintsova1 1Department of Psychological and Brain Sciences, University of Delaware Cardiovascular exercise and stimulating experiences in a complex environment have positive benefits on multiple measures of neuroplasticity within the rodent brain. This article will discuss the implementation of these interventions as a "superintervention" which combines wheel running and environmental complexity and will address the limitations of these interventions. Bioengineering Image-guided, Laser-based Fabrication of Vascular-derived Microfluidic Networks Keely A. Heintz1, David Mayerich2, John H. Slater1,3 1Department of Biomedical Engineering, University of Delaware, 2Department of Electrical and Computer Engineering, University of Houston, 3Delaware Biotechnology Institute This protocol outlines the implementation of image-guided, laser-based hydrogel degradation to fabricate vascular-derived, biomimetic microfluidic networks embedded in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. These biomimetic microfluidic systems may be useful for tissue engineering applications, generation of in vitro disease models, and fabrication of advanced "on-a-chip" devices. Chemistry Synthesis of Hierarchical ZnO/CdSSe Heterostructure Nanotrees Zhengxin Li1,2, Jesus Nieto-Pescador1,2, Alexander J. Carson1,2, Jolie C. Blake1,2, Lars Gundlach1,2 1Department of Chemistry & Biochemistry, University of Delaware, 2Department of Physics & Astronomy, University of Delaware Here, we prepare and characterize novel tree-like hierarchical ZnO/CdSSe nanostructures, where CdSSe branches are grown on vertically aligned ZnO nanowires. The resulting nanotrees are a potential material for solar energy conversion and other opto-electronic devices. Bioengineering Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications Lisa A. Sawicki1, April M. Kloxin1,2 1Department of Chemical and Biomolecular Engineering, University of Delaware, 2Department of Materials Science and Engineering, University of Delaware We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented. Neuroscience Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue Jaclyn M. Schwarz1 1Department of Psychological and Brain Sciences, University of Delaware The goal of this protocol is to use the fluorescence activated cell sorting (FACS) technique to sort specific types of neural cells for subsequent analysis of cell-type-specific gene expression, epigenetic markers, and or protein expression. Bioengineering Construction and Characterization of a Novel Vocal Fold Bioreactor Aidan B. Zerdoum1, Zhixiang Tong2, Brendan Bachman2, Xinqiao Jia1,2 1Biomedical Engineering Program, University of Delaware, 2Department of Materials Science and Engineering, Delaware Biotechnology Institute, University of Delaware A novel vocal fold bioreactor capable of delivering physiologically relevant, vibratory stimulation to cultured cells is constructed and characterized. This dynamic culture device, when combined with a fibrous poly(ε-caprolactone) scaffold, creates a vocal fold-mimetic environment that modulates the behaviors of mesenchymal stem cells. Engineering Measuring Material Microstructure Under Flow Using 1-2 Plane Flow-Small Angle Neutron Scattering A. Kate Gurnon1, P. Douglas Godfrin1, Norman J. Wagner1, Aaron P. R. Eberle1,2, Paul Butler2, Lionel Porcar1,3 1Center for Neutron Science, Department of Chemical and Biomolecular Engineering, University of Delaware, 2NIST Center for Neutron Research, National Institute of Standards and Technology, 3Institut Laue-Langevin A shear cell is developed for small-angle neutron scattering measurements in the velocity-velocity gradient plane of shear and is used to characterize complex fluids. Spatially resolved measurements in the velocity gradient direction are possible for studying shear-banding materials. Applications include investigations of colloidal dispersions, polymer solutions, and self-assembled structures. Biology High Throughput Microinjections of Sea Urchin Zygotes Nadezda A. Stepicheva1, Jia L. Song1 1Department of Biological Sciences, University of Delaware Microinjection is a common technique used to deliver DNA constructs, mRNAs, morpholino antisense oligonucleotides or other treatments into eggs, embryos, and cells of various species. Biology Peptides from Phage Display Library Modulate Gene Expression in Mesenchymal Cells and Potentiate Osteogenesis in Unicortical Bone Defects Gary Balian1, Gina Beck1, Vedavathi Madhu1, Robert Sikes2, Quanjun Cui3, Haixiang Liang1, Joshua Bush1 1Orthopaedics Research, University of Virginia, 2Biological Sciences, University of Delaware, 3Orthopaedic Surgery, University of Virginia A phage display library was used to identify peptide sequences that target bone. The objective was to investigate the effect of these peptides on mesenchymal cell differentiation and to determine their effect on bone regeneration. Biology RhoC GTPase Activation Assay Michelle Lucey1, Heather Unger1, Kenneth L. van Golen1 1Department of Biological Sciences, The Center for Translational Cancer Research, University of Delaware This protocol utilizes a pull down assay to determine the levels of active RhoC GTPase within cells.
