Carnegie Mellon University View Institution's Website 19 articles published in JoVE Biology Universal Molecular Retention with 11-Fold Expansion Microscopy Brendan R. Gallagher1, Aleksandra Klimas1, Zhangyu Cheng1, Yongxin Zhao1 1Department of Biological Sciences, Carnegie Mellon University Presented here is a new version of expansion microscopy (ExM), Magnify, that is modified for up to 11-fold expansion, conserving a comprehensive array of biomolecule classes, and is compatible with a broad range of tissue types. It enables the interrogation of the nanoscale configuration of biomolecules using conventional diffraction-limited microscopes. Bioengineering Mechanostimulation of Multicellular Organisms Through a High-Throughput Microfluidic Compression System Utku M. Sönmez1, Nolan Frey2, Jonathan S. Minden2, Philip R. LeDuc1 1Department of Mechanical Engineering, Carnegie Mellon University, 2Department of Biological Sciences, Carnegie Mellon University The present protocol describes the design, fabrication, and characterization of a microfluidic system capable of aligning, immobilizing, and precisely compressing hundreds of Drosophila melanogaster embryos with minimal user intervention. This system enables high-resolution imaging and recovery of samples for post-stimulation analysis and can be scaled to accommodate other multicellular biological systems. Engineering Microtensiometer for Confocal Microscopy Visualization of Dynamic Interfaces Steven V. Iasella1, Sourav Barman1, Clara Ciutara1, Boxun Huang1, Michael L. Davidson2, Joseph A. Zasadzinski1 1Department of Chemical Engineering and Materials Science, University of Minnesota, 2Department of Chemical Engineering, Carnegie Mellon University This manuscript describes the design and operation of a microtensiometer/confocal microscope to do simultaneous measurements of interfacial tension and surface dilatational rheology while visualizing the interfacial morphology. This provides the real-time construction of structure-property relationships of interfaces important in technology and physiology. Bioengineering A Large Animal Model for Pulmonary Hypertension and Right Ventricular Failure: Left Pulmonary Artery Ligation and Progressive Main Pulmonary Artery Banding in Sheep Rei Ukita1, John W. Stokes1, W. Kelly Wu1, Jennifer Talackine1, Nancy Cardwell1, Yatrik Patel1, Clayne Benson5, Caitlin T. Demarest1, Erika B. Rosenzweig3, Keith Cook2, Emily J. Tsai4, Matthew Bacchetta1,6 1Department of Thoracic Surgery, Vanderbilt University Medical Center, 2Department of Biomedical Engineering, Carnegie Mellon University, 3Department of Pediatrics, Columbia University Medical Center, Columbia University Irving Medical Center, 4Department of Medicine, Columbia University Medical Center, Columbia University Irving Medical Center, 5Department of Anesthesiology, Vanderbilt University Medical Center, 6Department of Biomedical Engineering, Vanderbilt University This manuscript describes the surgical technique and experimental approach to develop severe right ventricular pressure overload to model their adaptive and maladaptive phenotypes. Biology Chemical Dimerization-Induced Protein Condensates on Telomeres Rongwei Zhao1, David M. Chenoweth2, Huaiying Zhang1 1Department of Biological Sciences, Mellon College of Science, Carnegie Mellon University, 2Department of Chemistry, School of Arts and Sciences, University of Pennsylvania This protocol illustrates a chemically induced protein dimerization system to create condensates on chromatin. The formation of promyelocytic leukemia (PML) nuclear body on telomeres with chemical dimerizers is demonstrated. Droplet growth, dissolution, localization and composition are monitored with live cell imaging, immunofluorescence (IF) and fluorescence in situ hybridization (FISH). Bioengineering Self-Assembly of Gamma-Modified Peptide Nucleic Acids into Complex Nanostructures in Organic Solvent Mixtures Sriram Kumar1, Ying Liu1, Rebecca E. Taylor1,2,3 1Department of Mechanical Engineering, Carnegie Mellon University, 2Department of Biomedical Engineering, Carnegie Mellon University, 3Department of Electrical and Computer Engineering, Carnegie Mellon University This article provides protocols for the design and self-assembly of nanostructures from gamma-modified peptide nucleic acid oligomers in organic solvent mixtures. Immunology and Infection Clarifying and Imaging Candida albicans Biofilms Frederick Lanni1, Katherine Lagree1, Manning Y. Huang1, Lan Yan2, Carol A. Woolford1, Aaron P. Mitchell1 1Department of Biological Sciences, Carnegie Mellon University, 2Department of Pharmacology, Second Military Medical University To view and quantify the internal features of Candida albicans biofilms, we prepare fixed intact specimens that are clarified by refractive index matching. Then, optical sectioning microscopy can be used to obtain three-dimensional image data though the full thickness of the biofilm. Medicine Nanoscopic Imaging of Human Tissue Sections via Physical and Isotropic Expansion Aleksandra Klimas1, Octavian Bucur2, Brigdet Njeri1, Yongxin Zhao1 1Department of Biological Sciences, Carnegie Mellon University, 2QPathology Nanoscale imaging of clinical tissue samples can improve understanding of disease pathogenesis. Expansion pathology (ExPath) is a version of expansion microscopy (ExM), modified for compatibility with standard clinical tissue samples, to explore the nanoscale configuration of biomolecules using conventional diffraction limited microscopes. Neuroscience Translational Brain Mapping at the University of Rochester Medical Center: Preserving the Mind Through Personalized Brain Mapping Bradford Z. Mahon1,2, Jeffrey A. Mead3, Benjamin L Chernoff2, Maxwell H. Sims5, Frank E. Garcea4, Emily Prentiss5, Raouf Belkhir2, Sam J. Haber1, Sarah B. Gannon5, Steve Erickson5, Kelly A. Wright5, Michael Z. Schmidt5, Audrey Paulzak1, Vanessa C. Milano1, David A. Paul1, Kenneth Foxx1, Madalina Tivarus7,8, Jacob W. Nadler6, Jacqueline M Behr1, Susan O. Smith1, Yan Michael Li1, Kevin Walter1, Webster H. Pilcher1 1Department of Neurosurgery, University of Rochester Medical Center, 2Department of Psychology, Carnegie Mellon University, 3Public Relations and Communications, University of Rochester Medical Center, 4MOSS Rehabilitation Research Institute, Cognitive Neuroscience, 5University of Rochester Medical Center, 6Department of Anesthesiology and Perioperative Medicine, University of Rochester Medical Center, 7Department of Imaging Sciences, University of Rochester Medical Center, 8Department of Neuroscience, University of Rochester Medical Center This article provides an overview of a multi-modal brain mapping program designed to identify regions of the brain that support critical cognitive functions in individual neurosurgery patients. Bioengineering Three-dimensional Printing of Thermoplastic Materials to Create Automated Syringe Pumps with Feedback Control for Microfluidic Applications Ming-Cheng Chen*1, John R. Lake*1, Keith C. Heyde2, Warren C. Ruder1,2 1Department of Bioengineering, University of Pittsburgh, 2Department of Mechanical Engineering, Carnegie Mellon University Here we present a protocol to construct a pressure-controlled syringe pump to be used in microfluidic applications. This syringe pump is made from an additively manufactured body, off-the-shelf hardware, and open-source electronics. The resulting system is low-cost, straightforward to build, and delivers well-regulated fluid flow to enable rapid microfluidic research. Neuroscience Modeling Fast-scan Cyclic Voltammetry Data from Electrically Stimulated Dopamine Neurotransmission Data Using QNsim1.0 Rashed Harun1,2,3, Christine M. Grassi2, Miranda J. Munoz2,4, Amy K. Wagner1,2,3 1Center for Neuroscience, University of Pittsburgh, 2Department of Physical Medicine & Rehabilitation, University of Pittsburgh, School of Medicine, 3Safar Center for Resuscitation Research, University of Pittsburgh, 4Department of Biological Sciences, Mellon College of Science, Carnegie Mellon University Fast-scan cyclic voltammetry can monitor in vivo dopamine neurotransmission in the context of drugs, disease, and other experimental manipulations. This work describes the implementation of QNsim1.0, a software to model electrically stimulated dopamine responses according to the quantitative neurobiological model to quantify estimates of dopamine release and reuptake dynamics. Bioengineering Using Synthetic Biology to Engineer Living Cells That Interface with Programmable Materials Keith C. Heyde*1,2, Felicia Y. Scott*3, Sung-Ho Paek3, Ruihua Zhang3, Warren C. Ruder3,4 1Department of Mechanical Engineering, Carnegie Mellon University, 2Engineering Science and Mechanics Program, Virginia Polytechnic Institute and State University, 3Department of Biological Systems Engineering, Virginia Polytechnic Institute and State University, 4Department of Bioengineering, University of Pittsburgh This paper presents a series of protocols for developing engineered cells and functionalized surfaces that enable synthetically engineered E. coli to control and manipulate programmable material surfaces. Environment Use of a Battery of Chemical and Ecotoxicological Methods for the Assessment of the Efficacy of Wastewater Treatment Processes to Remove Estrogenic Potency Nicola Beresford1, Alice Baynes1, Rakesh Kanda1, Matthew R. Mills2, Karla Arias-Salazar2, Terrence J. Collins2, Susan Jobling1 1Institute of Environment Health and Societies, Brunel University London, 2Department of Chemistry, Carnegie Mellon University Endocrine Disrupting Compounds (EDC) pose a substantial risk to the aquatic environment. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. The methodology provided in this paper allows for an assessment of the efficacy and suitability of wastewater treatment processes with respect to EDC removal. Immunology and Infection Promoting 3-D Aggregation of FACS Purified Thymic Epithelial Cells with EAK 16-II/EAKIIH6 Self-assembling Hydrogel Asako Tajima1, Wen Liu2, Isha Pradhan1, Suzanne Bertera1, Robert A. Lakomy1, William A. Rudert1, Massimo Trucco1,3, Wilson S. Meng2, Yong Fan1,3 1Institute of Cellular Therapeutics, Allegheny Health Network, 2Division of Pharmaceutical Sciences, Mylan School of Pharmacy, Duquesne University, 3Department of Biological Sciences, Carnegie Mellon University This video demonstrates a protocol to enrich thymic epithelial cells (TECs) with density gradient for FACS isolation. It also shows the use of EAK16-II/EAKIIH6 peptides to promote the TEC aggregate formation. The microenvironments of EAK16-II/EAKIIH6 hydrogel provide the 3-D configuration necessary to maintain the survival and function of the TECs. Immunology and Infection Gene Expression Profiling of Infecting Microbes Using a Digital Bar-coding Platform Wenjie Xu1, Norma V. Solis2, Scott G. Filler2, Aaron P. Mitchell1 1Department of Biological Sciences, Carnegie Mellon University, 2Infectious Diseases, Harbor-UCLA Medical Center We developed a protocol that is fast, sensitive and reproducible for pathogen gene expression profiling during an infection. Biology Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy Amanda L. Soohoo1, Shanna L. Bowersox1, Manojkumar A. Puthenveedu1 1Department of Biological Sciences, Carnegie Mellon University Clathrin-mediated endocytosis, a rapid and highly dynamic process internalizes many proteins, including signaling receptors. The protocol described here directly visualizes the kinetics of individual endocytic events. This is essential for understanding how core members of the endocytic machinery coordinate with each other, and how protein cargo influence this process. Biology A Method for Microinjection of Patiria minata Zygotes Alys M. Cheatle Jarvela1, Veronica Hinman1 1Department of Biological Sciences, Carnegie Mellon University Methods that produce morphant embryos are essential to study developmental mechanisms and gene regulatory networks. The sea star Patiria miniata is an emerging model system for these studies. Here we present a protocol for obtaining gametes, producing cultures of embryos, and rapid microinjection of zygotes from this species. Bioengineering ECM Protein Nanofibers and Nanostructures Engineered Using Surface-initiated Assembly John M. Szymanski1, Quentin Jallerat1, Adam W. Feinberg1,2 1Department of Biomedical Engineering, Carnegie Mellon University, 2Department of Materials Science and Engineering, Carnegie Mellon University A method to obtain nanofibers and complex nanostructures from single or multiple extracellular matrix proteins is described. This method uses protein-surface interactions to create free-standing protein-based materials with tunable composition and architecture for use in a variety of tissue engineering and biotechnology applications. Neuroscience Co-analysis of Brain Structure and Function using fMRI and Diffusion-weighted Imaging Jeffrey S. Phillips1,2, Adam S. Greenberg1,3, John A. Pyles1,3, Sudhir K. Pathak1,4, Marlene Behrmann1,3, Walter Schneider1,3, Michael J. Tarr1,3 1Center for the Neural Basis of Cognition, 2Department of Psychology, University of Pittsburgh, 3Department of Psychology, Carnegie Mellon University, 4Department of Bioengineering, University of Pittsburgh We describe a novel approach for simultaneous analysis of brain function and structure using magnetic resonance imaging (MRI). We assess brain structure with high-resolution diffusion-weighted imaging and white-matter fiber tractography. Unlike standard structural MRI, these techniques allow us to directly relate anatomical connectivity to functional properties of brain networks.