Imperial College London View Institution's Website 69 articles published in JoVE Engineering Large Scale Energy Efficient Sensor Network Routing Using a Quantum Processor Unit Jie Chen1 1Imperial College London This study provides a method to use a quantum processor unit to compute the routes for various traffic dynamics that work to outperform classical methods in literature to maximize network lifetime. Biology Methods for Embedding Cell-Free Protein Synthesis Reactions in Macro-Scale Hydrogels Siji Kavil*1, Alex Laverick*1, Colette J. Whitfield1, Alice M. Banks2, Thomas P. Howard1 1School of Natural and Environmental Sciences, Newcastle University, 2Department of Life Sciences, Imperial College London Here, we present two protocols for embedding cell-free protein synthesis reactions in macro-scale hydrogel matrices without the need for an external liquid phase. Immunology and Infection Whole-Mount Fluorescence In Situ Hybridization to Study Spermatogenesis in the Anopheles Mosquito Matteo Vitale*1, Jiangtao Liang*2, Igor Sharakhov2, Federica Bernardini1 1Department of Life Sciences, Sir Alexander Fleming Building, Imperial College London, 2Department of Entomology, Virginia Tech Given their simple anatomy, Anopheles testes offer a good cytological model for studying spermatogenesis. This protocol describes whole-mount fluorescence in situ hybridization, a technique used to investigate this biological process, as well as the phenotype of transgenic strains harboring mutations in the genes involved in sperm production. Chemistry Screening of Coatings for an All-Solid-State Battery Using In Situ Transmission Electron Microscopy Shibarata Basak1,2, Junbeom Park1, Janghyun Jo2, Osmane Camara1, Amir H. Tavabi2, Hermann Tempel1, Hans Kungl1, Chandramohan George3, Rafal E. Dunin-Borkowski2, Joachim Mayer2,4, Rüdiger-A. Eichel1,5 1Institute of Energy and Climate Research - Fundamental Electrochemistry (IEK-9), Forschungszentrum Jülich GmbH, 2Ernst Ruska-Centre for Microscopy and Spectroscopy with Electrons, Forschungszentrum Jülich GmbH, 3Dyson School of Design Engineering, Imperial College London, 4Central Facility for Electron Microscopy (GFE), RWTH Aachen University, 5Institute of Physical Chemistry, RWTH Aachen University Utilizing the volume change of Si nanoparticles during (de)lithiation, the present protocol describes a screening method of potential coatings for all-solid-state batteries using in situ transmission electron microscopy. Biology Bottom-Up In Vitro Methods to Assay the Ultrastructural Organization, Membrane Reshaping, and Curvature Sensitivity Behavior of Septins Brieuc Chauvin*1, Koyomi Nakazawa*1, Alexandre Beber1,7, Aurélie Di Cicco1, Bassam Hajj1, François Iv2, Manos Mavrakis2, Gijsje H. Koenderink3, João T. Cabral4, Michaël Trichet5, Stéphanie Mangenot*6, Aurélie Bertin*1 1Laboratoire Physico Chimie Curie, Institut Curie, PSL Research University, Sorbonne Université, 2Institut Fresnel, CNRS UMR7249, Aix Marseille Univ, Centrale Marseille, 3Department of Bionanoscience, Kavli Institute of Nanoscience Delft, Delft University of Technology, 4Department of Chemical Engineering, Imperial College London, 5Sorbonne Université, CNRS, Institut de Biologie Paris-Seine (IBPS), Service de microscopie électronique (IBPS-SME), 6Laboratoire Matière et Systèmes Complexes (MSC), Université Paris Cité, 7Institute of Biotechnology, Czech Academy of Sciences, BIOCEV Septins are cytoskeletal proteins. They interact with lipid membranes and can sense but also generate membrane curvature at the micron scale. We describe in this protocol bottom-up in vitro methodologies for analyzing membrane deformations, curvature-sensitive septin binding, and septin filament ultrastructure. Neuroscience Preparation of Rat Sciatic Nerve for Ex Vivo Neurophysiology Adrien Rapeaux1,2,3,4, Omaer Syed5, Estelle Cuttaz5, Christopher A. R. Chapman5, Rylie A. Green5, Timothy G. Constandinou1,2,3,4 1Department of Electrical and Electronic Engineering, Imperial College London, 2Centre for Bioinspired Technology, Imperial College London, 3Care Research and Technology (CR&T), Imperial College London and the University of Surrey, 4Dementia Institute (UK DRI), 5Department of Bioengineering, Imperial College London This protocol describes the preparation of rat whole sciatic nerve tissue for ex vivo electrophysiological stimulation and recording in an environmentally-regulated, two-compartment, perfused saline bath. Bioengineering Microfluidic Tools for Probing Fungal-Microbial Interactions at the Cellular Level Emily Masters-Clark1, Amelia J. Clark1, Claire E. Stanley1 1Department of Bioengineering, Imperial College London Owing to the opacity of soil, interactions between its constituent microbes cannot easily be visualised with cellular resolution. Here, two microfluidic tools are presented, which offer new opportunities for investigating fungal-microbial interactions. The devices are versatile and simple to use, enabling high spatiotemporal control and high-resolution imaging at the cellular level. Bioengineering Rapid Antibody Glycoengineering in Chinese Hamster Ovary Cells Masue Marbiah*1,2, Pavlos Kotidis*1,3, Roberto Donini1,4, Itzcóatl A. Gómez5, Ioscani Jimenez del Val5, Stuart M. Haslam4, Karen M. Polizzi1,2, Cleo Kontoravdi1 1Department of Chemical Engineering, Imperial College London, 2Imperial College Centre for Synthetic Biology, Imperial College London, 3BioPharm Process Research, GlaxoSmithKline Research and Development, 4Department of Life Science, Imperial College London, 5School of Chemical & Bioprocess Engineering, University College Dublin The glycosylation pattern of an antibody determines its clinical performance, thus industrial and academic efforts to control glycosylation persist. Since typical glycoengineering campaigns are time- and labor-intensive, the generation of a rapid protocol to characterize the impact of glycosylation genes using transient silencing would prove useful. Medicine Therapy Interventions for Upper Limb Amputees Undergoing Selective Nerve Transfers Agnes Sturma1,2, Laura A. Hruby1,3, Anna Boesendorfer1, Clemens Gstoettner1, Dario Farina2, Oskar C. Aszmann1,4 1Clinical Laboratory for Bionic Extremity Reconstruction, Medical University of Vienna, 2Bioengineering Department, Imperial College London, 3Department of Orthopaedics and Trauma Surgery, Medical University of Vienna, 4Department of Plastic and Reconstructive Surgery, Medical University of Vienna This work presents a protocol to enhance prosthetic function after selective nerve transfer surgery. Rehabilitation interventions include patient information and selection, support of wound healing, cortical re-activation of sensory-motor areas of the upper limb, training of selective muscle activation, prosthetic handling in daily life, and regular follow-up assessments. Bioengineering A High-Yield Streptomyces Transcription-Translation Toolkit for Synthetic Biology and Natural Product Applications Ming Toh1,2,3,4, Kameshwari Chengan5, Tanith Hanson5, Paul S. Freemont1,2,3,4,6,7, Simon J. Moore5 1Centre for Synthetic Biology and Innovation, South Kensington Campus, 2Department of Medicine, South Kensington Campus, 3Section of Structural and Synthetic Biology, Department of Infectious Disease, Imperial College London, 4Sir Alexander Fleming Building, South Kensington Campus, 5School of Biosciences, Division of Natural Sciences, University of Kent, 6UK Dementia Research Institute Care Research and Technology Centre, Imperial College London; Hammersmith Campus, 7UK Innovation and Knowledge Centre for Synthetic Biology (SynbiCITE) and the London Biofoundry, Imperial College Translation & Innovation Hub This protocol details an enhanced method for synthesizing high yields of recombinant proteins from a Streptomyces venezuelae cell-free transcription-translation (TX-TL) system. Neuroscience Fluorescence-Activated Cell Sorting-Radioligand Treated Tissue (FACS-RTT) to Determine the Cellular Origin of Radioactive Signal Quentin Amossé1,2, Kelly Ceyzériat1,3,4, Stergios Tsartsalis1,5, Benjamin B. Tournier1,2, Philippe Millet1,2 1Division of Adult Psychiatry, Department of Psychiatry, University Hospitals of Geneva, 2Department of Psychiatry, University of Geneva, 3Division of Nuclear medicine and Molecular Imaging, University Hospitals of Geneva, 4Division of Radiation Oncology, Department of Oncology, University Hospitals of Geneva, 5Department of Brain Sciences, Faculty of Medicine, Imperial College London Fluorescence-Activated Cell Sorting-Radioligand Treated Tissue (FACS-RTT) is a powerful tool to study the role of the 18 kDa translocator protein or Serotonin 5HT2A-receptor expression in Alzheimer's Disease at a cellular scale. This protocol describes the ex-vivo application of FACS-RTT in the TgF344-AD rat model. Bioengineering Segmenting Growth of Endothelial Cells in 6-Well Plates on an Orbital Shaker for Mechanobiological Studies Kuin T. Pang*1,2, Mean Ghim*1, Mehwish Arshad1, Xiaomeng Wang2,3,4, Peter D. Weinberg1 1Department of Bioengineering, Imperial College London, 2Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), 3Lee Kong Chian School of Medicine, Nanyang Technological University Singapore, 4Singapore Eye Research Institute, The Academia This protocol describes a coating method to restrict endothelial cell growth to a specific region of a 6-well plate for shear stress application using the orbital shaker model. Biochemistry Preparation of Sample Support Films in Transmission Electron Microscopy using a Support Floatation Block Natàlia de Martín Garrido1, Kailash Ramlaul1, Christopher H. S. Aylett1 1Section of Structural & Synthetic Biology, Department of Infectious Diseases, Faculty of Medicine, Imperial College London, South Kensington Campus Sample preparation for cryo-electron microscopy (cryo-EM) is a significant bottleneck in the structure determination workflow of this method. Here, we provide detailed methods for using an easy-to-use, three-dimensionally printed block for the preparation of support films to stabilize samples for transmission EM studies. Environment Flash Infrared Annealing for Perovskite Solar Cell Processing Pui Sha Victoria Ling1,2, Anders Hagfeldt2, Sandy Sanchez2 1Department of Chemistry, Molecular Sciences Research Hub, Imperial College London, 2Laboratory of Photomolecular Science (LSPM), École Polytechnique Fédérale de Lausanne (EPFL) We describe a flash infrared annealing method used for the synthesis of perovskite and mesoscopic-TiO2 films. Annealing parameters are varied and optimized for processing on fluorine-doped tin oxide (FTO) glass and indium tin oxide-coated polyethylene terephthalate (ITO PET), subsequently giving devices power conversion efficiencies >20%. Cancer Research Isolation of Proximal Fluids to Investigate the Tumor Microenvironment of Pancreatic Adenocarcinoma Greta Donisi*1, Marialuisa Barbagallo*2, Giovanni Capretti1,3, Gennaro Nappo1,3, Panteleimon G. Takis4,5,6, Alessandro Zerbi1,3, Federica Marchesi2,7, Nina Cortese2 1Section of Pancreatic Surgery, Humanitas Clinical and Research Center-IRCCS, 2Department of Immunology and Inflammation, Humanitas Clinical and Research Center-IRCCS, 3Department of Biomedical Sciences, Humanitas University, 4Giotto Biotech S.R.L., 5Section of Bioanalytical Chemistry, Division of Systems Medicine, Department of Metabolism, Digestion and Reproduction, Imperial College London, 6National Phenome Centre, Department of Metabolism, Digestion and Reproduction, Faculty of Medicine, Imperial College London, 7Department of Medical Biotechnology and Translational Medicine, University of Milan Pancreatic juice is a precious source of biomarkers for human pancreatic cancer. We describe here a method for intraoperative collection procedure. To overcome the challenge of adopting this procedure in murine models, we suggest an alternative sample, tumor interstitial fluid, and describe here two protocols for its isolation. Cancer Research Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity Sarah Spear1,2, Iain A McNeish1, Melania Capasso2,3 1Division of Cancer, Department of Surgery and Cancer, Imperial College London, 2Centre for Cancer and Inflammation, Barts Cancer Institute, Queen Mary University of London, 3German Center for Neurodegenerative Diseases (DZNE) This protocol describes the surgical generation of orthotopic pancreatic tumors and the rapid digestion of freshly isolated murine pancreatic tumors. Following digestion, viable immune cell populations can be used for further downstream analysis, including ex vivo stimulation of T cells for intracellular cytokine detection by flow cytometry. Developmental Biology Cell-cell Fusion of Genome Edited Cell Lines for Perturbation of Cellular Structure and Function Robert Mahen1,2, Reiner Schulte3 1Photonics Group, Department of Physics, Imperial College London, 2The Medical Research Council Cancer Unit, Hutchison/MRC Research Centre, Cambridge Biomedical Campus, University of Cambridge, 3Cambridge Institute for Medical Research, Cambridge Biomedical Campus, University of Cambridge The purpose of this protocol is to fuse two different cell types to create hybrid cells. Fluorescence microscopy analysis of fused cells is used to track the cell of origin of cellular organelles. This assay can be used to explore how cellular structure and function respond to perturbation by cell fusion. Neuroscience Surface Electromyographic Biofeedback as a Rehabilitation Tool for Patients with Global Brachial Plexus Injury Receiving Bionic Reconstruction Laura A. Hruby1,2, Agnes Sturma1,3, Oskar C. Aszmann1,4 1Clinical Laboratory for Bionic Extremity Reconstruction, Medical University of Vienna, 2Department of Orthopaedics and Trauma Surgery, Medical University of Vienna, 3Department of Bioengineering, Imperial College London, 4Division of Plastic and Reconstructive Surgery, Department of Surgery, Medical University of Vienna Optimal functional outcomes after bionic reconstruction in patients with global brachial plexus injury depend on a structured rehabilitation protocol. Surface electromyographic guided training may improve the amplitude, separation and consistency of EMG signals, which - after elective amputation of a functionless hand - control and drive a prosthetic hand. Medicine Structured Motor Rehabilitation After Selective Nerve Transfers Agnes Sturma1,2,3, Laura A. Hruby1,4, Dario Farina2, Oskar C. Aszmann1,5 1Clinical Laboratory for Bionic Extremity Reconstruction, Medical University of Vienna, 2Bioengineering Department, Imperial College London, 3Master's Degree Program Health Assisting Engineering, University of Applied Sciences FH Campus Wien, 4Department of Orthopedics and Trauma Surgery, Medical University of Vienna, 5Division of Plastic and Reconstructive Surgery, Department of Surgery, Medical University of Vienna Here, we present a protocol for the motor rehabilitation of patients with severe nerve injuries and selective nerve transfer surgery. It aims at restoring the motor function proposing several stages in patient education, early-stage therapy after surgery and interventions for rehabilitation after successful re-innervation of the nerve’s target. Immunology and Infection Use of the Invertebrate Galleria mellonella as an Infection Model to Study the Mycobacterium tuberculosis Complex Masanori Asai*1, Yanwen Li*1, Jasmeet Singh Khara1,2, Camilla A. Gladstone1, Brian D. Robertson3, Paul R. Langford*1, Sandra M. Newton*1 1Section of Pediatric Infectious Diseases and Allergy, Department of Medicine, Imperial College London, 2Department of Pharmacy, National University of Singapore, 3MRC Centre for Molecular Bacteriology and Infection, Department of Medicine, Imperial College London Galleria mellonella was recently established as a reproducible, cheap, and ethically acceptable infection model for the Mycobacterium tuberculosis complex. Here we describe and demonstrate the steps taken to establish successful infection of G. mellonella with bioluminescent Mycobacterium bovis BCG lux. Genetics Transcription Start Site Mapping Using Super-low Input Carrier-CAGE Nevena Cvetesic1,2, Elena Pahita1,2, Boris Lenhard1,2,3 1Institute of Clinical Sciences, Faculty of Medicine, Imperial College London, 2MRC London Institute of Medical Sciences, 3Sars International Centre for Marine Molecular Biology, University of Bergen Cap Analysis of Gene Expression (CAGE) is a method for genome-wide quantitative mapping of mRNA 5’ends to capture RNA polymerase II transcription start sites at a single-nucleotide resolution. This work describes a low-input (SLIC-CAGE) protocol for generation of high-quality libraries using nanogram-amounts of total RNA. Immunology and Infection Use of Single Chain MHC Technology to Investigate Co-agonism in Human CD8+ T Cell Activation Xiang Zhao1, Maryam Hamidinia1, Joanna Ai Ling Choo1, Chien Tei Too1,2, Zi Zong Ho3, Ee Chee Ren4, Antonio Bertoletti3, Paul A. MacAry1,2,5, Keith G. Gould6, Joanna Brzostek1, Nicholas R.J. Gascoigne1,2,5 1Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, 2Immunology Programme, Life Sciences Institute, National University of Singapore, 3Emerging Infectious Diseases Program, Duke-NUS Graduate Medical School, 4Singapore Immunology Network, A*STAR, 5NUS Graduate School for Integrative Sciences and Engineering (NGS), National University of Singapore, 6Department of Immunology, Wright-Fleming Institute, Imperial College London This protocol describes the use of single chain MHC class I complexes to investigate molecular interactions in human CD8+ T cell activation: generation of engineered antigen presenting cells expressing single chain constructs, culture of human CD8+ T cell clone and T cell activation experiments. Immunology and Infection "Liver-on-a-Chip" Cultures of Primary Hepatocytes and Kupffer Cells for Hepatitis B Virus Infection Ana Maria Ortega-Prieto1, Jessica Katy Skelton1, Catherine Cherry1, Marco Antonio Briones-Orta1, Charlotte Alexandra Hateley1, Marcus Dorner1 1Section of Virology, Department of Medicine, Imperial College The goal of this protocol is to provide a step-by-step guide to perform 3-D "liver-on-a-chip" infection experiments with the hepatitis B virus. Neuroscience A Novel In Vitro Model of Blast Traumatic Brain Injury Rita Campos-Pires1,2, Amina Yonis1, Warren Macdonald2,3, Katie Harris1, Christopher J. Edge4,5, Peter F. Mahoney6, Robert Dickinson1,2 1Anaesthetics, Pain Medicine and Intensive Care Section, Department of Surgery and Cancer, Imperial College London, 2Royal British Legion Centre for Blast Injury Studies, Department of Bioengineering, Imperial College London, 3Department of Bioengineering, Imperial College London, 4Department of Life Sciences, Imperial College London, 5Department of Anaesthetics, Royal Berkshire Hospital NHS Foundation Trust, 6Royal Centre for Defence Medicine, Medical Directorate Joint Force Command This paper describes a novel model of primary blast traumatic brain injury. A compressed-air driven shock tube is used to expose in vitro mouse hippocampal slice cultures to a single shock wave. This is a simple and rapid protocol generating a reproducible brain tissue injury with a high throughput. Engineering Pore-scale Imaging and Characterization of Hydrocarbon Reservoir Rock Wettability at Subsurface Conditions Using X-ray Microtomography Amer M. Alhammadi1, Ahmed AlRatrout1, Branko Bijeljic1, Martin J. Blunt1 1Department of Earth Science and Engineering, Imperial College London This protocol is presented to characterize the complex wetting conditions of an opaque porous medium (hydrocarbon reservoir rock) using three-dimensional images obtained by X-ray microtomography at subsurface conditions. Engineering Film Control to Study Contributions of Waves to Droplet Impact Dynamics on Thin Flowing Liquid Films Idris T. Adebayo1, Omar K. Matar1 1Department of Chemical Engineering, Imperial College London A protocol to study the contributions of waves to droplet impact dynamics on flowing liquid films is presented. Cancer Research Counting Proteins in Single Cells with Addressable Droplet Microarrays Stelios Chatzimichail*1, Pashiini Supramaniam*1, Oscar Ces1, Ali Salehi-Reyhani1 1Institute of Chemical Biology, Department of Chemistry, Imperial College London Here we present addressable droplet microarrays (ADMs), a droplet array based method able to determine absolute protein abundance in single cells. We demonstrate the capability of ADMs to characterize the heterogeneity in expression of the tumor suppressor protein p53 in a human cancer cell line. Neuroscience Concurrent Recording of Co-localized Electroencephalography and Local Field Potential in Rodent Sungmin Kang1, Michael Bruyns-Haylett2, Yurie Hayashi1, Ying Zheng1 1School of Biological Sciences, Whiteknights, University of Reading, 2Department of Bioengineering, Imperial College This protocol describes a simple method for concurrent recording of co-localized electroencephalography (EEG) and multi-laminar local field potential in an anesthetized rat. A burr hole drilled in the skull for the insertion of a microelectrode is shown to produce negligible distortion of the EEG signal. Bioengineering Evaluating Primary Blast Effects In Vitro Niall J. Logan1, Hari Arora1, Claire A. Higgins1 1Department of Bioengineering, Imperial College London Understanding how cells are modulated by exposure to shock waves can help identify the mechanisms behind injuries triggered from blast events. This protocol uses custom-built shock tube equipment to apply shock waves at a range of pressures to cell monolayers and to identify the subsequent effects on cell viability. Chemistry Measurements of Long-range Electronic Correlations During Femtosecond Diffraction Experiments Performed on Nanocrystals of Buckminsterfullerene Rebecca A. Ryan1, Sophie Williams1, Andrew V. Martin1, Ruben A. Dilanian1, Connie Darmanin2, Corey T. Putkunz1, David Wood3, Victor A. Streltsov4, Michael W.M. Jones5, Naylyn Gaffney6, Felix Hofmann7, Garth J. Williams8, Sebastien Boutet9, Marc Messerschmidt10, M. Marvin Seibert11, Evan K. Curwood11, Eugeniu Balaur2, Andrew G. Peele5, Keith A. Nugent2, Harry M. Quiney1, Brian Abbey2 1ARC Centre of Excellence in Advanced Molecular Imaging, School of Physics, University of Melbourne, 2Australian Research Council (ARC) Centre of Excellence in Advanced Molecular Imaging, Department of Chemistry and Physics, La Trobe Institute for Molecular Sciences, La Trobe University, 3Department of Physics, Imperial College London, 4Florey Institute of Neuroscience and Mental Health, 5Science and Engineering Faculty, Queensland University of Technology, 6Swinburne University of Technology, 7Department of Engineering Science, University of Oxford, 8Brookhaven National Laboratory, 9Linac Coherent Light Source, SLAC National Accelerator Laboratory, 10BioXFEL Science and Technology Center, 11Laboratory of Molecular Biophysics, Department of Cell and Molecular Biology, Uppsala University, 12Australian Synchrotron We describe an experiment designed to probe the electronic damage induced in nanocrystals of Buckminsterfullerene (C60) by intense, femtosecond pulses of X-rays. The experiment found that, surprisingly, rather than being stochastic, the X-ray induced electron dynamics in C60 are highly correlated, extending over hundreds of unit cells within the crystals1. Environment Measurement of the Rheology of Crude Oil in Equilibrium with CO2 at Reservoir Conditions Ruien Hu1, John Crawshaw1 1Chemical Engineering Department, Imperial College London, Qatar Carbonate and Carbon Storage Research Centre A method for measuring the rheology of crude oil in equilibrium with carbon dioxide at reservoir conditions is presented. Engineering A Novel Biaxial Testing Apparatus for the Determination of Forming Limit under Hot Stamping Conditions Zhutao Shao1, Nan Li1 1Department of Mechanical Engineering, Imperial College London This protocol proposes a novel biaxial testing system used on a resistance heating uniaxial tensile test machine in order to determine the forming limit diagram (FLD) of sheet metals under hot stamping conditions. Engineering Dynamic Pore-scale Reservoir-condition Imaging of Reaction in Carbonates Using Synchrotron Fast Tomography Hannah P. Menke1, Matthew G. Andrew2, Joan Vila-Comamala3, Christoph Rau3, Martin J. Blunt1, Branko Bijeljic1 1Department of Earth Science and Engineering, Imperial College London, 2Carl Zeiss X-Ray Microscopy, 3Diamond Manchester Imaging Branchline (I13-2), Diamond Lightsource Synchrotron fast tomography was used to dynamically image dissolution of limestone in the presence of CO2-saturated brine at reservoir conditions. 100 scans were taken at a 6.1 µm resolution over a period of 2 h. Biology Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy Frederik Görlitz*1, Douglas J. Kelly*1, Sean C. Warren1, Dominic Alibhai2, Lucien West3, Sunil Kumar1, Yuriy Alexandrov1, Ian Munro1, Edwin Garcia1, James McGinty1, Clifford Talbot1, Remigiusz A. Serwa4, Emmanuelle Thinon4, Vincenzo da Paola3, Edward J. Murray5, Frank Stuhmeier6, Mark A. A. Neil1, Edward W. Tate4, Christopher Dunsby1,7, Paul M. W. French1 1Photonics Group, Department of Physics, Imperial College London, 2Institute for Chemical Biology, Department of Chemistry, Imperial College London, 3MRC Clinical Sciences Centre, Hammersmith Hospital, 4Chemical Biology Section, Department of Chemistry, Imperial College London, 5Retroscreen Virology Ltd, 6Pfizer Global Research and Development, Pfizer Limited, Sandwich, Kent, UK, 7Centre for Histopathology, Imperial College London We present an open source high content analysis (HCA) instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using Förster resonance energy transfer (FRET) based readouts. Data acquisition for this openFLIM-HCA instrument is controlled by software written in µManager and data analysis is undertaken in FLIMfit. Bioengineering Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells Sara Correia Carreira1, James P.K. Armstrong2, Mitsuhiro Okuda3,4, Annela M. Seddon1, Adam W. Perriman5, Walther Schwarzacher6 1Bristol Centre for Functional Nanomaterials, University of Bristol, 2Department of Materials, Imperial College London, 3Self Assembly Group, CIC nanoGUNE, 4Ikebasque, Basque Foundation for Science, 5School of Cellular and Molecular Medicine, University of Bristol, 6H.H. Wills Physics Laboratory, University of Bristol A protocol for the synthesis and cationization of cobalt-doped magnetoferritin is presented, as well as a method to rapidly magnetize stem cells with cationized magnetoferritin. Engineering Knowledge Based Cloud FE Simulation of Sheet Metal Forming Processes Du Zhou1, Xi Yuan1, Haoxiang Gao1, Ailing Wang1, Jun Liu1, Omer El Fakir1, Denis J. Politis1, Liliang Wang1, Jianguo Lin1 1Department of Mechanical Engineering, Imperial College London The following paper presents a novel FE simulation technique (KBC-FE), which reduces computational cost by performing simulations on a cloud computing environment, through the application of individual modules. Moreover, it establishes a seamless collaborative network between world leading scientists, enabling the integration of cutting edge knowledge modules into FE simulations. Neuroscience Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer Chun-Chieh Lin1, Olena Riabinina2, Christopher J. Potter1 1The Solomon H. Snyder Department of Neuroscience, Center for Sensory Biology, Johns Hopkins University School of Medicine, 2MRC Clinical Sciences Center, Imperial College London We describe here a behavioral setup and data analysis method for assaying olfactory responses of up to 100 vinegar flies (Drosophila melanogaster). This system may be used with single or multiple olfactory stimuli, and adaptable for optogenetic activation or silencing of neuronal subsets. Environment Pretreatment of Lignocellulosic Biomass with Low-cost Ionic Liquids Florence J. V. Gschwend1, Agnieszka Brandt1, Clementine L. Chambon1, Wei-Chien Tu1, Lisa Weigand1, Jason P. Hallett1 1Department of Chemical Engineering, Imperial College London The pretreatment of lignocellulosic biomass with protic low-cost ionic liquids is shown, resulting in a delignified cellulose-rich pulp and a purified lignin. The pulp gives rise to high glucose yields after enzymatic saccharification. Engineering Advanced Compositional Analysis of Nanoparticle-polymer Composites Using Direct Fluorescence Imaging Colin R. Crick*1, Sacha Noimark*2,3, William J. Peveler*3, Joseph C. Bear3, Aleksandar P. Ivanov1, Joshua B. Edel1, Ivan P. Parkin3 1Department of Chemistry, Imperial College London, 2Department of Medical Physics and Biomedical Engineering, University College London, 3Department of Chemistry, University College London Here we present a reliable method to monitor the incorporation of nanoparticles into a polymer host matrix via swell encapsulation. We show that the surface concentration of cadmium selenide quantum dots can be accurately visualized through cross-sectional fluorescence imaging. Medicine A Simple Device to Rapidly Prepare Whole Mounts of the Mouse Intestine Mitsuhiro Yoneda1, Alfredo A. Molinolo2, Jerrold M. Ward3, Shioko Kimura1, Robert A. Goodlad4 1National Cancer Institute, Laboratory of Metabolism, National Institutes of Health, 2National Institute of Dental and Craniofacial Research, Oral and Pharyngeal Cancer Branch, National Institutes of Health, 3Global VetPathology, 4Centre for Pathology, Hammersmith Hospital, Imperial College The use of a simple device to cut and ‘roll’ mouse intestines to rapidly prepare whole mount preparations is described. Engineering A Method for Studying the Temperature Dependence of Dynamic Fracture and Fragmentation David R. Jones1, David J. Chapman1, Daniel E. Eakins1 1Institute of Shock Physics, Imperial College London Fracture and fragmentation are late stage phenomena in dynamic loading scenarios and are typically studied using explosives. We present a technique for driving expansion using a gas gun which uniquely enables control of both loading rate and sample temperature. Chemistry The Synthesis, Characterization and Reactivity of a Series of Ruthenium N-triphosPh Complexes Andreas Phanopoulos1, Nicholas Long1, Philip Miller1 1Department of Chemistry, Imperial College London Ruthenium phosphine complexes are widely used for homogeneous catalytic reactions such as hydrogenations. The synthesis of a series of novel tridentate ruthenium complexes bearing the N-triphos ligand N(CH2PPh2)3 is reported. Additionally, the stoichiometric reaction of a dihydride Ru–N-triphos complex with levulinic acid is described. Medicine Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines Ylenia Lombardo*1, Alexander de Giorgio*1, Charles R. Coombes1, Justin Stebbing1, Leandro Castellano1 1Department of Surgery and Cancer, Imperial College London Floating mammosphere assays can investigate the subset of stem-like breast cancer cells that survive in suspension conditions and show enhanced tumorigenesis when implanted into mice. This protocol provides a convenient in vitro measure of sphere-forming ability, a proxy for in vivo tumorigenesis, while facilitating analysis of the stem-associated transcriptional landscape. Neuroscience Analysis of Gene Expression Changes in the Rat Hippocampus After Deep Brain Stimulation of the Anterior Thalamic Nucleus Tharakeswari Selvakumar1, Kambiz N. Alavian2, Travis Tierney1 1 The mechanism underlying the therapeutic effects of Deep Brain Stimulation (DBS) surgery needs investigation. The methods presented in this manuscript describe an experimental approach to examine the cellular events triggered by DBS by analyzing the gene expression profile of candidate genes that can facilitate neurogenesis post DBS surgery. Engineering Reservoir Condition Pore-scale Imaging of Multiple Fluid Phases Using X-ray Microtomography Matthew Andrew1, Branko Bijeljic1, Martin Blunt1 1Department of Earth Science and Engineering, Imperial College London We present a methodology for the imaging of multiple fluid phases at reservoir conditions by the use of x-ray microtomography. We show some representative results of capillary trapping in a carbonate rock sample. Neuroscience Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains Maria Weinert1, Tharakeswari Selvakumar2, Travis S. Tierney2, Kambiz N. Alavian1,3 1Division of Brain Sciences, Department of Medicine, Imperial College London, 2 The causes of degeneration of midbrain dopaminergic neurons during Parkinson’s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration. Immunology and Infection Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions Maria J. Mazon Moya1, Emma Colucci-Guyon2, Serge Mostowy1 1Section of Microbiology, MRC Centre for Molecular Bacteriology and Infection, Imperial College London, 2Département de Biologie du Développement et des Cellules Souches, Institut Pasteur, Unité Macrophages et Développement de l'Immunité To counteract pathogen dissemination, host cells reorganize their cytoskeleton to compartmentalize bacteria and induce autophagy. Using Shigella infection of tissue culture cells, host and pathogen determinants underlying this process are identified and characterized. Using zebrafish models of Shigella infection, the role of discovered molecules and mechanisms are investigated in vivo. Biology In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow Mark K. Scott1, Olufolake Akinduro2, Cristina Lo Celso2 1Department of Life Science and Facility for Imaging by Light Microscopy, Imperial College London, 2Department of Life Sciences, Imperial College London The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement. Bioengineering Manufacturing Of Robust Natural Fiber Preforms Utilizing Bacterial Cellulose as Binder Koon-Yang Lee1,2, Siti Rosminah Shamsuddin3, Marta Fortea-Verdejo1, Alexander Bismarck1,3 1Polymer and Composite Engineering (PaCE) Group, Institute of Materials Chemistry and Research, University of Vienna, 2Department of Chemical Engineering, University College London, 3Polymer and Composite Engineering (PaCE) Group, Department of Chemical Engineering, Imperial College London We present a novel method of manufacturing rigid and robust short natural fiber preforms using a papermaking process. Bacterial cellulose acts simultaneously as the binder for the loose fibers and provides rigidity to the fiber preforms. These preforms can be infused with a resin to produce truly green hierarchical composites. Chemistry Synthesis and Purification of Iodoaziridines Involving Quantitative Selection of the Optimal Stationary Phase for Chromatography Tom Boultwood1, Dominic P. Affron1, James A. Bull1 1Department of Chemistry, Imperial College London A protocol for the diastereoselective one-pot preparation of cis-N-Ts-iodoaziridines is described. The generation of diiodomethyllithium, addition to N-Ts aldimines and cyclization of the amino gem-diiodide intermediate to iodoaziridines is demonstrated. Also included is a protocol to rapidly and quantitatively assess the most appropriate stationary phase for purification by chromatography. Bioengineering FIBS-enabled Noninvasive Metabolic Profiling Alireza Behjousiar1,2, Antony Constantinou2, Karen M. Polizzi2, Cleo Kontoravdi1 1Centre for Process Systems Engineering, Department of Chemical Engineering and Chemical Technology, Imperial College London, 2Centre for Synthetic Biology and Innovation, Division of Molecular Biosciences, Imperial College London A description of how to calibrate Förster Resonance Energy Transfer integrated biological sensors (FIBS) for in situ metabolic profiling is presented. The FIBS can be used to measure intracellular levels of metabolites noninvasively aiding in the development of metabolic models and high throughput screening of bioprocess conditions. Medicine Intramyocardial Cell Delivery: Observations in Murine Hearts Tommaso Poggioli1,2, Padmini Sarathchandra1,2, Nadia Rosenthal2,3, Maria P. Santini1,2 1Magdi Yacoub Institute, Imperial College London, 2National Heart and Lung Institute, Imperial College London, 3Australian Regenerative Medicine Institute, Monash University Intramyocardial cell delivery in murine models of cardiovascular diseases, such as hypertension or myocardial infarction, is widely used to test the therapeutic potential of different cell types in regenerative studies. Therefore, a detailed description and a clear visualization of this surgical procedure will help to define the limits and advantages of cardiovascular cell therapeutic analyses in small rodents. Immunology and Infection Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection Clare R. Harding1, Gunnar N. Schroeder1, James W. Collins1, Gad Frankel1 1Center for Molecular Bacteriology and Infection, Imperial College London The larva of the wax moth Galleria mellonella was recently established as an in vivo model to study Legionella pneumophila infection. Here, we demonstrate fundamental techniques to characterize the pathogenesis of Legionella in the larvae, including inoculation, measurement of bacterial virulence and replication as well as extraction and analysis of infected hemocytes. Immunology and Infection 4D Multimodality Imaging of Citrobacter rodentium Infections in Mice James William Collins1, Jeffrey A Meganck2, Chaincy Kuo2, Kevin P Francis2, Gad Frankel1 1MRC Centre for Molecular Bacteriology and Infection, Division of Cell & Molecular Biology, Imperial College London, 2Preclinical Imaging, Caliper- A PerkinElmer Company Multi-modality imaging is a valuable approach for studying bacterial colonization in small animal models. This protocol outlines infection of mice with bioluminescent Citrobacter rodentium and the longitudinal monitoring of bacterial colonization using composite 3D diffuse light imaging tomography with μCT imaging to create a 4D movie of C. rodentium infection. Immunology and Infection A Visual Assay to Monitor T6SS-mediated Bacterial Competition Abderrahman Hachani1, Nadine S. Lossi1, Alain Filloux1 1MRC Centre for Molecular Bacteriology and Infection, Division of Cell and Molecular Biology, Imperial College London We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms. Medicine Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens Dorothée Sturm1,2, Lorella Marselli3, Florian Ehehalt1,2, Daniela Richter1, Marius Distler2, Stephan Kersting1,2, Robert Grützmann2, Krister Bokvist4, Philippe Froguel5, Robin Liechti6, Anne Jörns7, Paolo Meda8, Gustavo Bruno Baretton9, Hans-Detlev Saeger2, Anke M. Schulte10, Piero Marchetti3, Michele Solimena1 1Molecular Diabetology, Paul Langerhans Institute Dresden, 2Department of GI-, Thoracic- and Vascular Surgery, University Hospital Carl Gustav Carus, University of Technology Dresden, 3Department of Endocrinology and Metabolism, Metabolic Unit University of Pisa, 4Labs DC0522, Lilly Corporate Center, 5Genomics, Faculty of Medicine Imperial College London, 6Vital-IT, SIB Swiss Institute of Bioinformatics, 7Clinical Biochemistry, Hannover Medical School, 8Cell Physiology and Metabolism, Medical School, University of Geneva, 9Department of Pathology, University Hospital Carl Gustav Carus, University of Technology Dresden, 10R&D DIAB Division / Translational Medicine, Sanofi-Aventis Laser microdissection is a technique that allows the recovery of selected cells from minute amounts of parenchyma. Here we describe a protocol for acquiring human pancreatic islets from surgical specimens to be used for transcriptomic studies. Our protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their collection. Biology High-throughput Purification of Affinity-tagged Recombinant Proteins Simone C. Wiesler1, Robert O.J. Weinzierl1 1Department of Life Sciences, Imperial College London We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format. Immunology and Infection Reverse Genetics Mediated Recovery of Infectious Murine Norovirus Armando Arias*1, Luis Ureña*1, Lucy Thorne1, Muhammad A. Yunus1, Ian Goodfellow1 1Section of Virology, Imperial College London Noroviruses are a major cause of gastroenteritis yet molecular techniques for their characterisation are still relatively new. Here we report two different reverse genetics approaches for the efficient recovery of murine norovirus (MNV), the only member of this genus which can be propagated in cell culture. Medicine Roux-en-Y Gastric Bypass Operation in Rats Marco Bueter1,2, Kathrin Abegg2,3, Florian Seyfried4, Thomas A. Lutz2,3, Carel W. le Roux4 1Department of Surgery, University Hospital Zürich, 2Zürich Centre for Integrative Human Physiology, University of Zürich, 3Institute of Veterinary Physiology, Vetsuisse Faculty, University of Zürich, 4Imperial Weight Centre, Department of Investigative Medicine, Imperial College London Numerous studies using gastric bypass rat models have been recently conducted to uncover the underlying physiological mechanisms of Roux-en-Y gastric bypass operations. This article aims to demonstrate and discuss the technical and experimental details of our published gastric bypass rat model to understand advantages and limitations of this experimental tool. Medicine The Use of Pharmacological-challenge fMRI in Pre-clinical Research: Application to the 5-HT System Anne Klomp1, Jordi L. Tremoleda2, Anouk Schrantee1, Willy Gsell2, Liesbeth Reneman1 1Department of Radiology, Brain Imaging Center, Academic Medical Center Amsterdam, 2Biological Imaging Centre, MRC Clinical Sciences Centre, Imperial College London The goal of this technique is to assess serotonin (5-HT) neurotransmitter function in the live and free-breathing animal with pharmacological magnetic resonance imaging (phMRI) and an intravenous challenge with a selective serotonin reuptake inhibitor (SSRI), fluoxetine. Bioengineering Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis Teresa Mortera-Blanco1, Maria Rende1, Hugo Macedo1, Serene Farah1, Alexander Bismarck1, Athanasios Mantalaris1, Nicki Panoskaltsis2 1Department of Chemical Engineering and Chemical Technology, South Kensington campus, Imperial College London, 2Department of Hematology, Northwick Park & St. Mark's campus, Imperial College London A 3D culture system for hematopoiesis is described using human cord blood and leukemic bone marrow cells. The method is based on the use of a porous synthetic polyurethane scaffold coated with extracellular matrix proteins. This scaffold is adaptable to accommodate a wide range of cells. Biology Identification of Protein Interacting Partners Using Tandem Affinity Purification Dalan Bailey*1, Luis Urena*1, Lucy Thorne1, Ian Goodfellow1 1Section of Virology, Department of Medicine, Imperial College London Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein. Bioengineering Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells Klaus Suhling1, James A. Levitt1, Pei- Hua Chung1, Marina. K. Kuimova2, Gokhan Yahioglu3 1Department of Physics, King's College London, 2Department of Chemistry, Imperial College London, 3PhotoBiotics Ltd Fluorescence Lifetime Imaging (FLIM) has emerged as a key technique to image the environment and interaction of specific proteins and dyes in living cells. FLIM of fluorescent molecular rotors allows mapping of viscosity in living cells. Medicine Implantation of a Carotid Cuff for Triggering Shear-stress Induced Atherosclerosis in Mice Michael T. Kuhlmann1, Simon Cuhlmann2,3, Irmgard Hoppe1, Rob Krams3, Paul C. Evans2, Gustav J. Strijkers4, Klaas Nicolay4, Sven Hermann1, Michael Schäfers1 1European Institute for Molecular Imaging, Westfälische Wilhelms-University Münster, 2British Heart Foundation Cardiovascular Sciences Unit, Imperial College London, 3Department of Bioengineering, Imperial College London, 4Biomedical Engineering, Eindhoven University of Technology The constricting cuff presented in this article is designed to induce atherosclerosis in the murine common carotid artery. Due to the conical shape of its inner lumen the implanted cuff generates well-defined regions of low, high and oscillatory shear stress triggering the development of atherosclerotic lesions of different inflammatory phenotypes. Bioengineering Fluorescence detection methods for microfluidic droplet platforms Xavier Casadevall i Solvas1, Xize Niu1, Katherine Leeper1, Soongwon Cho1, Soo-Ik Chang2, Joshua B. Edel1, Andrew J. deMello3 1Department of Chemistry, Imperial College London, 2Department of Biochemistry, Protein Chip Research Center, Chungbuk National University, 3Department of Chemistry and Applied Biosciences, Institute for Chemical and Bioengineering, ETH Zurich Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized. Immunology and Infection A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb (BCG lux/M.Tb lux) Sandra Newton1, Adrian Martineau2, Beate Kampmann1 1Department of Paediatrics, Imperial College London, 2Centre for Health Sciences, Barts & The London School of Medicine and Dentistry We describe an alternative approach to the enumeration of mycobacteria in vitro, which uses reporter-gene tagged mycobacteria instead of colony-forming units (CFU). “Survival” of organisms as well as host response-markers are measured simultaneously, providing a low-cost, versatile and functional system for studies of host/pathogen interactions in the context of tuberculosis. Neuroscience An Experimental Platform to Study the Closed-loop Performance of Brain-machine Interfaces Naveed Ejaz1, Kris D. Peterson1, Holger G. Krapp1 1Department of Bioengineering, Imperial College London We use a closed-loop fly-machine interface to investigate general principles in neuronal control. Biology Detection of Signaling Effector-Complexes Downstream of BMP4 Using in situ PLA, a Proximity Ligation Assay Efstathia Thymiakou1, Vasso Episkopou1 1Medical Research Council, Clinical Sciences Centre, Imperial College, Hammersmith Hospital Here we show how to use Proximity Ligation Assay (PLA), with a combination of antibodies to visualize Bone Morphogenetic Protein (BMP) signaling in fixed cells. This technique allowed us to follow the nuclear accumulation of endogenous BMP activated effector-complexes and quantify their levels over time under BMP4 stimulation. Biology The MODS method for diagnosis of tuberculosis and multidrug resistant tuberculosis Mark F Brady1, Jorge Coronel2, Robert H Gilman3, David AJ Moore4 1The Warren Alpert Medical School of Brown University, 2Laboratorio de Investigacion de Enfermedades Infecciosas, Universidad Peruana Cayetano Heredia, 3Department of International Health, Johns Hopkins Bloomberg School of Public Health, 4Wellcome Trust Centre for Clinical Tropical Medicine, Imperial College London The microscopic-observation drug-susceptibility (MODS) assay is a low-cost, low-tech tool for high-performance detection of tuberculosis (TB) and multidrug-resistant tuberculosis (MDRTB). This video describes the MODS liquid media culture method.