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Imperial College London

63 ARTICLES PUBLISHED IN JoVE

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Biology

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)
Cristina Lo Celso 1, David Scadden 1
1Center for Regenerative Medicine, Massachusetts General Hospital, Harvard Stem Cell Institute, Harvard Medical School

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

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Neuroscience

An Experimental Platform to Study the Closed-loop Performance of Brain-machine Interfaces
Naveed Ejaz 1, Kris D. Peterson 1, Holger G. Krapp 1
1Department of Bioengineering, Imperial College London

We use a closed-loop fly-machine interface to investigate general principles in neuronal control.

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Bioengineering

Fluorescence detection methods for microfluidic droplet platforms
Xavier Casadevall i Solvas 1, Xize Niu 1, Katherine Leeper 1, Soongwon Cho 1, Soo-Ik Chang 2, Joshua B. Edel 1, Andrew J. deMello 3
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.

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Bioengineering

Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis
Teresa Mortera-Blanco 1, Maria Rende 1, Hugo Macedo 1, Serene Farah 1, Alexander Bismarck 1, Athanasios Mantalaris 1, Nicki Panoskaltsis 2
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.

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Neuroscience

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes
Silvio Sacchetti 1, Kambiz N. Alavian 1, Emma Lazrove 1, Elizabeth A. Jonas 1
1Department of Internal Medicine, Yale University

A method to isolate submitochondrial vesicles enriched in F1FO ATP synthase complexes from rat brain is described. These vesicles allow the study of the activity of F1FO ATPase complex and its modulation using the technique of patch clamp recording.

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Immunology and Infection

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice
James William Collins 1, Jeffrey A Meganck 2, Chaincy Kuo 2, Kevin P Francis 2, Gad Frankel 1
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.

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Immunology and Infection

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection
Clare R. Harding 1, Gunnar N. Schroeder 1, James W. Collins 1, Gad Frankel 1
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.

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Medicine

Intramyocardial Cell Delivery: Observations in Murine Hearts
Tommaso Poggioli 1,2, Padmini Sarathchandra 1,2, Nadia Rosenthal 2,3, Maria P. Santini 1,2
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.

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Bioengineering

FIBS-enabled Noninvasive Metabolic Profiling
Alireza Behjousiar 1,2, Antony Constantinou 2, Karen M. Polizzi 2, Cleo Kontoravdi 1
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.

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Bioengineering

Manufacturing Of Robust Natural Fiber Preforms Utilizing Bacterial Cellulose as Binder
Koon-Yang Lee 1,2, Siti Rosminah Shamsuddin 3, Marta Fortea-Verdejo 1, Alexander Bismarck 1,3
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.

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Immunology and Infection

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions
Maria J. Mazon Moya 1, Emma Colucci-Guyon 2, Serge Mostowy 1
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.

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Chemistry

Synthesis and Purification of Iodoaziridines Involving Quantitative Selection of the Optimal Stationary Phase for Chromatography
Tom Boultwood 1, Dominic P. Affron 1, James A. Bull 1
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.

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Biology

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow
Mark K. Scott 1, Olufolake Akinduro 2, Cristina Lo Celso 2
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.

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Engineering

Reservoir Condition Pore-scale Imaging of Multiple Fluid Phases Using X-ray Microtomography
Matthew Andrew 1, Branko Bijeljic 1, Martin Blunt 1
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.

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Neuroscience

Analysis of Gene Expression Changes in the Rat Hippocampus After Deep Brain Stimulation of the Anterior Thalamic Nucleus
Tharakeswari Selvakumar 1, Kambiz N. Alavian 2, Travis Tierney 1
1Department of Neurosurgery, Brigham & Women's Hospital, Harvard Medical School, 2Division of Brain Sciences, Department of Medicine, Imperial College London

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.

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Education

A Method for Studying the Temperature Dependence of Dynamic Fracture and Fragmentation
David R. Jones 1, David J. Chapman 1, Daniel E. Eakins 1
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.

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Neuroscience

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains
Maria Weinert 1, Tharakeswari Selvakumar 2, Travis S. Tierney 2, Kambiz N. Alavian 1,3
1Division of Brain Sciences, Department of Medicine, Imperial College London, 2Department of Neurosurgery, Brigham and Women's Hospital, Harvard Medical School, 3Department of Internal Medicine, Endocrinology, Yale University School of Medicine

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.

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Medicine

Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines
Ylenia Lombardo *1, Alexander de Giorgio *1, Charles R. Coombes 1, Justin Stebbing 1, Leandro Castellano 1
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.

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Chemistry

The Synthesis, Characterization and Reactivity of a Series of Ruthenium N-triphosPh Complexes
Andreas Phanopoulos 1, Nicholas Long 1, Philip Miller 1
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.

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Behavior

A Structured Rehabilitation Protocol for Improved Multifunctional Prosthetic Control: A Case Study
Aidan Dominic Roche 1,2, Ivan Vujaklija 3,4, Sebastian Amsüss 3,4, Agnes Sturma 1,5, Peter Göbel 6, Dario Farina 3,4, Oskar C. Aszmann 1,2
1Christian Doppler Laboratory for Restoration of Extremity Function, 2Department of Surgery, Division of Plastic and Reconstructive Surgery, Medical University of Vienna, 3Department of Neurorehabilitation Engineering, Bernstein Focus Neurotechnology Göttingen, 4University Medical Center Göttingen, Georg-August University, 5University of Applied Sciences FH Campus Wien, 6Research & Development, Otto Bock Healthcare Products GmbH

As prosthetic development moves towards the goal of natural control, harnessing amputees’ inherent ability to learn new motor skills may enable proficiency. This manuscript describes a structured rehabilitation protocol, which includes imitation, repetition, and reinforcement learning strategies, for improved multifunctional prosthetic control.

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Bioengineering

Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography
Dustin R. Morado 1, Bo Hu 1, Jun Liu 1
1Department of Pathology and Laboratory Medicine, University of Texas Health Science Center at Houston

We present a protocol on how to utilize high-throughput cryo-electron tomography to determine high resolution in situ structures of molecular machines. The protocol permits large amounts of data to be processed, avoids common bottlenecks and reduces resource downtime, allowing the user to focus on important biological questions.

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Engineering

Dynamic Pore-scale Reservoir-condition Imaging of Reaction in Carbonates Using Synchrotron Fast Tomography
Hannah P. Menke 1, Matthew G. Andrew 2, Joan Vila-Comamala 3, Christoph Rau 3, Martin J. Blunt 1, Branko Bijeljic 1
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.

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JoVE Core

Knowledge Based Cloud FE Simulation of Sheet Metal Forming Processes
Du Zhou 1, Xi Yuan 1, Haoxiang Gao 1, Ailing Wang 1, Jun Liu 1, Omer El Fakir 1, Denis J. Politis 1, Liliang Wang 1, Jianguo Lin 1
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.

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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. Bear 3, Aleksandar P. Ivanov 1, Joshua B. Edel 1, Ivan P. Parkin 3
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.

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Environment

Pretreatment of Lignocellulosic Biomass with Low-cost Ionic Liquids
Florence J. V. Gschwend 1, Agnieszka Brandt 1, Clementine L. Chambon 1, Wei-Chien Tu 1, Lisa Weigand 1, Jason P. Hallett 1
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.

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Neuroscience

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer
Chun-Chieh Lin 1, Olena Riabinina 2, Christopher J. Potter 1
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.

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Bioengineering

Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells
Sara Correia Carreira 1, James P.K. Armstrong 2, Mitsuhiro Okuda 3,4, Annela M. Seddon 1, Adam W. Perriman 5, Walther Schwarzacher 6
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.

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Biology

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy
Frederik Görlitz *1, Douglas J. Kelly *1, Sean C. Warren 1, Dominic Alibhai 2, Lucien West 3, Sunil Kumar 1, Yuriy Alexandrov 1, Ian Munro 1, Edwin Garcia 1, James McGinty 1, Clifford Talbot 1, Remigiusz A. Serwa 4, Emmanuelle Thinon 4, Vincenzo da Paola 3, Edward J. Murray 5, Frank Stuhmeier 6, Mark A. A. Neil 1, Edward W. Tate 4, Christopher Dunsby 1,7, Paul M. W. French 1
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.

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JoVE Core

A Novel Biaxial Testing Apparatus for the Determination of Forming Limit under Hot Stamping Conditions
Zhutao Shao 1, Nan Li 1
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.

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Bioengineering

Evaluating Primary Blast Effects In Vitro
Niall J. Logan 1, Hari Arora 1, Claire A. Higgins 1
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.

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Developmental Biology

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity
Shen Zhang 1, Yonggen Wu 1, Yimin Weng 2, Zhihui Xu 1, Wenmin Chen 3, Dahan Zheng 4, Wei Lin 5, Jun Liu 6, Ying Zhou 1,7
1Reproductive Medicine Center, The First Affiliated Hospital of Wenzhou Medical University, 2Department of Orthopaedics, The Second Affiliated Hospital of Wenzhou Medical University, 3Department of Obstetrics, The First Affiliated Hospital of Wenzhou Medical University, 4School of Laboratory Medicine and Life Science, Wenzhou Medical University, 5School of Pharmaceutical Science, Wenzhou Medical University, 6Stem Cells and Genetic Engineering Group, AgriBioscience Research Centre, Department of Economic Development, Jobs, Transport and Resources, 7Department of Histology and Embryology, Wenzhou Medical University

A highly promising technique to generate tissue constructs without using matrix is to culture cells in a simulated microgravity condition. Using a rotary culture system, we examined ovarian follicle growth and oocyte maturation in terms of follicle survival, morphology, growth, and oocyte function under the simulated microgravity condition.

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Cancer Research

Counting Proteins in Single Cells with Addressable Droplet Microarrays
Stelios Chatzimichail *1, Pashiini Supramaniam *1, Oscar Ces 1, Ali Salehi-Reyhani 1
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.

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Chemistry

Measurements of Long-range Electronic Correlations During Femtosecond Diffraction Experiments Performed on Nanocrystals of Buckminsterfullerene
Rebecca A. Ryan 1, Sophie Williams 1, Andrew V. Martin 1, Ruben A. Dilanian 1, Connie Darmanin 2, Corey T. Putkunz 1, David Wood 3, Victor A. Streltsov 4, Michael W.M. Jones 5, Naylyn Gaffney 6, Felix Hofmann 7, Garth J. Williams 8, Sebastien Boutet 9, Marc Messerschmidt 10, M. Marvin Seibert 11, Evan K. Curwood 11, Eugeniu Balaur 2, Andrew G. Peele 5, Keith A. Nugent 2, Harry M. Quiney 1, Brian Abbey 2
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.

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Bioengineering

Optimized Setup and Protocol for Magnetic Domain Imaging with In Situ Hysteresis Measurement
Jun Liu 1, John Wilson 2, Claire Davis 1, Anthony Peyton 2
1Advanced Steel Research Centre, Warwick Manufacturing Group, University of Warwick, 2School of Electrical and Electronic Engineering, University of Manchester

This paper elaborates the sample and sensor preparation procedures and the protocols for using the test rig particularly for dynamic domain imaging with in situ BH measurements in order to achieve optimal domain pattern quality and accurate BH measurements.

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Engineering

Film Control to Study Contributions of Waves to Droplet Impact Dynamics on Thin Flowing Liquid Films
Idris T. Adebayo 1, Omar K. Matar 1
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.

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Engineering

Pore-scale Imaging and Characterization of Hydrocarbon Reservoir Rock Wettability at Subsurface Conditions Using X-ray Microtomography
Amer M. Alhammadi 1, Ahmed AlRatrout 1, Branko Bijeljic 1, Martin J. Blunt 1
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.

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Neuroscience

A Novel In Vitro Model of Blast Traumatic Brain Injury
Rita Campos-Pires 1,2, Amina Yonis 1, Warren Macdonald 2,3, Katie Harris 1, Christopher J. Edge 4,5, Peter F. Mahoney 6, Robert Dickinson 1,2
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.

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Immunology and Infection

Use of Single Chain MHC Technology to Investigate Co-agonism in Human CD8+ T Cell Activation
Xiang Zhao 1, Maryam Hamidinia 1, Joanna Ai Ling Choo 1, Chien Tei Too 1,2, Zi Zong Ho 3, Ee Chee Ren 4, Antonio Bertoletti 3, Paul A. MacAry 1,2,5, Keith G. Gould 6, Joanna Brzostek 1, Nicholas R.J. Gascoigne 1,2,5
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.

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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 Khara 1,2, Camilla A. Gladstone 1, Brian D. Robertson 3, 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.

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Genetics

Transcription Start Site Mapping Using Super-low Input Carrier-CAGE
Nevena Cvetesic 1,2, Elena Pahita 1,2, Boris Lenhard 1,2,3
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.

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Neuroscience

Surface Electromyographic Biofeedback as a Rehabilitation Tool for Patients with Global Brachial Plexus Injury Receiving Bionic Reconstruction
Laura A. Hruby 1,2, Agnes Sturma 1,3, Oskar C. Aszmann 1,4
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.

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Medicine

Structured Motor Rehabilitation After Selective Nerve Transfers
Agnes Sturma 1,2,3, Laura A. Hruby 1,4, Dario Farina 2, Oskar C. Aszmann 1,5
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.

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Bioengineering

Electrowetting-based Digital Microfluidics Platform for Automated Enzyme-linked Immunosorbent Assay
Nikolay Dimov 1, Martin B. McDonnell 1, Ian Munro 1, Daniel K. McCluskey 1, Ian D. Johnston 1, Christabel K. L. Tan 1, Loïc Coudron 1
1School of Engineering and Computer Science, University of Hertfordshire

Electrowetting-based digital microfluidic is a technique that utilizes a voltage-driven change in the apparent contact angle of a microliter-volume droplet to facilitate its manipulation. Combining this with functionalized magnetic beads enables the integration of multiple laboratory unit operations for sample preparation and identification of pathogens using Enzyme-linked Immunosorbent Assay (ELISA).

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Developmental Biology

Cell-cell Fusion of Genome Edited Cell Lines for Perturbation of Cellular Structure and Function
Robert Mahen 1,2, Reiner Schulte 3
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.

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Cancer Research

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity
Sarah Spear 1,2, Iain A McNeish 1, Melania Capasso 2,3
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.

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Immunology and Infection

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System
David Chetrit 1, Donghyun Park 1,2, Bo Hu 3, Jun Liu 1,2, Craig R. Roy 1
1Department of Microbial Pathogenesis, Boyer Center for Molecular Medicine, Yale University School of Medicine, 2Microbial Sciences Institute, Yale University, 3Department of Microbiology and Molecular Genetics, McGovern Medical School, The University of Texas Health Science Center at Houston

Imaging of bacterial cells is an emerging systems biology approach focused on defining static and dynamic processes that dictate the function of large macromolecular machines. Here, integration of quantitative live cell imaging and cryo-electron tomography is used to study Legionella pneumophila type IV secretion system architecture and functions.

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Medicine

Applying a Three-dimensional Uniaxial Mechanical Stimulation Bioreactor System to Induce Tenogenic Differentiation of Tendon-Derived Stem Cells
Ziming Chen *1, Peilin Chen *1, Rui Ruan *1, Lianzhi Chen 1, Jun Yuan 1, David Wood 1, Tao Wang 1, Ming Hao Zheng 1
1Centre of Orthopaedic Translational Research, Medical School, University of Western Australia

A three-dimensional uniaxial mechanical stimulation bioreactor system is an ideal bioreactor for tenogenic-specific differentiation of tendon-derived stem cells and neo-tendon formation.

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Cancer Research

Isolation of Proximal Fluids to Investigate the Tumor Microenvironment of Pancreatic Adenocarcinoma
Greta Donisi *1, Marialuisa Barbagallo *2, Giovanni Capretti 1,3, Gennaro Nappo 1,3, Panteleimon G. Takis 4,5,6, Alessandro Zerbi 1,3, Federica Marchesi 2,7, Nina Cortese 2
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.

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Environment

Flash Infrared Annealing for Perovskite Solar Cell Processing
Pui Sha Victoria Ling 1,2, Anders Hagfeldt 2, Sandy Sanchez 2
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%.

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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 Arshad 1, Xiaomeng Wang 2,3,4, Peter D. Weinberg 1
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.

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Cancer Research

Probing Metabolism and Viscosity of Cancer Cells using Fluorescence Lifetime Imaging Microscopy
Liubov Shimolina 1, Maria Lukina 1, Vladislav Shcheslavskiy 1,2, Vadim Elagin 1, Varvara Dudenkova 1, Nadezhda Ignatova 1, Marina K. Kuimova 3, Marina Shirmanova 1
1Institute of Experimental Oncology and Biomedical Technologies, Privolzhsky Research Medical University, 2Becker & Hickl GmbH, 3Department of Chemistry, Imperial College London

Here, we demonstrate the use of fluorescence lifetime imaging microscopy (FLIM) to sequentially image cellular metabolism and plasma membrane viscosity in live cancer cell culture. Metabolic assessments are performed by detecting endogenous fluorescence. Viscosity is measured using a fluorescent molecular rotor.

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Neuroscience

Fluorescence-Activated Cell Sorting-Radioligand Treated Tissue (FACS-RTT) to Determine the Cellular Origin of Radioactive Signal
Quentin Amossé 1,2, Kelly Ceyzériat 1,3,4, Stergios Tsartsalis 1,5, Benjamin B. Tournier 1,2, Philippe Millet 1,2
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.

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JoVE Core

Therapy Interventions for Upper Limb Amputees Undergoing Selective Nerve Transfers
Agnes Sturma 1,2, Laura A. Hruby 1,3, Anna Boesendorfer 1, Clemens Gstoettner 1, Dario Farina 2, Oskar C. Aszmann 1,4
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.

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Bioengineering

A High-Yield Streptomyces Transcription-Translation Toolkit for Synthetic Biology and Natural Product Applications
Ming Toh 1,2,3,4, Kameshwari Chengan 5, Tanith Hanson 5, Paul S. Freemont 1,2,3,4,6,7, Simon J. Moore 5
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.

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Neuroscience

Preparation of Rat Sciatic Nerve for Ex Vivo Neurophysiology
Adrien Rapeaux 1,2,3,4, Omaer Syed 5, Estelle Cuttaz 5, Christopher A. R. Chapman 5, Rylie A. Green 5, Timothy G. Constandinou 1,2,3,4
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.

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Bioengineering

Rapid Antibody Glycoengineering in Chinese Hamster Ovary Cells
Masue Marbiah *1,2, Pavlos Kotidis *1,3, Roberto Donini 1,4, Itzcóatl A. Gómez 5, Ioscani Jimenez del Val 5, Stuart M. Haslam 4, Karen M. Polizzi 1,2, Cleo Kontoravdi 1
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.

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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 Beber 1,7, Aurélie Di Cicco 1, Bassam Hajj 1, François Iv 2, Manos Mavrakis 2, Gijsje H. Koenderink 3, João T. Cabral 4, Michaël Trichet 5, 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.

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Bioengineering

Microfluidic Tools for Probing Fungal-Microbial Interactions at the Cellular Level
Emily Masters-Clark 1, Amelia J. Clark 1, Claire E. Stanley 1
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.

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Chemistry

Screening of Coatings for an All-Solid-State Battery Using In Situ Transmission Electron Microscopy
Shibarata Basak 1,2, Junbeom Park 1, Janghyun Jo 2, Osmane Camara 1, Amir H. Tavabi 2, Hermann Tempel 1, Hans Kungl 1, Chandramohan George 3, Rafal E. Dunin-Borkowski 2, Joachim Mayer 2,4, Rüdiger-A. Eichel 1,5
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.

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Engineering

Large Scale Energy Efficient Sensor Network Routing Using a Quantum Processor Unit
Jie Chen 1
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.

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Immunology and Infection

Whole-Mount Fluorescence In Situ Hybridization to Study Spermatogenesis in the Anopheles Mosquito
Matteo Vitale *1, Jiangtao Liang *2, Igor Sharakhov 2, Federica Bernardini 1
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.

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Biology

Methods for Embedding Cell-Free Protein Synthesis Reactions in Macro-Scale Hydrogels
Siji Kavil *1, Alex Laverick *1, Colette J. Whitfield 1, Alice M. Banks 2, Thomas P. Howard 1
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.

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Bioengineering

Synthetic Condensates and Cell-Like Architectures from Amphiphilic DNA Nanostructures
Layla Malouf 1,2, Diana A. Tanase 1,2, Lorenzo Di Michele 1,2,3
1Department of Chemical Engineering and Biotechnology, University of Cambridge, 2Department of Chemistry, Imperial College London, 3fabriCELL, Imperial College London

We present a protocol for preparing synthetic biomolecular condensates consisting of amphiphilic DNA nanostars starting from their constituent DNA oligonucleotides. Condensates are produced from either a single nanostar component or two components and are modified to sustain in vitro transcription of RNA from an embedded DNA template.

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