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Massachusetts Institute of Technology

74 ARTICLES PUBLISHED IN JoVE

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Biology

CD4+ T-Lymphocyte Capture Using a Disposable Microfluidic Chip for HIV
Sang Jun Moon 1, Richard Lin 2, Utkan Demirci 1
1Bio-Acoustic-MEMS Laboratory in Medicine (BAMM), HST-Center for Bioengineering, Brigham and Women's Hospital, 2Massachusetts Institute of Technology

CD4+ T-Lymphocyte Capture Using a Disposable Microfluidic Chip for HIV

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Biology

Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4
G. Grant Welstead 1, Tobias Brambrink 1, Rudolf Jaenisch 1
1Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology

This video shows the procedure for generating induced pluripotent stem cells using inducible lentivirus that express Oct4, Sox2, c-Myc and Klf4.

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Biology

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy
Ellie Graeden 1,2, Hazel Sive 1,2
1Department of Biology, MIT - Massachusetts Institute of Technology, 2Whitehead Institute for Biomedical Research, MIT - Massachusetts Institute of Technology

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

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Biology

Zebrafish Brain Ventricle Injection
Jennifer H. Gutzman 1, Hazel Sive 1,2
1Whitehead Institute for Biochemical Research, 2MIT - Massachusetts Institute of Technology

After neural tube formation, the neuroepithelium constricts and folds while the tube fills with embryonic cerebrospinal fluid (eCSF) to form the embryonic brain ventricles. We developed this ventricle injection technique to better visualize the fluid filled space in contrast to the neuroepithelial shape in a live embryo.

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Neuroscience

Scalable Fluidic Injector Arrays for Viral Targeting of Intact 3-D Brain Circuits
Stephanie Chan 1, Jacob Bernstein 1, Edward Boyden 1
1Biological Engineering, Brain and Cognitive Sciences, and McGovern Institute, Massachusetts Institute of Technology

Controlling and analyzing neural circuits in vivo would be facilitated by a technology for delivery of viruses and other reagents to desired 3-dimensional sets of brain regions. We demonstrate customized fluidic injector array fabrication, and delivery of virally-encoded optical sensitizers, enabling optical manipulation of complex brain circuits.

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Biology

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.
Nynke L. van Berkum *1, Erez Lieberman-Aiden *2,3,4,5, Louise Williams *2, Maxim Imakaev 6, Andreas Gnirke 2, Leonid A. Mirny 3,6, Job Dekker 1, Eric S. Lander 2,7,8
1Program in Gene Function and Expression, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 2Broad Institute of Harvard and Massachusetts Institute of Technology, 3Division of Health Sciences and Technology, Massachusetts Institute of Technology, 4Program for Evolutionary Dynamics, Department of Organismic and Evolutionary Biology, Department of Mathematics, Harvard University , 5Department of Applied Mathematics, Harvard University , 6Department of Physics, Massachusetts Institute of Technology, 7Department of Systems Biology, Harvard Medical School, 8Department of Biology, Massachusetts Institute of Technology

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

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Immunology and Infection

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT
Oktay Kirak 1, Eva-Maria Frickel 1, Gijsbert M. Grotenbreg 1,2, Heikyung Suh 1, Rudolf Jaenisch 1,3, Hidde L. Ploegh 1,3
1Whitehead Institute for Biomedical Research, 2Departments of Microbiology and Biological Sciences, National University of Singapore, 3Department of Biology, Massachusetts Institute of Technology

We demonstrate here that epigenetic reprogramming via Somatic Cell Nuclear Transfer (SCNT) can be used as a tool to generate mouse models with pre-defined T cell receptor (TCR) specificities. These transnuclear mice express the corresponding TCR from their endogenous locus under the control of the endogenous promoter.

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Bioengineering

Studying Cell Rolling Trajectories on Asymmetric Receptor Patterns
Chia-Hua Lee 1, Suman Bose 2, Krystyn J. Van Vliet 1, Jeffrey M. Karp 3, Rohit Karnik 2
1Department of Materials Science and Engineering, MIT - Massachusetts Institute of Technology, 2Department of Mechanical Engineering, MIT - Massachusetts Institute of Technology, 3HST Center for Biomedical Engineering and Harvard Stem Cell Institute, Brigham and Women's Hospital and Harvard Medical School

We describe a protocol to observe and analyze cell rolling trajectories on asymmetric receptor-patterned substrates. The resulting data are useful for engineering of receptor-patterned substrates for label-free cell separation and analysis.

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Immunology and Infection

Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging
Joel R. Meyerson 1,2, Tommi A. White 1, Donald Bliss 3, Amy Moran 3, Alberto Bartesaghi 1, Mario J. Borgnia 1, M. Jason V. de la Cruz 1, David Schauder 1, Lisa M. Hartnell 1, Rachna Nandwani 1,4, Moez Dawood 5, Brianna Kim 6, Jun Hong Kim 7, John Sununu 8, Lisa Yang 9, Siddhant Bhatia 10, Carolyn Subramaniam 1, Darrell E. Hurt 11, Laurent Gaudreault 12, Sriram Subramaniam 1
1Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 2The Medical Research Council Mitochondrial Biology Unit, University of Cambridge , 3National Library of Medicine, National Institutes of Health, 4Massachusetts Institute of Technology, 5William Fremd High School, 6University of Virginia , 7Duke University , 8Yale University, 9University of Notre Dame , 10Washington University in St. Louis , 11Bioinformatics and Computational Biosciences Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 12Thomas Jefferson High School for Science and Technology

The protocol describes a high-throughput approach to determining structures of membrane proteins using cryo-electron tomography and 3D image processing. It covers the details of specimen preparation, data collection, data processing and interpretation, and concludes with the production of a representative target for the approach, the HIV-1 Envelope glycoprotein. These computational procedures are designed in a way that enables researchers and students to work remotely and contribute to data processing and structural analysis.

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Bioengineering

Polymer Microarrays for High Throughput Discovery of Biomaterials
Andrew L. Hook 1, Chien-Yi Chang 2, Jing Yang 1, David J. Scurr 1, Robert Langer 3, Daniel G. Anderson 3, Steve Atkinson 2, Paul Williams 2, Martyn C. Davies 1, Morgan R. Alexander 1
1Laboratory of Biophysics and Surface Analysis, University of Nottingham , 2School of Molecular Medical Sciences, University of Nottingham , 3David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

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Neuroscience

Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat
Elizabeth D. Kirby 1, Kelly Jensen 2, Ki A. Goosens 3, Daniela Kaufer 1,4
1Helen Wills Neuroscience Institute, University of California Berkeley, 2Office of Laboratory Animal Care, University of California Berkeley, 3McGovern Institute for Brain Research & The Department of Brain and Cognitive Science, Massachusetts Institute of Technology, 4Integrative Biology Department, University of California Berkeley

Targeted ablation of specific brain region(s) by infusion of an excitotoxin using stereotaxic coordinates is described. This technique could also be adapted for infusion of other chemicals into the rat brain.

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Neuroscience

An Assay for Permeability of the Zebrafish Embryonic Neuroepithelium
Jessica T. Chang 1,2, Hazel Sive 1,2
1Department of Biology, Massachusetts Institute of Technology, 2Whitehead Institute of Biomedical Research

We describe a live whole animal quantitative measurement for permeability of the embryonic zebrafish brain. The technique analyzes the ability to retain cerebrospinal fluid and molecules of different molecular weights within the neural tube lumen and quantifies their movement out of the ventricles. This method is useful for determining differences in epithelial permeability and maturation during development and disease.

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Neuroscience

Manual Drainage of the Zebrafish Embryonic Brain Ventricles
Jessica T. Chang 1, Hazel Sive 1
1Department of Biology, Whitehead Institute of Biomedical Research, Massachusetts Institute of Technology

We present a method to collect cerebrospinal fluid (CSF) and to create a system which lacks CSF within the embryonic zebrafish brain ventricular system. This allows for further examination of CSF composition and its requirement during embryonic brain development.

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Biology

The Logic, Experimental Steps, and Potential of Heterologous Natural Product Biosynthesis Featuring the Complex Antibiotic Erythromycin A Produced Through E. coli
Ming Jiang 1, Haoran Zhang 2, Blaine A. Pfeifer 1
1Chemical and Biological Engineering Department, State University of New York at Buffalo, 2Chemical Engineering Department, Massachusetts Institute of Technology

The heterologous biosynthesis of erythromycin A through E. coli includes the following experimental steps: 1) genetic transfer; 2) heterologous reconstitution; and 3) product analysis. Each step will be explained in the context of the motivation, potential, and challenges in producing therapeutic natural products using E. coli as a surrogate host.

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JoVE Journal

Micro 3D Printing Using a Digital Projector and its Application in the Study of Soft Materials Mechanics
Howon Lee 1, Nicholas X. Fang 1
1Department of Mechanical Engineering, Massachusetts Institute of Technology

We demonstrate controlled pattern transformation of swelling gel tubes by elastic instability. A simple projection micro stereo-lithography setup is built using an off-the-shelf digital data projector to fabricate three-dimensional polymeric structures in a layer-by-layer fashion. Swelling hydrogel tubes under mechanical constraint display various circumferential buckling modes depending on dimension.

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Engineering

Solid-state Graft Copolymer Electrolytes for Lithium Battery Applications
Qichao Hu 1, Antonio Caputo 2, Donald R. Sadoway 1
1Materials Science and Engineering, Massachusetts Institute of Technology, 2Materials Processing Center, Massachusetts Institute of Technology

Lithium ion batteries employ flammable and volatile organic electrolytes that are suitable for ambient temperature applications. A safer alternative to organic electrolytes are solid polymer batteries. Solid polymer batteries operate safely at high temperatures (>120 °C), thus making them applicable to high temperature applications such as deep oil drilling and hybrid electric vehicles. This paper will discuss (a) the polymer synthesis, (b) the polymer conduction mechanism, and (c) provide temperature cycling for both solid polymer and organic electrolytes.

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Education

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans
Elizabeth C. Pino 1, Christopher M. Webster 1, Christopher E. Carr 2, Alexander A. Soukas 1
1Center for Human Genetic Research and Department of Medicine, Massachusetts General Hospital and Harvard Medical School, 2Department of Earth, Atmospheric, and Planetary Sciences, Massachusetts Institute of Technology

We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.

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Biology

Acquiring Fluorescence Time-lapse Movies of Budding Yeast and Analyzing Single-cell Dynamics using GRAFTS
Christopher J. Zopf 1, Narendra Maheshri 1
1Department of Chemical Engineering, Massachusetts Institute of Technology

We present a simple protocol to obtain fluorescence microscopy movies of growing yeast cells, and a GUI-based software package to extract single-cell time series data. The analysis includes automated lineage and division time assignment integrated with visual inspection and manual curation of tracked data.

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Immunology and Infection

Measuring Growth and Gene Expression Dynamics of Tumor-Targeted S. Typhimurium Bacteria
Tal Danino *1, Arthur Prindle *2, Jeff Hasty 2,3,4, Sangeeta Bhatia 1,5,6,7,8
1Health Sciences and Technology, Massachusetts Institute of Technology, 2Department of Bioengineering, University of California, San Diego , 3Biocircuits Institute, University of California, San Diego , 4Molecular Biology Section, Division of Biological Science, University of California, San Diego , 5Broad Institute of Harvard and MIT, 6Department of Medicine, Brigham and Women's Hospital, 7Electrical Engineering and Computer Science and David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 8Howard Hughes Medical Institute

The goal of these experiments is to generate quantitative time-course data on the growth and gene expression dynamics of attenuated S. typhimurium bacterial colonies growing inside tumors. This video covers tumor cell preparation and implantation, bacteria preparation and injection, whole-animal luminescence imaging, tumor excision, and bacterial colony counting.

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Bioengineering

CometChip: A High-throughput 96-Well Platform for Measuring DNA Damage in Microarrayed Human Cells
Jing Ge *1, Somsak Prasongtanakij *2, David K. Wood 3, David M. Weingeist 1, Jessica Fessler 1, Panida Navasummrit 2, Mathuros Ruchirawat 2, Bevin P. Engelward 1
1Department of Biological Engineering, Massachusetts Institute of Technology, 2Environmental Toxicology, Chulabhorn Graduate Institute, 3Department of Biomedical Engineering, University of Minnesota

We describe here a platform that allows comet assay detection of DNA damage with unprecedented throughput. The device patterns mammalian cells into a microarray and enables parallel processing of 96 samples. The approach facilitates analysis of base level DNA damage, exposure-induced DNA damage and DNA repair kinetics.

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JoVE Core

Scanning-probe Single-electron Capacitance Spectroscopy
Kathleen A. Walsh 1, Megan E. Romanowich 1, Morewell Gasseller 1,2, Irma Kuljanishvili 1,3, Raymond Ashoori 4, Stuart Tessmer 1
1Department of Physics and Astronomy, Michigan State University, 2Department of Chemistry & Biochemistry/Physics, Mercyhurst University, 3Department of Physics, Saint Louis University, 4Department of Physics, Massachusetts Institute of Technology

Scanning-probe single-electron capacitance spectroscopy facilitates the study of single-electron motion in localized subsurface regions. A sensitive charge-detection circuit is incorporated into a cryogenic scanning probe microscope to investigate small systems of dopant atoms beneath the surface of semiconductor samples.

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Biology

Facial Transplants in Xenopus laevis Embryos
Laura A. Jacox *1,3, Amanda J. Dickinson *4, Hazel Sive 2,3
1Biological Sciences in Dental Medicine, Harvard University, 2Biology Department, Massachusetts Institute of Technology, 3Whitehead Institute, Massachusetts Institute of Technology, 4Biology Department, Virginia Commonwealth University

A technique for transplanting "Extreme Anterior Domain" facial tissue between Xenopus laevis embryos has been developed. Tissue can be moved from one gene expression background into another, allowing the study of local requirements for craniofacial development and for signaling interactions between facial regions.

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Biology

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology
Zachary Z. Sun *1, Clarmyra A. Hayes *2, Jonghyeon Shin 3, Filippo Caschera 4, Richard M. Murray 2, Vincent Noireaux 4
1Department of Biology, California Institute of Technology, 2Department of Bioengineering, California Institute of Technology, 3Synthetic Biology Center, Department of Bioengineering, Massachusetts Institute of Technology, 4School of Physics and Astronomy, University of Minnesota

This five-day protocol outlines all steps, equipment, and supplemental software necessary for creating and running an efficient endogenous Escherichia coli based TX-TL cell-free expression system from scratch. With reagents, the protocol takes 8 hours or less to setup a reaction, collect, and process data.

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Bioengineering

Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System
Oren Levy 1,2,3,4,5, Priya Anandakumaran 1,2,3,4,5, Jessica Ngai 1,2,3,4,5, Rohit Karnik 6, Jeffrey M. Karp 1,2,3,4,5
1Division of Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital, 2Center for Regenerative Therapeutics, Brigham and Women's Hospital, 3Harvard Medical School, Harvard University, 4Harvard Stem Cell Institute, Harvard University, 5Harvard-MIT Division of Health Sciences and Technology, 6Department of Mechanical Engineering, Massachusetts Institute of Technology

This study used a multi-well plate microfluidic system, significantly increasing throughput of cell rolling studies under physiologically relevant shear flow. Given the importance of cell rolling in the multi-step cell homing cascade and the importance of cell homing following systemic delivery of exogenous populations of cells in patients, this system offers potential as a screening platform to improve cell-based therapy.

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Chemistry

Particles without a Box: Brush-first Synthesis of Photodegradable PEG Star Polymers under Ambient Conditions
Jenny Liu 1, Angela Xiaodi Gao 1, Jeremiah A. Johnson 1
1Department of Chemistry, Massachusetts Institute of Technology

Poly(ethylene glycol) (PEG) brush-arm star polymers (BASPs) with narrow mass distributions and tunable nanoscopic sizes are synthesized in via ring opening metathesis polymerization (ROMP) of a PEG-norbornene macromonomer followed by transfer of portions of the resulting living brush initiator to vials containing varied amounts of a rigid, photo-cleavable bis-norbornene crosslinker.

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Neuroscience

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices
Martina Sonego *1, Ya Zhou *1, Madeleine Julie Oudin 2, Patrick Doherty 1, Giovanna Lalli 1
1Wolfson Centre for Age-Related Diseases, King's College London, 2David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology

Neuroblast migration is a fundamental event in postnatal neurogenesis. We describe a protocol for efficient labeling of neuroblasts by in vivo postnatal electroporation and subsequent visualization of their migration using time-lapse imaging of acute brain slices. We include a description for the quantitative analysis of neuroblast dynamics by video tracking.

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Bioengineering

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform
Armon Sharei 1, Nahyun Cho 1, Shirley Mao 1, Emily Jackson 1, Roberta Poceviciute 1, Andrea Adamo 1, Janet Zoldan 2, Robert Langer 1,2, Klavs F Jensen 1
1Department of Chemical Engineering, Massachusetts Institute of Technology, 2David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

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Biology

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle
William C.W. Chen 1, Arman Saparov 2,3, Mirko Corselli 4, Mihaela Crisan 5, Bo Zheng 6, Bruno Péault 7,8, Johnny Huard 9
1Stem Cell Research Center, Department of Bioengineering and Orthopedic Surgery, University of Pittsburgh, 2Department of Orthopedic Surgery, University of Pittsburgh, 3Nazarbayev University Research and Innovation System, Nazarbayev University, 4Department of Orthopaedic Surgery, UCLA Orthopaedic Hospital and the Orthopaedic Hospital Research Center, University of California at Los Angeles, 5Department of Cell Biology, Erasmus MC Stem Cell Institute, 6OHSU Center for Regenerative Medicine, Oregon Health & Science University, 7Centre for Cardiovascular Science and MRC Centre for Regenerative Medicine, Queen's Medical Research Institute and University of Edinburgh, 8David Geffen School of Medicine and the Orthopaedic Hospital Research Center, University of California at Los Angeles, 9Stem Cell Research Center, Department of Orthopedic Surgery and McGowan Institute for Regenerative Medicine, University of Pittsburgh

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

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Neuroscience

In vivo Optogenetic Stimulation of the Rodent Central Nervous System
Michelle M. Sidor 1, Thomas J. Davidson 2, Kay M. Tye 3, Melissa R. Warden 4, Karl Diesseroth 2,5, Colleen A. McClung 1
1Department of Psychiatry, University of Pittsburgh Medical Center, 2Department of Bioengineering, Stanford University, 3Department of Brain and Cognitive Sciences, Picower Institute for Learning and Memory, Massachusetts Institute of Technology, 4Department of Neurobiology and Behavior, Cornell University, 5Department of Psychiatry and Behavioral Sciences, Stanford University

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

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Neuroscience

Design and Fabrication of Ultralight Weight, Adjustable Multi-electrode Probes for Electrophysiological Recordings in Mice
Philip M. Brunetti *1, Ralf D. Wimmer *1, Li Liang 1, Joshua H. Siegle 2, Jakob Voigts 2, Matthew Wilson 2, Michael M. Halassa 1
1The Neuroscience Institute, New York University Langone Medical Center, 2Department of Brain and Cognitive Science, Massachusetts Institute of Technology

Understanding the neural substrates of behavior requires brain circuit ensemble recording. Because of its genetic tractability, the mouse offers a model for circuit dissection and disease mimicry. Here, a method of designing and fabricating miniaturized probes is described that is suitable for targeting deep brain structure in the mouse.

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JoVE Core

Micro-masonry for 3D Additive Micromanufacturing
Hohyun Keum 1, Seok Kim 1
1Mechanical Science and Engineering, University of Illinois at Urbana-Champaign

This paper introduces a 3D additive micromanufacturing strategy (termed ‘micro-masonry’) for the flexible fabrication of microelectromechanical system (MEMS) structures and devices. This approach involves transfer printing-based assembly of micro/nanoscale materials in conjunction with rapid thermal annealing-enabled material bonding techniques.

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Engineering

Making Record-efficiency SnS Solar Cells by Thermal Evaporation and Atomic Layer Deposition
Rafael Jaramillo 2,4, Vera Steinmann 1,2, Chuanxi Yang 3, Katy Hartman 2,4, Rupak Chakraborty 1,2, Jeremy R. Poindexter 2,4, Mariela Lizet Castillo 2, Roy Gordon 5, Tonio Buonassisi 1,2
1Department of Mechanical Engineering, Massachusetts Institute of Technology, 2Laboratory for Manufacturing and Productivity, Massachusetts Institute of Technology, 3School of Engineering and Applied Sciences, Harvard University, 4Department of Materials Science and Engineering, Massachusetts Institute of Technology, 5Department of Chemistry & Chemical Biology, Harvard University

Tin sulfide (SnS) is a candidate material for Earth-abundant, non-toxic solar cells. Here, we demonstrate the fabrication procedure of the SnS solar cells employing atomic layer deposition, which yields 4.36% certified power conversion efficiency, and thermal evaporation which yields 3.88%.

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Biology

Sampling Blood from the Lateral Tail Vein of the Rat
Graham Lee 1, Ki A. Goosens 1,2
1McGovern Institute for Brain Research, Massachusetts Institute of Technology, 2Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology

Blood samples are useful for assessing biomarkers of physiological states or disease in vivo. Here we describe the methodology to sample blood from the lateral tail vein in the rat. This method provides rapid samples with minimal pain and invasiveness.

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Biology

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays
Leanna S. Morinishi 1, Paul Blainey 2
1Broad Institute, 2Department of Biological Engineering, Massachusetts Institute of Technology

We describe an endpoint digital assay for quantifying nucleic acids with a simplified (analog) readout. We measure bulk fluorescence of droplet-based digital assays using a standard qPCR machine rather than specialized instrumentation and confirm our results by microscopy.

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Biology

Experimental Multiscale Methodology for Predicting Material Fouling Resistance
Pavlina Karafillis 1, Abdulla Alhajri 2, Vikash Mishra 3, Leigh Lin 1, Rasheed Auguste 2, Gabrielle Ledoux 2, Alan Schwartzman 4, Ekaterina Paramonova 2, Michael P. Short 2
1Department of Mechanical Engineering, Massachusetts Institute of Technology, 2Department of Nuclear Science and Engineering, Massachusetts Institute of Technology, 3Department of Mechanical Engineering, University of Arkansas, 4Department of Materials Science and Engineering, Massachusetts Institute of Technology

Fouling, or corrosion product deposition, plagues numerous fields of energy production. At its core is adhesion between dissimilar materials. Understanding and controlling adhesion could reduce or eliminate fouling. This paper demonstrates pool boiling experiments (macroscale) and atomic force microscope force-spectroscopy measurements (microscale), which when in agreement, indicate fouling-resistance.

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Biology

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis
Alexandra Naba 1, Karl R. Clauser 2, Richard O. Hynes 1
1Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 2Proteomics Platform, Broad Institute

This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry.

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Chemistry

Reverse Microemulsion-mediated Synthesis of Monometallic and Bimetallic Early Transition Metal Carbide and Nitride Nanoparticles
Sean T. Hunt 1, Yuriy Román-Leshkov 1
1Chemical Engineering, Massachusetts Institute of Technology

A “removable ceramic coating method” is presented in visual format for the synthesis of non-sintered and metal-terminated monometallic and bimetallic early transition metal carbide and nitride nanoparticles with tunable sizes and crystal structures.

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Bioengineering

High Speed Sub-GHz Spectrometer for Brillouin Scattering Analysis
Kim V. Berghaus 1, Seok H. Yun 2,3, Giuliano Scarcelli 1
1Fischell Department of Bioengineering, University of Maryland, 2Wellman Center for Photomedicine, Harvard Medical School, Massachusetts General Hospital, 3The Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology

Here we present a protocol to build a rapid Brillouin spectrometer. Cascading virtually imaged phase array (VIPA) etalons achieve a measurement speed more than 1,000 times faster than traditional scanning Fabry-Perot spectrometers. This improvement provides the means for Brillouin analysis of tissue and biomaterials at low power levels in vivo.

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Medicine

A Multimodal Imaging- and Stimulation-based Method of Evaluating Connectivity-related Brain Excitability in Patients with Epilepsy
Mouhsin M. Shafi 1,2,3, Susan Whitfield-Gabrieli 4, Catherine J. Chu 1,5, Alvaro Pascual-Leone 1,2,3, Bernard S. Chang 1,2
1Department of Neurology, Harvard Medical School, 2Department of Neurology, Beth Israel Deaconess Medical Center, 3Berenson-Allen Center for Noninvasive Brain Stimulation, Beth Israel Deaconess Medical Center, 4Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, 5Department of Neurology, Massachusetts General Hospital

Resting-state functional-connectivity MRI has identified abnormalities in patients with a wide range of neuropsychiatric disorders, including epilepsy due to malformations of cortical development. Transcranial Magnetic Stimulation in combination with EEG can demonstrate that patients with epilepsy have cortical hyperexcitability in regions with abnormal connectivity.

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Engineering

Creating Sub-50 Nm Nanofluidic Junctions in PDMS Microfluidic Chip via Self-Assembly Process of Colloidal Particles
Xi Wei 1,2, Abeer Syed 1, Pan Mao 3, Jongyoon Han 4, Yong-Ak Song 1,2
1Division of Engineering, New York University Abu Dhabi (NYUAD), 2Department of Chemical and Biomolecular Engineering, New York University Tandon School of Engineering, 3Newomics, Inc., 4Department of Electrical Engineering and Computer Science, Department of Biological Engineering, MIT

We propose a simple self-assembly technique of silica colloidal nanoparticles to create a nanofluidic junction between two microchannels in polydimethylsiloxane (PDMS). Using this technique, a nanoporous bead membrane with a pore size down to ~45 nm was built inside a microchannel and applied to electrokinetic preconcentration of DNA samples.

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Neuroscience

Characterizing Multiscale Mechanical Properties of Brain Tissue Using Atomic Force Microscopy, Impact Indentation, and Rheometry
Elizabeth Peruski Canovic 1, Bo Qing 2, Aleksandar S. Mijailovic 3, Anna Jagielska 2, Matthew J. Whitfield 2, Elyza Kelly 4, Daria Turner 4, Mustafa Sahin 4, Krystyn J. Van Vliet 1,2
1Department of Materials Science and Engineering, Massachusetts Institute of Technology, 2Department of Biological Engineering, Massachusetts Institute of Technology, 3Department of Mechanical Engineering, Massachusetts Institute of Technology, 4Department of Neurology, The F.M. Kirby Neurobiology Center, Boston Children’s Hospital, Harvard Medical School

We present a set of techniques to characterize the viscoelastic mechanical properties of brain at the micro-, meso-, and macro-scales.

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Developmental Biology

Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue
James E. Baily 1, William C.W. Chen 2,3, Nusrat Khan 4, Iain R. Murray 4, Zaniah N. González Galofre 4, Johnny Huard 5,6, Bruno Péault 4,7
1Centre for Cardiovascular Science, Queen's Medical Research Institute, University of Edinburgh, 2Department of Bioengineering and Orthopaedic Surgery, University of Pittsburgh, 3Research Laboratory of Electronics and Department of Biological Engineering, Massachusetts Institute of Technology, 4MRC Centre for Regenerative Medicine, University of Edinburgh, 5Stem Cell Research Center, Department of Orthopaedic Surgery, University of Pittsburgh, 6Department of Orthopaedic Surgery, University of Texas Health Science Center at Houston, 7Department of Orthopaedic Surgery, UCLA Orthopaedic Hospital, David Geffen School of Medicine, University of California at Los Angeles

Human cardiac tissue harbours multipotent perivascular precursor cell populations that may be suitable for myocardial regeneration. The technique described here allows for the simultaneous isolation and purification of two multipotent stromal cell populations associated with native blood vessels, i.e. CD146+CD34- pericytes and CD34+CD146- adventitial cells, from the human myocardium.

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Engineering

Electrospray Deposition of Uniform Thickness Ge23Sb7S70 and As40S60 Chalcogenide Glass Films
Spencer Novak 1, Pao-Tai Lin 2,3, Cheng Li 4, Nikolay Borodinov 1, Zhaohong Han 5, Corentin Monmeyran 5, Neil Patel 5, Qingyang Du 5, Marcin Malinowski 4, Sasan Fathpour 4, Chatdanai Lumdee 4, Chi Xu 4, Pieter G. Kik 4, Weiwei Deng 6, Juejun Hu 7, Anuradha Agarwal 7, Igor Luzinov 1, Kathleen Richardson 4
1Department of Materials Science and Engineering, Clemson University, 2Department of Materials Science and Engineering, Texas A&M University, 3Department of Electrical and Computer Engineering, Texas A&M University, 4College of Optics and Photonics, Center for Research and Education in Optics and Lasers (CREOL), University of Central Florida, 5Department of Materials Science and Engineering, Massachusetts Institute of Technology, 6Department of Mechanical Engineering, Virginia Polytechnic Institute, 7Microphotonics Center, Massachusetts Institute of Technology

A method of uniform thickness solution-derived chalcogenide glass film deposition is demonstrated using computer numerical controlled motion of a single-nozzle electrospray.

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Engineering

Applying X-ray Imaging Crystal Spectroscopy for Use as a High Temperature Plasma Diagnostic
Norman M. Cao 1, Andrés M. Mier Valdivia 2, John E. Rice 3
1Department of Nuclear Science and Engineering, Massachusetts Institute of Technology, 2Department of Physics, Massachusetts Institute of Technology, 3Plasma Science and Fusion Center, Massachusetts Institute of Technology

X-ray spectra provide a wealth of information on high temperature plasmas. This manuscript presents the operation of a high wavelength resolution spatially imaging X-ray spectrometer used to view hydrogen- and helium-like ions of medium atomic number elements in a tokamak plasma.

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Genetics

Detection of Copy Number Alterations Using Single Cell Sequencing
Kristin A. Knouse 1,2,3, Jie Wu 4, Austin Hendricks 5
1Koch Institute for Integrative Cancer Research, Department of Biology, Massachusetts Institute of Technology, 2Howard Hughes Medical Institute, 3Division of Health Sciences and Technology, Harvard Medical School, 4The Barbara K. Ostrom (1978) Bioinformatics and Computing Facility in the Swanson Biotechnology Center, Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 5BioMicro Center, Department of Biology, Massachusetts Institute of Technology

Single cell sequencing is an increasingly popular and accessible tool for addressing genomic changes at high resolution. We provide a protocol that uses single cell sequencing to identify copy number alterations in single cells.

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Biochemistry

A Simple, Robust, and High Throughput Single Molecule Flow Stretching Assay Implementation for Studying Transport of Molecules Along DNA
Kan Xiong 1,2, Paul C. Blainey 1,2
1Broad Institute, Massachusetts Institute of Technology and Harvard Medical School, 2Dept. of Biological Engineering, Massachusetts Institute of Technology

This protocol demonstrates a simple, robust and high throughput single molecule flow-stretching assay for studying one-dimensional (1D) diffusion of molecules along DNA.

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Behavior

Large Volume, Behaviorally-relevant Illumination for Optogenetics in Non-human Primates
Leah C Acker 1,2,3, Erica N. Pino 1,4,5, Edward S. Boyden 1,6,7, Robert Desimone 1,7
1McGovern Institute, Massachusetts Institute of Technology, 2Harvard-MIT Division of Heath Sciences and Technology, 3Department of Anesthesiology, Duke University Medical Center, 4Department of Biology, Massachusetts Institute of Technology, 5Division of Pharmacoengineering and Molecular Pharmaceutics, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, 6Media Lab and Department of Biological Engineering, Massachusetts Institute of Technology, 7Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology

A protocol to build a tissue penetrating illuminator for delivering light over large volumes with minimal diameter is presented.

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Chemistry

Utilization of Stop-flow Micro-tubing Reactors for the Development of Organic Transformations
Ren Wei Toh 1, Jie Sheng Li 1, Jie Wu 1
1Department of Chemistry, National University of Singapore

A protocol for organic reaction screening using stop-flow micro-tubing (SFMT) reactors employing gaseous reactants and/or visible-light mediated reactions is presented.

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Cancer Research

Generation and Isolation of Cell Cycle-arrested Cells with Complex Karyotypes
Ruoxi W. Wang *1, Emily MacDuffie *1, Stefano Santaguida 1
1Koch Institute for Integrative Cancer Research at MIT, Department of Biology, Massachusetts Institute of Technology

Aneuploidy leads to genome instability, which eventually produces cell cycle-arrested cells with complex karyotypes. This paper provides a simple and convenient method to isolate aneuploid cells with complex karyotypes that cease to divide.

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Biology

Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-β Signaling
Hiroshi I. Suzuki 1, Masafumi Horie 2,3, Hajime Mihira 4, Akira Saito 2
1David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 2Department of Respiratory Medicine, Graduate School of Medicine, The University of Tokyo, 3Hastings Center for Pulmonary Research, Division of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, Keck School of Medicine, University of Southern California, 4Department of Molecular Pathology, Graduate School of Medicine, The University of Tokyo

A protocol for in vitro induction of endothelial-mesenchymal transition (EndMT), which is useful for investigating cellular signaling pathways involved in EndMT, is described. In this experimental model, EndMT is induced by treatment with TGF-β in MS-1 endothelial cells.

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Engineering

A Modular Microfluidic Technology for Systematic Studies of Colloidal Semiconductor Nanocrystals
Robert W Epps 1, Kobi C Felton 1, Connor W Coley 2, Milad Abolhasani 1
1Chemical and Biomolecular Engineering, North Carolina State University, 2Chemical Engineering, Massachusetts Institute of Technology

Detailed herein are the operation and assembly protocols of a modular microfluidic screening platform for the systematic characterization of colloidal semiconductor nanocrystal syntheses. Through fully adjustable system arrangements, highly efficient spectra collection may be carried out across 4 orders of magnitude reaction time scales within a mass transfer-controlled sampling space.

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Immunology and Infection

Label-free Neutrophil Enrichment from Patient-derived Airway Secretion Using Closed-loop Inertial Microfluidics
Hyunryul Ryu 1, Kyungyong Choi 1,2, Yanyan Qu 3, Taehong Kwon 1,2, Janet S. Lee 3,5, Jongyoon Han 1,2,4
1Research Laboratory of Electronics, Massachusetts Institute of Technology, 2Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, 3Department of Medicine, University of Pittsburgh, 4Department of Biological Engineering, Massachusetts Institute of Technology, 5Vascular Medicine Institute, University of Pittsburgh

In this research, we demonstrate a label-free neutrophil separation method from clinical airway secretions using closed-loop operation of spiral inertial microfluidics. The proposed method would expand the clinical in vitro assays for various respiratory diseases.

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Biochemistry

Analyzing Dynamic Protein Complexes Assembled On and Released From Biolayer Interferometry Biosensor Using Mass Spectrometry and Electron Microscopy
Alexandra J. Machen 1, Pierce T. O'Neil 1, Bradley L. Pentelute 2, Maria T. Villar 1, Antonio Artigues 1, Mark T. Fisher 1
1Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, 2Department of Chemistry, Massachusetts Institute of Technology

Here we present a protocol to monitor the assembly and disassembly of the anthrax toxin using biolayer interferometry (BLI). Following assembly/disassembly on the biosensor surface, the large protein complexes are released from the surface for visualization and identification of components of the complexes using electron microscopy and mass spectrometry, respectively.

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JoVE Journal

CRISPR Guide RNA Cloning for Mammalian Systems
Sathiji Nageshwaran *1,2, Alejandro Chavez *1,2,3, Nan Cher Yeo 1,2, Xiaoge Guo 1,2, Alissa Lance-Byrne 1, Angela Tung 1, James J. Collins 1,4,5,6,7, George M. Church 1,2
1Wyss Institute for Biologically Inspired Engineering, Harvard University, 2Department of Genetics, Harvard Medical School, 3Department of Pathology, Massachusetts General Hospital, 4Institute for Medical Engineering & Science, Massachusetts Institute of Technology, 5Synthetic Biology Center, Massachusetts Institute of Technology, 6Department of Biological Engineering, Massachusetts Institute of Technology, 7Broad Institute

Here, a simple, efficient, and cost-effective method of sgRNA cloning is outlined.

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Engineering

Multiscale Structures Aggregated by Imprinted Nanofibers for Functional Surfaces
Yeonho Jeong *1, Seok Kim *2, Nicholas Xuanlai Fang 2, Seunghang Shin 1, Hyunmin Choi 1, Seonjun Kim 1, Sin Kwon 3, Young Tae Cho 1
1Department of Mechanical Engineering, Changwon National University, 2Department of Mechanical Engineering, Massachusetts Institute of Technology, 3Printed Electronics Research Team, Korea Institute of Machinery and Materials

Presented is an easy method to fabricate nano-micro multiscale structures, for functional surfaces, by aggregating nanofibers fabricated using an anodic aluminum oxide filter.

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Engineering

Theoretical Calculation and Experimental Verification for Dislocation Reduction in Germanium Epitaxial Layers with Semicylindrical Voids on Silicon
Motoki Yako 1, Yasuhiko Ishikawa 2, Eiji Abe 1, Kazumi Wada 1,3
1Department of Materials Engineering, The University of Tokyo, 2Department of Electrical and Electronic Information Engineering, Toyohashi University of Technology, 3Department of Materials Science and Engineering, Massachusetts Institute of Technology

Theoretical calculation and experimental verification are proposed for a reduction of threading dislocation (TD) density in germanium epitaxial layers with semicylindrical voids on silicon. Calculations based on the interaction of TDs and surface via image force, TD measurements, and transmission electron microscope observations of TDs are presented.

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Cancer Research

The Clinical Application of Tumor Treating Fields Therapy in Glioblastoma
Mercedes M. Riley 1,2, Pyay San 1, Edwin Lok 1, Kenneth D. Swanson 1, Eric T. Wong 1
1Brain Tumor Center and Neuro-Oncology Unit, Department of Neurology, Harvard Medical School, Beth Israel Deaconess Medical Center, 2Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology

Glioblastoma is the most common and aggressive primary brain malignancy in adults, with most tumors recurring after initial treatment. Tumor Treating Fields (TTFields) therapy is the newest treatment modality for glioblastoma. Here, we describe the proper application of TTFields-transducer arrays on patients and discuss theory and aspects of treatment.

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Behavior

Modified Blood Collection from Tail Veins of Non-anesthetized Mice with a Vacuum Blood Collection System and Eyeglass Magnifier
Xiaoyan Liu *1, Huanhuan Li *1, Jie Wu 1, Yijun Yu 1, Mingjing Zhang 1, Wusong Zou 1, Ye Gu 1
1Heart Center, Wuhan Fourth Hospital, Puai Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology

This study reports blood sampling from tail vein in mice using a vacuum extraction tube system with eyeglass magnifier. Our method is easy to practice and could be used for repeat blood sampling in mice.

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Developmental Biology

An In Vitro 3D Model and Computational Pipeline to Quantify the Vasculogenic Potential of iPSC-Derived Endothelial Progenitors
Cody O. Crosby 1, Janet Zoldan 1
1Department of Biomedical Engineering, University of Texas at Austin

Endothelial progenitors derived from induced pluripotent stem cells (iPSC-EPs) have the potential to revolutionize cardiovascular disease treatments and to enable the creation of more faithful cardiovascular disease models. Herein, the encapsulation of iPSC-EPs in three-dimensional (3D) collagen microenvironments and a quantitative analysis of these cells’ vasculogenic potential are described.

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Bioengineering

High-resolution Imaging of Nuclear Dynamics in Live Cells under Uniaxial Tensile Strain
Ekta Makhija 1, Anna Jagielska 1,2, Krystyn J. Van Vliet 1,2,3
1BioSystems and Micromechanics Interdisciplinary Research Group, Singapore-MIT Alliance for Research and Technology, CREATE, 2Department of Material Science and Engineering, Massachusetts Institute of Technology, 3Department of Biological Engineering, Massachusetts Institute of Technology

Using a previously designed device to apply mechanical strain to adherent cells, this paper describes a redesigned substratum geometry and a customized apparatus for high-resolution single-cell imaging of strained cells with a 100x oil immersion objective.

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Chemistry

Facile Synthesis of Colloidal Lead Halide Perovskite Nanoplatelets via Ligand-Assisted Reprecipitation
Seung Kyun Ha 1, William A Tisdale 1
1Department of Chemical Engineering, Massachusetts Institute of Technology

This work demonstrates facile room-temperature synthesis of colloidal quantum-confined lead halide perovskite nanoplatelets by ligand-assisted reprecipitation method. Synthesized nanoplatelets show spectrally narrow optical features and continuous spectral tunability throughout the visible range by varying the composition and thicknesses.

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Genetics

Engineering Intracellular Protein Sensors in Mammalian Cells
Marina Duhkinova 1, Claudia Crina 1, Ron Weiss 2, Velia Siciliano 1
1Istituto Italiano di Tecnologia, 2Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology

Here, we present a protocol for engineering genetically-encoded intracellular protein sensor-actuator(s). The device specifically detects target proteins through intracellular antibodies (intrabodies) and responds by switching on gene transcriptional output. A general framework is built to rapidly replace intrabodies, enabling rapid detection of any desired protein, without altering the general architecture.

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Engineering

Lumped-Parameter and Finite Element Modeling of Heart Failure with Preserved Ejection Fraction
Luca Rosalia *1,2, Caglar Ozturk *1, Ellen T. Roche 1,2,3
1Institute for Medical Engineering and Science, Massachusetts Institute of Technology, 2Health Science and Technology Program, Harvard/Massachusetts Institute of Technology, 3Department of Mechanical Engineering, Massachusetts Institute of Technology

This work introduces two computational models of heart failure with preserved ejection fraction based on a lumped-parameter approach and finite element analysis. These models are used to evaluate the changes in the hemodynamics of the left ventricle and related vasculature induced by pressure overload and diminished ventricular compliance.

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Cancer Research

Longitudinal Intravital Imaging Through Clear Silicone Windows
Laura Maiorino *1,2,3, Margaret Shevik *1,4,5, José M. Adrover 1, Xiao Han 1,6, Elias Georgas 7,8, John Erby Wilkinson 9, Harrison Seidner 1, Leonie Foerschner 1, David A. Tuveson 1, Yi-Xian Qin 8, Mikala Egeblad 1
1Cold Spring Harbor Laboratory, 2Cold Spring Harbor Laboratory School of Biological Sciences, 3Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 4Medical Scientist Training Program, School of Medicine, Stony Brook University, 5Graduate Program in Pharmacology, Stony Brook University, 6Graduate Program in Genetics, Stony Brook University, 7Graduate Program in Biomedical Engineering, Stony Brook University, 8Department of Biomedical Engineering, Stony Brook University, 9Department of Pathology, University of Michigan

An approach is here presented for long-term intravital imaging using optically clear, silicone windows that can be glued directly to the tissue/organ of interest and the skin. These windows are cheaper and more versatile than others currently used in the field, and the surgical insertion causes limited inflammation and distress to the animals.

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Biochemistry

Measuring Mitochondrial Electron Transfer Complexes in Previously Frozen Cardiac Tissue from the Offspring of Sow: A Model to Assess Exercise-Induced Mitochondrial Bioenergetics Changes
Daniel Barrera 1, Sierra Upton 2, Megan Rauch 3, Tara Notarianni 4, Ki Suk Eum 5, Megan Liberty 6, Sarmila Venkoba Sah 7, Robert Liu 8, Sean Newcomer 9, Linda E. May 10, Emre Agbas 11, Jessica Sage 12, Edina Kosa 7, Abdulbaki Agbas 7,13
1AdventHelath, 2University of Illinois Chicago School of Public Health, 3Lincoln Memorial University, 4Children’s Mercy Hospital, 5Scripps Clinic, 6Cincinnati Children’s Hospital Medical Center, 7Kansas City University, 8Roblex Rex Veterans Affairs Medical Center, 9California State University San Marcos, 10East Carolina University, 11Massachusetts Institute of Technology, 12Boehringer Ingelheim Norway KS, 13Heartland Center for Mitochondrial Medicine

Preparation of mitochondria-enriched samples from previously frozen archived solid tissues allowed the investigators to perform both functional and analytical assessments of mitochondria in various experimental modalities. This study demonstrates how to prepare mitochondria-enriched preparations from frozen heart tissue and perform analytical assessments of mitochondria.

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Medicine

A Retrograde Implantation Approach for Peritoneal Dialysis Catheter Placement in Mice
Saran Lotfollahzadeh 1, Mengwei Zhang 1, Marc Arthur Napoleon 1, Wenqing Yin 1, Josephine Orrick 1, Nagla Elzind 1, Austin Morrissey 1, Isaac E. Sellinger 1, Lauren D. Stern 1, Mostafa Belghasem 2, Jean M. Francis 1, Vipul C. Chitalia 1,3,4
1Renal Section, Department of Medicine, Boston University Aram V. Chobanian & Edward Avedisian School of Medicine, 2Department of Biomedical Science, Kaiser Permanente Bernard J. Tyson School of Medicine, 3Veterans Affairs Boston Healthcare System, 4Institute of Medical Engineering and Sciences, Massachusetts Institute of Technology

This article describes modifications of a procedure to implant a peritoneal dialysis catheter in a murine model to avoid major technical issues observed with the conventional techniques.

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Bioengineering

Rapid Development of Cell State Identification Circuits with Poly-Transfection
Noreen Wauford 1, Ross Jones 1,2, Charles Van De Mark 3, Ron Weiss 1,3
1Department of Biological Engineering, Massachusetts Institute of Technology, 2School of Biological Engineering, University of British Columbia, 3Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology

Complex genetic circuits are time-consuming to design, test, and optimize. To facilitate this process, mammalian cells are transfected in a way that allows the testing of multiple stoichiometries of circuit components in a single well. This protocol outlines the steps for experimental planning, transfection, and data analysis.

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CryoEM Status Update and Innovative Developments
Abigail K. R. Lytton-Jean 1, David V. Mankus 1, Margaret E. Bisher 1
1Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology

CryoEM Status Update and Innovative Developments

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Engineering

Active Probe Atomic Force Microscopy with Quattro-Parallel Cantilever Arrays for High-Throughput Large-Scale Sample Inspection
Fangzhou Xia 1, Kamal Youcef-Toumi 1, Thomas Sattel 2, Eberhard Manske 3, Ivo W. Rangelow 4,5
1Mechatronics Research Lab, Department of Mechanical Engineering, Massachusetts Institute of Technology, 2Mechatronics Group, Department of Mechanical Engineering, Ilmenau University of Technology, 3Production and Precision Measurement Technology Group, Institute of Process Measurement and Sensor Technology, Ilmenau University of Technology, 4Nanoscale Systems Group, Institute of Process Measurement and Sensor Technology, Ilmenau University of Technology, 5nano analytik GmbH

Large-scale sample inspection with nanoscale resolution has a wide range of applications, especially for nanofabricated semiconductor wafers. Atomic force microscopes can be a great tool for this purpose, but are limited by their imaging speed. This work utilizes parallel active cantilever arrays in AFMs to enable high-throughput and large-scale inspections.

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Cancer Research

Venous Thrombosis Assay in a Mouse Model of Cancer
Saran Lotfollahzadeh 1, Xiaosheng Yang 1, David Jasen Wu Wong 1, Jingyan Han 2, Francesca Seta 2, Suvranu Ganguli 3, Asha Jose 1, Katya Ravid 4,5, Vipul C. Chitalia 1,6,7,8
1Renal Section, Department of Medicine, Chobanian and Avedisian School of Medicine, Boston University, 2Vascular Biology Section, Department of Medicine, Chobanian and Avedisian School of Medicine, Boston University, 3Division of Interventional Radiology, Department of Radiology, Chobanian and Avedisian School of Medicine, Boston University, 4Department of Medicine, Whitaker Cardiovascular Institute, Chobanian and Avedisian School of Medicine, Boston University, 5Department of Biochemistry and Cell Biology, Chobanian and Avedisian School of Medicine, Boston University, 6Veterans Affairs Boston Healthcare System, 7Institute of Medical Engineering and Sciences, Massachusetts Institute of Technology, 8Center of Cross-Organ Vascular Pathology, Department of Medicine, Chobanian and Avedisian School of Medicine, Boston University

This article aims to present an optimized method for assessing venous thrombosis in a mouse cancer model, using vascular clips to achieve venous ligation. Optimization minimizes variability in thrombosis-related measurements and enhances relevance to human cancer-associated venous thrombosis.

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Bioengineering

A Practical Guide to Phage- and Robotics-Assisted Near-Continuous Evolution
Samir Aoudjane 1, Stefan Golas 2, Osaid Ather 1, Michael J. Hammerling 3, Erika DeBenedictis 1
1The Francis Crick Institute, 2Massachusetts Institute of Technology, 3Future House

Phage- and Robotics-assisted Near-continuous Evolution (PRANCE) is a technique for rapid, robust protein evolution. Robotics allows the parallelization of experiments, real-time monitoring, and feedback control.

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Biochemistry

Application of Monolayer Graphene to Cryo-Electron Microscopy Grids for High-resolution Structure Determination
Andrew V. Grassetti *1, Mira B. May *1, Joseph H. Davis 1,2
1Department of Biology, Massachusetts Institute of Technology, 2Program in Computational and Systems Biology, Massachusetts Institute of Technology

The application of support layers to cryogenic electron microscopy (cryoEM) grids can increase particle density, limit interactions with the air-water interface, reduce beam-induced motion, and improve the distribution of particle orientations. This paper describes a robust protocol for coating cryoEM grids with a monolayer of graphene for improved cryo-sample preparation.

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Bioengineering

CRISPR-Cas-mediated Multianalyte Synthetic Urine Biomarker Test for Portable Diagnostics
Audrey E. Van Heest 1, Feiyang Deng 1, Renee T. Zhao 2, Nour Saida Harzallah 2,3, Heather E. Fleming 3,4, Sangeeta N. Bhatia 2,3,4,5,6,7,8, Liangliang Hao 1,2,3
1Department of Biomedical Engineering, Boston University, 2Institute for Medical Engineering and Science, Massachusetts Institute of Technology, 3Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 4Howard Hughes Medical Institute, 5Broad Institute of Massachusetts Institute of Technology and Harvard, 6Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, 7Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, 8Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School

This protocol describes a CRISPR-Cas-mediated, multianalyte synthetic urine biomarker test that enables point-of-care cancer diagnostics through the ex vivo analysis of tumor-associated protease activities.

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Genetics

AQRNA-seq for Quantifying Small RNAs
Ruixi Chen 1, Daniel Yim 2, Peter C. Dedon 1,2
1Department of Biological Engineering, Massachusetts Institute of Technology, 2Singapore-MIT Alliance for Research and Technology Antimicrobial Resistance Interdisciplinary Research Group

Absolute quantification RNA sequencing (AQRNA-seq) is a technology developed to quantify the landscape of all small RNAs in biological mixtures. Here, both the library preparation and data processing steps of AQRNA-seq are demonstrated, quantifying changes in the transfer RNA (tRNA) pool in Mycobacterium bovis BCG during starvation-induced dormancy.

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