1Department of Molecular and Cell Biology, Life Technologies, 2Life Technologies
This protocol describes how to perform cell viability and fluorescence expression assays using the Tali Image-Based Cytometer.
Published November 17, 2011. Keywords: Cell Biology, cytometry, imaging, image-based, cell viability, apoptosis, cell counting, expression assay, Dead-Cell Red, propidium iodide, PI, GFP expression, RFP expression, cell analysis, fluorescence protein expression
JoVE Immunology and Infection
1Applied Physiology Laboratory, University of North Texas
This method demonstrates a technique for assessing granulocyte function by simultaneously measuring phagocytosis of bacteria and oxidative burst. Image-based flow cytometry allowed for the identification of three distinct subsets of activated granulocytes that all differed in their relative functional capacity.
Published December 26, 2014. Keywords: Immunology, Imaging Flow Cytometry, Phagocytosis, Oxidative Burst, Granulocyte Activity, Innate Immunity, FlowSight, Amnis
1Cancer Biology, UCL Cancer Institute
Presented is a flexible informatics workflow enabling multiplexed image-based analysis of fluorescently labeled cells. The workflow quantifies nuclear and cytoplasmic markers and computes marker translocation between these compartments. Procedures are provided for perturbation of cells using siRNA and reliable methodology for marker detection by indirect immunofluorescence in 96-well formats.
Published December 16, 2014. Keywords: Cellular Biology, Image analysis, High-content analysis, Screening, Microscopy, Individual cell analysis, Multiplexed assays
JoVE Immunology and Infection
1Inserm U1019 - CNRS UMR 8024, Institut Pasteur de Lille, Université de Lille
Here, we describe a phenotypic assay applicable to the High-throughput/High-content screens of small-interfering synthetic RNA (siRNA), chemical compound, and Mycobacterium tuberculosis mutant libraries. This method relies on the detection of fluorescently labeled Mycobacterium tuberculosis within fluorescently labeled host cell using automated confocal microscopy.
Published January 17, 2014. Keywords: Infection, Mycobacterium tuberculosis, High-content/High-throughput screening, chemogenomics, Drug Discovery, siRNA library, automated confocal microscopy, image-based analysis
JoVE Developmental Biology
1Centre of Human and Aerospace Physiological Sciences, King's College London, 2Wellcome Trust-Medical Research Council, Cambridge Stem Cell Institute
The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.
Published January 12, 2015. Keywords: Developmental Biology, Stem cell Biology, Tissue Engineering, Stem Cells, Satellite Cells, Skeletal Muscle, Adipocytes, Myogenic Cells, Myoblasts, Fibroblasts, Magnetic Activated Cell Sorting, Image Analysis
1Green Center for Systems Biology, UT Southwestern Medical Center, 2Advanced Imaging Research Center, UT Southwestern Medical Center, 3Princeton University
Here we describe a workflow for rapidly analyzing and exploring collections of fluorescence microscopy images using PhenoRipper, a recently developed image-analysis platform.
Published March 19, 2014. Keywords: Basic Protocol, PhenoRipper, fluorescence microscopy, image analysis, High-content analysis, high-throughput screening, Open-source, Phenotype
1Institute for Computational Medicine and the Department of Biomedical Engineering, Johns Hopkins University
A methodology to estimate ventricular fiber orientations from in vivo images of patient heart geometries for personalized modeling is described. Validation of the methodology performed using normal and failing canine hearts demonstrate that that there are no significant differences between estimated and acquired fiber orientations at a clinically observable level.
Published January 8, 2013. Keywords: Bioengineering, Biomedical Engineering, Medicine, Anatomy, Physiology, Cardiology, Myocytes, Cardiac, Image Processing, Computer-Assisted, Magnetic Resonance Imaging, MRI, Diffusion Magnetic Resonance Imaging, Cardiac Electrophysiology, computerized simulation (general), mathematical modeling (systems analysis), Cardiomyocyte, biomedical image processing, patient-specific modeling, Electrophysiology, simulation
JoVE Immunology and Infection
1Department of Biology, Merrimack College, 2Center for Biotechnology and Biomedical Sciences, Merrimack College, 3Department of Technology R&D, Nexcelom Bioscience LLC
Here, we demonstrate how image cytometry can be used for quantification of pathogenic fungi in association with host cells in culture. This technique can be used as an alternative to CFU enumeration.
Published June 19, 2013. Keywords: Infection, Microbiology, Infectious Diseases, Medicine, Immunology, Cellular Biology, Molecular Biology, Genetics, Pathology, Mycology, Bacteria, Macrophages, Fungi, Candida, Candida albicans, yeast, Histoplasma, Image cytometry, macrophage, fungus, propidium iodide, acridine orange, Cellometer Vision, cell, imaging, cell culture
1Department of Medical Biophysics, University of Toronto, 2Platform Biological Sciences, Sunnybrook Research Institute, 3Department of Laboratory Medicine and Pathobiology, University of Toronto, 4Physical Sciences, Sunnybrook Research Institute, 5Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 6Manitoba Institute of Cell Biology, University of Manitoba
We present an image registration approach for 3-dimensional (3D) histology volume reconstruction, which facilitates the study of the changes of an organ at the level of macrostructures made up of cells . Using this approach, we studied the 3D changes between wild-type and Igfbp7-null mammary glands.
Published July 26, 2014. Keywords: Bioengineering, Histology Volume Reconstruction, Transgenic Mouse Model, Image Registration, Digital Histology, Image Processing, Mouse Mammary Gland
1Department of Chemical and Biological Engineering, Iowa State University, 2Department of Genetics, Development and Cell Biology, Iowa State University, 3Biology Program, Iowa State University
This study describes an experimental platform to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.
Published January 4, 2015. Keywords: Medicine, Mesenchymal stem cells, high throughput screening, genetic modification, cell tracking, neurotrophic factors, high content screening, HCS, neuroprotection
JoVE Immunology and Infection
1Microbiology Unit, Department of Biochemistry, University of Oxford, 2Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford
Photoactivated localization microscopy (PALM) combined with single-molecule tracking allows direct observation and quantification of protein-DNA interactions in live Escherichia coli cells.
Published March 10, 2014. Keywords: Immunology, Super-resolution microscopy, single-particle tracking, Live-cell imaging, DNA-binding proteins, DNA repair, molecular diffusion
1Department of Clinical Genetics, VU University Medical Center, 2Center for Neurogenomics and Cognitive Research, Neuroscience Campus Amsterdam
We describe a methodology combining automated cell culturing with high-content imaging to visualize and quantify multiple cellular processes and structures, in a high-throughput manner. Such methods can aid in the further functional annotation of genomes as well as identify disease gene networks and potential drug targets.
Published January 6, 2012. Keywords: Medicine, High-throughput screening, high-content screening, neurodegeneration, automated cell culturing, Parkinson’s disease
1Faculty of Engineering, Bar-Ilan University, 210 Nachum Street, Kfar Saba, Israel
A method for overcoming the optical diffraction limit is presented. The method includes a two-step process: optical phase retrieval using iterative Gerchberg-Saxton algorithm, and imaging system shifting followed by repetition of the first step. A synthetically increased lens aperture is generated along the direction of movement, yielding higher imaging resolution.
Published February 12, 2014. Keywords: Physics, Superresolution, Fourier optics, Remote Sensing and Sensors, Digital Image Processing, optics, resolution
1Department of Pharmacology and Toxicology, University of Alabama at Birmingham
Described here is a protocol for semi-automated imaging of tissue-specific fluorescence in zebrafish embryos.
Published May 17, 2014. Keywords: Developmental Biology, zebrafish, Imaging, fluorescence microscopy, estrogen, developmental biology, endocrine disrupting compounds
1Department of Medicine, University of Chicago, 2Laboratory of Biological Modeling, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 3Department of Surgery, University of Chicago, 4Diabetes Division, University of Massachusetts
Novel computer-assisted methods of large-scale procurement and analysis of immunohistochemically stained pancreatic specimens are described: (1) Virtual Slice capture of the entire section; (2) Mass analysis of large-scale data; (3) Reconstruction of 2D Virtual Slices; (4) 3D islet mapping; and (5) Mathematical analysis.
Published March 4, 2011. Keywords: Cellular Biology, beta-cells, islets, large-scale analysis, pancreas
1Pharmacology and Chemical Biology, University of Pittsburgh Drug Discovery Institute, 2Department of Microbiology and Molecular Genetics, University of Pittsburgh, 3Department of Pharmaceutical Sciences, University of Pittsburgh, 4Department of Chemistry, University of Pittsburgh
We report the development of a system for automated imaging and analysis of zebrafish transgenic embryos in multiwell plates. This demonstrates the ability to measure dose dependent effects of a small molecule, BCI, on Fibroblast Growth Factor reporter gene expression and provide technology for establishing high-throughput zebrafish chemical screens.
Published June 24, 2010. Keywords: Cellular Biology, Zebrafish, Chemical Screens, Cognition Network Technology, Fibroblast Growth Factor, (E)-2-benzylidene-3-(cyclohexylamino)-2, 3-dihydro-1H-inden-1-one (BCI), Tg(dusp6:d2EGFP)
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.
Published October 16, 2013. Keywords: Bioengineering, Microfluidics, Endothelial Cells, Leukocyte Rolling, HL-60 cells, TNF-α, P-selectin, E-selectin
1Department of Biological Sciences, University of Notre Dame
The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.
Published November 8, 2014. Keywords: Developmental Biology, zebrafish, chemical genetics, chemical screen, in vivo small molecule screen, drug discovery, whole mount in situ hybridization (WISH), high-throughput screening (HTS), high-content screening (HCS)
1Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, 2Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 3Department of Physics, Jilin University
We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the λ repressor CI 1.
Published March 15, 2013. Keywords: Biophysics, Biochemistry, Genetics, Chemistry, Molecular Biology, Cellular Biology, Microbiology, Proteins, Single molecule, fluorescence protein, protein expression, cotranslational activation, CoTrAC, cell culture, fluorescent microscopy, imaging, translational activation, systems biology
1Department of Biochemistry and Molecular Medicine, University of California, Davis, 2NSF Center for Biophotonics Science & Technology, University of California, Davis, 3University of Tromsø, 4Department of Surgery (Division of Surgical Oncology), University of California, Davis, 5UC Davis Comprehensive Cancer Center, University of California, Davis, 6Department of Biological Chemistry, University of California, Davis
Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.
Published May 3, 2013. Keywords: Cellular Biology, Biochemistry, Molecular Biology, Medicine, Cancer Biology, Biophysics, Chemical Biology, Proteins, Microscopy, Fluorescence, autophagy, arginine deiminase, prostate cancer, deconvolution microscopy, super-resolution structured-illumination microscopy, live cell imaging, tumors, autophagosomes, lysosomes, cells, cell culture, microscopy, imaging, visualization
1Materials Engineering Division, Lawrence Livermore National Laboratory, 2UCSF Center for Integrative Neuroscience and the Department of Physiology, University of California, San Francisco
Insertion of flexible neural microelectrode probes is enabled by attaching probes to rigid stiffeners with polyethylene glycol (PEG). A unique assembly process ensures uniform and repeatable attachment. After insertion into tissue, the PEG dissolves and the stiffener is extracted. An in vitro test method evaluates the technique in agarose gel.
Published September 27, 2013. Keywords: Bioengineering, Nervous System Diseases, Surgical Procedures, Operative, Investigative Techniques, Nonmetallic Materials, Engineering (General), neural interfaces, polymer neural probes, surgical insertion, polyethylene glycol, microelectrode arrays, chronic implantation
JoVE Immunology and Infection
1Department of Oncology, INSERM U661, Functional Genomic Institute, 2Department of Anatomy, University of California
By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.
Published October 6, 2014. Keywords: Cancer Biology, intravital imaging, spinning disk confocal, ApcMin/+ mice, colorectal cancer, tumor, myeloid cells
1Department of Physics, University of Massachusetts Boston
Particle-tracking microrheology can be used to non-destructively quantify and spatially map changes in extracellular matrix mechanical properties in 3D tumor models.
Published June 10, 2014. Keywords: Bioengineering, viscoelasticity, mechanobiology, extracellular matrix (ECM), matrix remodeling, 3D tumor models, tumor microenvironment, stroma, matrix metalloprotease (MMP), epithelial-mesenchymal transition (EMT)
1Department of Surgery, Duke University Medical Center, 2Department of Biomedical Engineering, Duke University, 3School of Medicine, University of Pennsylvania, 4Department of Medicine, Division of Cardiology, Duke University Medical Center
We are describing a method to subject adherent cells to laminar flow shear stress in a sterile continuous flow circuit. The cells' adhesion, morphology can be studied through the transparent chamber, samples obtained from the circuit for metabolite analysis and cells harvested after shear exposure for future experiments or culture.
Published January 17, 2012. Keywords: Bioengineering, Fluid Shear Stress, Shear Stress, Shear Force, Endothelium, Endothelial Progenitor Cells, Flow Chamber, Laminar Flow, Flow Circuit, Continuous Flow, Cell Adhesion
1Department of Biomedical Engineering, Eindhoven University of Technology
This model system starts from a myofibroblast-populated fibrin gel that can be used to study endogenous collagen (re)organization real-time in a nondestructive manner. The model system is very tunable, as it can be used with different cell sources, medium additives, and can be adapted easily to specific needs.
Published October 28, 2013. Keywords: Bioengineering, Connective Tissue, Myofibroblasts, Heart Valves, Heart Valve Diseases, Mechanotransduction, Cellular, Adaptation, Biological, Cellular Microenvironment, collagen remodeling, fibrin-based tissues, tissue engineering, cardiovascular
1Institut für Geophysik und extraterrestrische Physik, Technische Universität Braunschweig
We present a technique to achieve low-velocity to intermediate-velocity collisions between fragile dust aggregates in the laboratory. For this purpose, two vacuum drop-tower setups have been developed that allow collision velocities between <0.01 and ~10 m/sec. The collision events are recorded by high-speed imaging.
Published June 5, 2014. Keywords: Physics, astrophysics, planet formation, collisions, granular matter, high-speed imaging, microgravity drop tower
1Medications Development, Ernest Gallo Clinic and Research Center, University of California, San Francisco, 2Clinical Pharmacology and Experimental Therapeutics, University of California, San Francisco, 3Translational Research Institute and the Institute for Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Australia
We developed a software platform that utilizes Imaris Neuroscience, ImarisXT and MATLAB to measure the changes in morphology of an undefined shape taken from three-dimensional confocal fluorescence of single cells. This novel approach can be used to quantify changes in cell shape following receptor activation and therefore represents a possible additional tool for drug discovery.
Published August 31, 2012. Keywords: Cellular Biology, 3-dimensional, microscopy, quantification, morphometric, single-cell, cell dynamics
JoVE Immunology and Infection
1Department of Surgery, The Ohio State University Medical Center
Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.
Published August 29, 2012. Keywords: Immunology, Infection, Anatomy, T cells, bone marrow transplant, immunology, cell purification, x-ray irradiation, tail vein injection, bioluminescent imaging
1Department of Mechanical Engineering, University of Michigan, 2Department of Biomedical Engineering, University of Michigan, 3Department of Biomedical Engineering, Institute for Cellular and Molecular Biology, The University of Texas at Austin, 4Department of Bioengineering, University of California, Berkeley, 5Physical Biosciences Division, Lawrence Berkeley National Laboratory
Microfluidic jetting against a droplet interface lipid bilayer provides a reliable way to generate vesicles with control over membrane asymmetry, incorporation of transmembrane proteins, and encapsulation of material. This technique can be applied to study a variety of biological systems where compartmentalized biomolecules are desired.
Published February 21, 2014. Keywords: Bioengineering, Microfluidic jetting, synthetic biology, vesicle encapsulation, lipid bilayer, biochemical reconstitution, giant unilamellar vesicles
1Institute of Toxicology and Genetics, Karlsruhe Institute of Technology - Campus North, 2Institute of Organic Chemistry, Karlsruhe Institute of Technology - Campus North, 3Institute of Organic Chemistry, Karlsruhe Institute of Technology - Campus South
We describe the procedure and data analysis of a chemical screening system for glucocorticoid stress hormone signaling using zebrafish larvae: the Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY (GRIZLY) assay. The assay sensitively and specifically detects effects on glucocorticoid signaling by compounds that require metabolization or affect endogenous glucocorticoid production.
Published September 10, 2013. Keywords: Developmental Biology, Biochemistry, Vertebrates, Zebrafish, environmental effects (biological and animal), genetics (animal), life sciences, animal biology, animal models, biochemistry, bioengineering (general), Hormones, Hormone Substitutes, and Hormone Antagonists, zebrafish, Danio rerio, chemical screening, luciferase, glucocorticoid, stress, high-throughput screening, receiver operating characteristic curve, in vivo, animal model
1Department of Biomedical Engineering, Washington University, 2Institute for Information Transmission Problems, Russian Academy of Sciences, 3Department of Mechanical Engineering and Materials Science, Washington University
This article describes surface labeling and ex ovo tissue culture in the early chick embryo. Techniques amenable to time-lapse bright field, fluorescence, and optical coherence tomography imaging are presented. Tracking surface labels with high spatiotemporal resolution enables kinematic quantities such as morphogenetic strains (deformations) to be calculated in both two and three dimensions.
Published October 17, 2011. Keywords: Developmental Biology, biomechanics, labeling, chick embryo, morphogenesis, time-lapse imaging, optical coherence tomography, strain analysis
1Faculty of Health Sciences, University of Southampton, 2Electronics and Computer Sciences, University of Southampton
This report presents details of how to adopt the acromion marker cluster method of obtaining scapular kinematics when using a passive marker motion capture device. As has been described in the literature, this method provides a robust, non-invasive, three-dimensional, dynamic and valid measurement of scapular kinematics, minimizing skin movement artifact.
Published February 10, 2015. Keywords: Medicine, Scapula, kinematics, reliability, acromion marker cluster
1Department of Psychiatry, Washington University School of Medicine, 2Department of Anatomy, Washington University School of Medicine, 3Department of Neurobiology, Washington University School of Medicine
Glutamatergic synapses can switch from an active mode to a silent mode. We demonstrate that presynaptic activity status in dissociated culture of rodent neurons is visualized using a fixable form of the FM1-43 dye to visualize active synapses and immunostaining with vGluT-1 antibody to visualize all glutamate synapses.
Published January 4, 2010. Keywords: Neurobiology, glutamate, synaptic plasticity, cAMP, excitotoxicity, homeostasis, FM1-43, presynaptic plasticity
1Department of Horticulture and Crop Science, The Ohio State University
Tomato Analyzer (TA) quantifies attributes of two dimensional shapes and color in a reproducible and accurate manner. A step-by-step procedure for obtaining high quality digitalized images of tomato fruit, morphological and color analyses of these images and several applications using the data generated through this software are described.
Published March 16, 2010. Keywords: Plant Biology, morphology, color, image processing, quantitative trait loci, software
1Institute of Biophysics, Johannes Kepler Universitat Linz
This video shows experiments with subsequent analysis of protein-protein interactions by the use of micro-patterned surfaces. The approach offers the possibility to detect protein interactions in living cells and combines high throughput capabilities with the possibility to extract quantitative information.
Published March 19, 2010. Keywords: Bioengineering, protein-protein interactions, quantification, in-vivo, micro-contact-printing, micro-patterned surfaces
1Department of Mechanical Engineering, Brigham Young University, 2Naval Undersea Warfare Center, Newport, RI
A technique for performing quantitative three-dimensional (3D) imaging for a range of fluid flows is presented. Using concepts from the area of Light Field Imaging, we reconstruct 3D volumes from arrays of images. Our 3D results span a broad range including velocity fields and multi-phase bubble size distributions.
Published March 6, 2013. Keywords: Physics, Mechanical Engineering, Fluid Mechanics, Engineering, synthetic aperture imaging, light field, camera array, particle image velocimetry, three dimensional, vector fields, image processing, auto calibration, vocal chords, bubbles, flow, fluids
JoVE Application Notes
Moxi Z is the only automated cell counter that combines the Coulter Principle typically used in high-end cell counters with a patented thin-film sensor technology to allow for highly accurate (> 95%) and repeatable particle counting and sizing for a broad range of cell types - from mammalian cells to cells as small as wine yeast and more. Since today's workflows demand accurate quality control of samples, determining cell counts precisely has a significant impact on outcomes and downstream costs.
Published August 2, 2012. Keywords: Advertisement, Cellular Biology, Molecular Biology, cell counting, coulter counting, cell culture health assessment, particle sizing, mammalian cells, Moxi Z
1Department of Biomedical Science, Cornell University, 2Department of Ecology and Evolutionary Biology, Cornell University, 3Cornell University Museum of Vertebrates, 4Department of Computer Science, Cornell University
We present a non-destructive method for sampling spatial variation in the direction of light scattered from structurally complex materials. By keeping the material intact, we preserve gross-scale scattering behavior, while concurrently capturing fine-scale directional contributions with high-resolution imaging. Results are visualized in software at biologically-relevant positions and scales.
Published May 20, 2013. Keywords: Biophysics, Molecular Biology, Biomedical Engineering, Physics, Computer Science, surface properties (nonmetallic materials), optical imaging devices (design and techniques), optical measuring instruments (design and techniques), light scattering, optical materials, optical properties, Optics, feathers, light scattering, reflectance, transmittance, color, iridescence, specular, diffuse, goniometer, C. cupreus, imaging, visualization