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October, 2006
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Immunology and Infection
Developmental Biology
 JoVE Biology

Assessment of GFP Expression and Viability Using the Tali Image-Based Cytometer

1Department of Molecular and Cell Biology, Life Technologies, 2Life Technologies

JoVE 3659

This protocol describes how to perform cell viability and fluorescence expression assays using the Tali Image-Based Cytometer.

 JoVE Immunology and Infection

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

1Applied Physiology Laboratory, University of North Texas

JoVE 52201

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.

 JoVE Biology

Workflow for High-content, Individual Cell Quantification of Fluorescent Markers from Universal Microscope Data, Supported by Open Source Software

1Cancer Biology, UCL Cancer Institute

JoVE 51882

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.

 JoVE Immunology and Infection

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

1Inserm U1019 - CNRS UMR 8024, Institut Pasteur de Lille, Université de Lille

JoVE 51114

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.

 JoVE Developmental Biology

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

1Centre of Human and Aerospace Physiological Sciences, King's College London, 2Wellcome Trust-Medical Research Council, Cambridge Stem Cell Institute

JoVE 52049

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.

 JoVE Biology

Rapid Analysis and Exploration of Fluorescence Microscopy Images

1Green Center for Systems Biology, UT Southwestern Medical Center, 2Advanced Imaging Research Center, UT Southwestern Medical Center, 3Princeton University

JoVE 51280

Here we describe a workflow for rapidly analyzing and exploring collections of fluorescence microscopy images using PhenoRipper, a recently developed image-analysis platform.

 JoVE Bioengineering

Patient-specific Modeling of the Heart: Estimation of Ventricular Fiber Orientations

1Institute for Computational Medicine and the Department of Biomedical Engineering, Johns Hopkins University

JoVE 50125

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.

 JoVE Immunology and Infection

Use of Image Cytometry for Quantification of Pathogenic Fungi in Association with Host Cells

1Department of Biology, Merrimack College, 2Center for Biotechnology and Biomedical Sciences, Merrimack College, 3Department of Technology R&D, Nexcelom Bioscience LLC

JoVE 50599

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.

 JoVE Biology

Reconstruction of 3-Dimensional Histology Volume and its Application to Study Mouse Mammary Glands

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

JoVE 51325

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.

 JoVE Medicine

High Throughput Characterization of Adult Stem Cells Engineered for Delivery of Therapeutic Factors for Neuroprotective Strategies

1Department of Chemical and Biological Engineering, Iowa State University, 2Department of Genetics, Development and Cell Biology, Iowa State University, 3Biology Program, Iowa State University

JoVE 52242

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.

 JoVE Immunology and Infection

Visualizing Protein-DNA Interactions in Live Bacterial Cells Using Photoactivated Single-molecule Tracking

1Microbiology Unit, Department of Biochemistry, University of Oxford, 2Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford

JoVE 51177

Photoactivated localization microscopy (PALM) combined with single-molecule tracking allows direct observation and quantification of protein-DNA interactions in live Escherichia coli cells.

 JoVE Medicine

High Content Screening in Neurodegenerative Diseases

1Department of Clinical Genetics, VU University Medical Center, 2Center for Neurogenomics and Cognitive Research, Neuroscience Campus Amsterdam

JoVE 3452

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.

 JoVE Engineering

Time Multiplexing Super Resolving Technique for Imaging from a Moving Platform

1Faculty of Engineering, Bar-Ilan University, 210 Nachum Street, Kfar Saba, Israel

JoVE 51148

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.

 JoVE Biology

Computer-assisted Large-scale Visualization and Quantification of Pancreatic Islet Mass, Size Distribution and Architecture

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

JoVE 2471

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.

 JoVE Biology

Development of automated imaging and analysis for zebrafish chemical screens.

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

JoVE 1900

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.

 JoVE Bioengineering

Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System

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

JoVE 50866

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.

 JoVE Biology

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

1Department of Biological Sciences, University of Notre Dame

JoVE 52063

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.

 JoVE Biology

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

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

JoVE 50042

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.

 JoVE Biology

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

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

JoVE 50047

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.

 JoVE Bioengineering

Insertion of Flexible Neural Probes Using Rigid Stiffeners Attached with Biodissolvable Adhesive

1Materials Engineering Division, Lawrence Livermore National Laboratory, 2UCSF Center for Integrative Neuroscience and the Department of Physiology, University of California, San Francisco

JoVE 50609

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.

 JoVE Immunology and Infection

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

1Department of Oncology, INSERM U661, Functional Genomic Institute, 2Department of Anatomy, University of California

JoVE 51916

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.

 JoVE Bioengineering

Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology

1Department of Physics, University of Massachusetts Boston

JoVE 51302

Particle-tracking microrheology can be used to non-destructively quantify and spatially map changes in extracellular matrix mechanical properties in 3D tumor models.

 JoVE Bioengineering

Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress

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

JoVE 3349

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.

 JoVE Bioengineering

Engineering Fibrin-based Tissue Constructs from Myofibroblasts and Application of Constraints and Strain to Induce Cell and Collagen Reorganization

1Department of Biomedical Engineering, Eindhoven University of Technology

JoVE 51009

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.

 JoVE Engineering

Laboratory Drop Towers for the Experimental Simulation of Dust-aggregate Collisions in the Early Solar System

1Institut für Geophysik und extraterrestrische Physik, Technische Universität Braunschweig

JoVE 51541

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.

 JoVE Biology

An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

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

JoVE 4233

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.

 JoVE Immunology and Infection

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

1Department of Surgery, The Ohio State University Medical Center

JoVE 3697

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.

 JoVE Bioengineering

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

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

JoVE 51510

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.

 JoVE Biology

A Chemical Screening Procedure for Glucocorticoid Signaling with a Zebrafish Larva Luciferase Reporter System

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

JoVE 50439

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.

 JoVE Biology

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo

1Department of Biomedical Engineering, Washington University, 2Institute for Information Transmission Problems, Russian Academy of Sciences, 3Department of Mechanical Engineering and Materials Science, Washington University

JoVE 3129

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.

 JoVE Medicine

Measurement of Dynamic Scapular Kinematics Using an Acromion Marker Cluster to Minimize Skin Movement Artifact

1Faculty of Health Sciences, University of Southampton, 2Electronics and Computer Sciences, University of Southampton

JoVE 51717

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.

 JoVE Biology

Presynaptically Silent Synapses Studied with Light Microscopy

1Department of Psychiatry, Washington University School of Medicine, 2Department of Anatomy, Washington University School of Medicine, 3Department of Neurobiology, Washington University School of Medicine

JoVE 1676

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.

 JoVE Biology

Tomato Analyzer: A Useful Software Application to Collect Accurate and Detailed Morphological and Colorimetric Data from Two-dimensional Objects

1Department of Horticulture and Crop Science, The Ohio State University

JoVE 1856

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.

 JoVE Biology

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

1Institute of Biophysics, Johannes Kepler Universitat Linz

JoVE 1969

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.

 JoVE Engineering

Determining 3D Flow Fields via Multi-camera Light Field Imaging

1Department of Mechanical Engineering, Brigham Young University, 2Naval Undersea Warfare Center, Newport, RI

JoVE 4325

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.

 JoVE Application Notes


JoVE 5015

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.

 JoVE Engineering

Measuring Spatially- and Directionally-varying Light Scattering from Biological Material

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

JoVE 50254

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.

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